A method for detecting the activity of anti-sclerostin antibodies based on a dual-cell system

Abstract

The present invention relates to a method for detecting cell activity of an anti-sclerostin (SOST) antibody or sclerostin, the method comprising: co-culturing a cell line capable of stably expressing Wnt1 protein and a cell line capable of expressing Luciferase protein under the regulation of TCF / LEF elements to detect the activity of the anti-SOST antibody or SOST. The molecular mechanism of action (MOA) of the present invention is clear, high in specificity, high in sensitivity, and good in robustness, and is of great significance for anti-SOST antibody drug screening, quality control, and clinical application.

Description

Technical Field

[0001] The present invention relates to the field of biological drug activity detection, in particular to a method for detecting cell activity of an anti-sclerostin (SOST) monoclonal antibody, and also to a method for detecting cell activity of sclerostin. Background Art

[0002] The WNT signaling pathway is a highly conserved signal transduction pathway in evolution that regulates a variety of cell functions during human growth and adulthood. It regulates cell proliferation, cell fate decision, apoptosis, cell migration, and cell polarity during growth, and maintains stem cell function in adulthood. Therefore, inappropriate activation of the WNT pathway will cause a variety of human diseases.

[0003] Sclerostin (SOST) is a glycoprotein with 190 amino acid residues secreted by the SOST gene. It contains a cysteine ​​ring domain and belongs to the DAN / Cerberus protein family. Studies have shown that SOST regulates bone formation by binding to low-density lipoprotein receptors LRP5 / 6 to inhibit WNT signal transduction, making it a popular target for the treatment of osteoporosis. Currently, many companies have developed monoclonal antibody drugs for sclerostin protein, and the marketed product includes Amgen's Romosozumab.

[0004] Biological activity testing is a necessary step in the development and production of monoclonal antibody drugs. The methods reported in the literature for testing the in vitro biological activity of anti-SOST antibodies include: (1) MC3T3-E1 cell line calcification experiment; (2) C2C12 cell line alkaline phosphatase test; (3) HEK-293T cell line transiently transfected with Wnt1, LRP6 and TCF / LEF-Luciferase genes for testing; (4) HEK-293T cell line, screening for cell lines stably expressing Wnt1, LRP6 and TCF / LEF-Luciferase genes for testing.

[0005] The MC3T3-E1 cell line is a preosteoblast isolated from the parietal bone of newborn C57BL / 6 mice, and it has been confirmed that it has the characteristics of osteoblast phenotypic differentiation. This unterminally differentiated preosteoblast will differentiate into osteoblasts in an osteogenic induction medium containing ascorbic acid, dexamethasone, and sodium β-glycerophosphate, generating calcified nodule deposits. Studies have shown that SOST can significantly inhibit the calcification process of MC3T3-E1 cells, and anti-SOST antibodies can effectively relieve the inhibition. The MC3T3-E1 cell line in the differentiation process was induced by the combination of anti-SOST antibodies with different concentration gradients and a certain concentration of SOST, and the biological activity of anti-SOST antibodies was reflected by detecting the calcium ion concentration. The disadvantages of this method are: (1) the experimental cycle is too long (7 days of culture + 21 days of calcification induction), which is not suitable for high-throughput antibody screening and product batch release; (2) the detection window is small (about 4 times the SOST inhibition window) and the sensitivity is low; (3) the robustness is poor, and a variety of environmental factors or experimental conditions affect the calcification results, such as serum batch, frequency and force of incubator door opening and closing, inducer, etc.

[0006] C2C12, mouse myoblasts. A certain concentration of Wnt3a conditioned medium can activate the Wnt / β-catein signaling pathway, causing β-catein to bind to the TCF / LEF transcription element and initiate alkaline phosphatase expression. SOST can bind to the Wnt protein co-receptor LRP5 / 6, inhibit the Wnt / β-catein signaling pathway, and significantly reduce the expression of alkaline phosphatase. Different concentration gradients of anti-SOST antibodies were used together with a certain concentration of SOST and Wnt3a conditioned medium or Wnt3a recombinant protein to act on C2C12 cell lines, and the biological activity of anti-SOST antibodies was reflected by the alkaline phosphatase level. The disadvantages of this method are: (1) C2C12 cells are prone to differentiation and produce higher levels of alkaline phosphatase, resulting in a higher background value; (2) Wnt3a and SOST proteins have different LRP5 / 6 binding sites and do not have a direct competitive relationship; (3) The detection window is too small (SOST inhibition window is 8-10 times, and antibody release inhibition window is 1.5-2 times), and the sensitivity is low; (4) The robustness is poor and is easily affected by the number of cell passages.

[0007] Transient transfection detection method based on HEK-293T cells. HEK-293T cells were used as host cells, and Wnt1 protein, LRP6 and TCF / LEF-Luciferase expression vectors were transiently transfected. After 24 hours of transient transfection, they were treated with a certain concentration of SOST and anti-SOST antibodies with different concentration gradients. The Luciferase expression level was detected after 18-24 hours. The biological activity of the anti-SOST antibody was reflected by the Luciferase response level. The disadvantages of this method are: (1) The operation is cumbersome and the detection results are affected by the transfection efficiency; (2) The cell viability is reduced after transient transfection, which affects the expression level of the reporter gene; (3) Wnt1 has been overexpressed before the action of SOST, resulting in a high background value and a small detection window; (4) The experimental cost is high, and sufficient high-purity plasmids need to be prepared before each test, which is not suitable for high-throughput antibody screening and product release.

[0008] A method for detecting stable expression cell lines based on HEK-293T cells. CN108866002A discloses obtaining a HEK-293T cell line that stably expresses Wnt1 protein, LRP6 receptor and TCF / LEF-Luciferase by limiting dilution or flow cytometry screening technology, treating with anti-SOST antibodies of different concentration gradients and a certain concentration of SOST, and detecting the Luciferase expression level for 18-24 hours. The biological activity of the anti-SOST antibody is reflected by the Luciferase response level. The stable cell line method avoids the shortcomings of transient transfection, such as cumbersome operation and high cost, but still has the following shortcomings: (1) LRP6 receptor and Wnt1 protein are continuously overexpressed in the target cells, which is not consistent with the human osteoporosis pathological model; (2) LRP6 receptor and Wnt1 protein are continuously overexpressed in the target cells, resulting in a high background value of Luciferase and a small detection window (2-4 times the antibody inhibition window); (3) The detection sensitivity is low, and a low SOST level has no inhibitory effect on the signaling pathway; (4) The long-term stability of the cell line constructed by the chemical transfection method is poor.

[0009] Therefore, it is urgent to develop a cell activity detection method for anti-sclerostin antibodies with a clear molecular mechanism of action, high specificity, high sensitivity, good robustness, and simple operation, which is of great significance for anti-SOST antibody drug screening, quality control, and clinical application. Summary of the invention

[0010] Based on the above-mentioned deficiencies in the prior art, the present invention uses a lentiviral infection method to construct cell lines that stably express Wnt1 protein and express Luciferase under the control of TCF / LEF elements, and detects the cell activity of anti-SOST antibodies by co-culturing the two cell lines at a certain cell density and mixing ratio. The method has high detection sensitivity, high signal-to-noise ratio, good robustness, short detection cycle, and simple operation, and can be applied to molecular screening of anti-SOST antibodies and batch release of antibody production processes.

[0011] The present invention provides a method for detecting the cell activity of an anti-SOST antibody or SOST, the method comprising: co-culturing a cell line that can stably express a Wnt1 protein and a cell line that can express a reporter gene under the regulation of a TCF / LEF element to detect the cell activity of the anti-SOST antibody or SOST; wherein the reporter gene is selected from a Luciferase protein or a fluorescent protein, preferably a Luciferase protein. As an exemplary illustration, the fluorescent protein includes but is not limited to a green fluorescent protein (GFP) and a red fluorescent protein (RFP).

[0012] In the method of the present invention, as one of the embodiments, the Wnt1 protein is expressed by the lentiviral expression vector plenti-CN-Wnt1, which is the nucleotide sequence shown in SEQ ID NO: 3; the TCF / LEF element and Luciferase protein are expressed by the lentiviral expression vector plenti-TCF / LEF-Luciferase, which is the nucleotide sequence shown in SEQ ID NO: 6.

[0013] In the method of the present invention, as one of the embodiments, the cell line includes but is not limited to 293 cell subtypes such as HEK-293T and HEK-293 or other human adherent cells. As an exemplary illustration, the cell line is selected from 293 cell subtypes or other human adherent cells, preferably HEK-293T or HEK-293, and more preferably HEK-293T cell line.

[0014] In the method of the present invention, as one of the embodiments, the method further comprises: the cell line expressing Wnt1 protein is 293T-Wnt1, the cell line expressing Luciferase protein is 293T-TCFLuc, and the 293T-TCFLuc cell seeding density is 10000 to 30000 cells / well, preferably 30000 cells / well.

[0015] In the method of the present invention, as one of the embodiments, the method further comprises: the mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 10 to 0.5:1, preferably 10 to 5:1, and more preferably 5:1.

[0016] In the method of the present invention, as one of the embodiments, the 293T-TCFLuc cell seeding density is 30,000 cells / well, and the mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 5:1.

[0017] In the method of the present invention, as one of the embodiments, the method further comprises:

[0018] (1) preparing a solution of recombinant human SOST protein and the anti-SOST antibody to be tested, or preparing a solution of the SOST protein to be tested;

[0019] (2) 293T-TCFLuc and 293T-Wnt1 cell treatment and 96-well plate seeding;

[0020] (3) adding the mixture of the recombinant human SOST protein and the anti-SOST antibody solution to be tested or the SOST protein solution to be tested in step (1) to the 96-well plate inoculated in step (2) and returning the plate to the CO2 incubator for incubation;

[0021] (4) Detecting the fluorescence response value of step (3) using a luciferase detection kit, and calculating the EC50 value of the anti-SOST antibody to be tested or the IC50 value of the SOST protein to be tested.

[0022] In the method of the present invention, as one of the embodiments, the step (1) further comprises:

[0023] Preparation of recombinant human SOST protein and anti-SOST antibody solution:

[0024] (1) Take the anti-SOST antibody solution to be tested, dilute it to 1 mg / ml with DMEM complete medium containing FBS, and then dilute it stepwise to 0.625, 1.25, 2.5, 5.0, 10, 20, 40, 60, 80, 120, 160, 240 μg / ml;

[0025] (2) taking the recombinant human SOST protein (rhSOST) stock solution, diluting it to 5-20 μg / ml, preferably 10 μg / ml, with DMEM complete medium containing FBS;

[0026] (3) taking a certain volume of rhSOST dilution and mixing it with an equal volume of anti-SOST antibody solution of different concentration gradients, and incubating at room temperature for 30-60 minutes, preferably 40 minutes, or

[0027] Preparation of SOST protein solution to be tested:

[0028] The recombinant human SOST protein (rhSOST) stock solution was taken and gradiently diluted to 0.008, 0.020, 0.051, 0.128, 0.32, 0.8, 2.0, 5.0, 10, 20, 40, 80 μg / ml with DMEM complete medium containing FBS, and incubated at room temperature for use.

[0029] In the method of the present invention, as one of the embodiments, the step (2) further comprises:

[0030] (1) Take 293T-Wnt1 cells and 293T-TCFLuc cells with good growth status and cell confluence of more than 90%, treat them according to the cell passaging method, centrifuge the cell suspension, remove the supernatant, add DMEM complete medium containing FBS to resuspend, and take the cell suspension to count;

[0031] (2) Dilute and mix the cells according to the conditions that the 293T-TCFLuc cell seeding density is 10,000 to 30,000 cells / well, the mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 10 to 5:1, and the actual required cell amount. The preferred conditions are that the 293T-TCFLuc cell seeding density is 30,000 cells / well, and the mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 5:1. After the two cell lines are diluted and mixed according to the above settings, 50 μl of cell dilution solution / well are inoculated into a white transparent bottom 96-well plate.

[0032] In the method of the present invention, as one of the embodiments, step (3) further comprises: adding the mixed solution of rhSOST and the anti-SOST antibody to be tested or the SOST protein solution to be tested prepared and incubated in step (1) at 50ul / well to the 96-well plate inoculated with cells in step (2), and returning it to the 5% CO2 incubator for incubation for 18-20h.

[0033] In the method of the present invention, as one of the embodiments, the step (4) further comprises: using a luciferase detection kit to detect the fluorescence response value of the 96-well plate after the incubation in step (3), using SoftMax or other data analysis software to process the raw data, and calculating the EC50 value of the anti-SOST antibody to be tested or the IC50 value of the SOST protein to be tested.

[0034] In the method of the present invention, as one of the embodiments, the method further comprises:

[0035] (1) Preparation of recombinant human SOST protein and anti-SOST antibody solution: dilute the anti-SOST antibody solution to 1 mg / ml with DMEM complete medium containing FBS, and then dilute to 0.625, 1.25, 2.5, 5.0, 10, 20, 40, 60, 80, 120, 160, 240 μg / ml;

[0036] The recombinant human SOST protein (rhSOST) stock solution was taken and diluted to 10 μg / ml with DMEM complete medium containing FBS;

[0037] Take a certain volume of rhSOST dilution and mix it with an equal volume of anti-SOST antibody solution of different concentration gradients, and incubate at room temperature for 40 minutes, or

[0038] Preparation of the SOST protein solution to be tested: Take the recombinant human SOST protein (rhSOST) stock solution and dilute it to 0.008, 0.020, 0.051, 0.128, 0.32, 0.8, 2.0, 5.0, 10, 20, 40, 80 μg / ml with DMEM complete medium containing FBS. Incubate at room temperature for later use;

[0039] (2) Treatment of 293T-TCFLuc and 293T-Wnt1 cells and inoculation of 96-well plates:

[0040] Take 293T-Wnt1 cells and 293T-TCFLuc cells with good growth status and cell confluence of more than 90%, treat them according to the cell passaging method, centrifuge the cell suspension, remove the supernatant, add an appropriate amount of DMEM complete medium containing FBS to resuspend, and take the cell suspension to count;

[0041] The cells were diluted and mixed according to the conditions that the 293T-TCFLuc cell seeding density was 30,000 cells / well, the mixing ratio of 293T-TCFLuc and 293T-Wnt1 was 5:1, and the actual required cell amount; after the two cell lines were diluted and mixed according to the above settings, 50 μl of cell dilution solution / well was inoculated into a white transparent bottom 96-well plate;

[0042] (3) Add 50ul / well of the rhSOST prepared and incubated in step (1) and the anti-SOST antibody mixture or the SOST protein solution to be tested into the 96-well plate seeded with cells in step (2), and return to the 5% CO2 incubator for incubation for 18-20h;

[0043] (4) Using a luciferase detection kit to detect the fluorescence response value of the 96-well plate after the incubation in step (3), using SoftMax or other data analysis software to process the raw data, and calculating the EC50 value of the anti-SOST antibody to be tested or the IC50 value of the SOST protein to be tested.

[0044] In the method of the present invention, as one of the embodiments, the anti-SOST antibodies to be tested include but are not limited to Romosozumab.

[0045] The present invention also provides a cell line 293T-Wnt1 stably expressing Wnt1 protein in any of the aforementioned methods, wherein the cell line is constructed by lentivirus infection method, and the vector expressing Wnt1 protein is a recombinant lentivirus expression vector plenti-CN-Wnt1, which has the nucleotide sequence shown in SEQ ID NO: 3.

[0046] The present invention also provides a cell line 293T-TCFLuc expressing Luciferase under the regulation of the TCF / LEF element in any of the aforementioned methods, wherein the cell line is constructed by lentiviral infection method, and the vector expressing the TCF / LEF regulatory element and the Luciferase fluorescent protein is a recombinant lentiviral expression vector plenti-TCF / LEF-Luciferase, which has the nucleotide sequence shown in SEQ ID NO: 6.

[0047] The present invention also provides the use of the aforementioned cell line in detection of β-Catein / Wnt signaling pathway, preferably in detection of anti-SOST antibody or SOST cell activity.

[0048] In the application described in the present invention, as one of the implementation schemes, the cell line 293T-Wnt1 stably expressing the Wnt1 protein and the cell line 293T-TCFLuc expressing the Luciferase protein under the regulation of the TCF / LEF element are co-cultured to detect the cell activity of the anti-SOST antibody or SOST.

[0049] In the application of the present invention, as one of the embodiments, the 293T-TCFLuc cell seeding density is 10,000 to 30,000 cells / well, preferably 30,000 cells / well, and the mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 10 to 0.5:1, preferably 10 to 5:1, and further preferably 5:1.

[0050] In the method of the present invention, as one of the embodiments, the 293T-Wnt1 cell line and the 293T-TCF / Luc cell line are prepared according to the following method:

[0051] 1) Construction of plenti-CN-Wnt1 and plenti-TCF / LEF-Luciferase

[0052] The rmWnt1 protein expression gene sequence shown in SEQ ID NO: 1 was taken to obtain a recombinant vector pUC57-Wnt1 containing the rmWnt1 protein expression gene; the pUC57-Wnt1 vector and the plenti-CN-Stat6 vector shown in SEQ ID NO: 2 were double-digested with NheI and XhoI; the digested products were gel-recovered and purified to obtain a rmWnt1 gene fragment with sticky ends of NheI and XhoI restriction sites and a plenti-CN linearized vector; the gene fragment and the linearized vector were connected with T4 ligase to obtain a lentiviral recombinant expression vector plenti-CN-Wnt1 expressing the Wnt1 protein shown in SEQ ID NO: 3;

[0053] The TCF / LEF transcriptional regulatory gene sequence shown in SEQ ID NO: 4 was taken to obtain a recombinant vector pUC57-TCF / LEF containing the TCF / LEF gene; the pUC57-TCF / LEF vector and the plenti-S6RE-Luciferase vector shown in SEQ ID NO: 5 were double-digested with ClaI and PstI; the digested products were gel-recovered and purified to obtain a TCF / LEF gene fragment with sticky ends of ClaI and PstI restriction sites and a plenti-Luciferase linearized vector; the gene fragment and the linearized vector were connected with T4 ligase to obtain a lentiviral recombinant expression vector plenti-TCF / LEF-Luciferase for expressing Luciferase fluorescent protein regulated by TCF / LEF as shown in SEQ ID NO: 6;

[0054] 2) Construction of 293T-Wnt1, 293T-TCFLuc and 293T-TCF-Wnt1 / Luc cell lines

[0055] Lentiviral packaging:

[0056] HEK-293T cells were revived and passaged 2-3 times before lentiviral packaging experiments.

[0057] (1) One day before lentiviral packaging, HEK-239T cells in good condition with a confluence of more than 90% were inoculated into T25 culture flasks for subculture at a subculture density of 2 to 4 × 10 5 cells / ml, preferably 3×10 5cells / ml; the subculture volume is 5-10ml, preferably 8ml. The culture medium is DMEM culture medium containing 5-10% FBS, preferably 10% FBS. The cells after subculture inoculation are placed in a CO2 incubator for culture, and the incubator is set to 5% CO2 and 37°C.

[0058] (2) On the day of transfection, the cell confluence should be 70-90%, preferably 80%, and the cells should be in good condition when observed under a microscope.

[0059] (3) Add the target protein expression plasmid and the lentiviral packaging auxiliary plasmid to the serum-free DMEM culture medium in a certain ratio and gently mix; the target protein expression plasmid is plenti-CN-Wnt1 or plenti-TCF / LEF-Luciferase lentiviral recombinant expression vector, the lentiviral packaging auxiliary plasmid is preferably psPAX and pMD2.G, and the mixing ratio of the target protein expression plasmid and the auxiliary plasmid psPAX and pMD2.G is 2:1:1, 5:3:3 or 5:2:3, preferably 5:3:3.

[0060] (4) Then, a certain amount of transfection reagent is added and gently mixed, and the mixture is incubated at room temperature to form a DNA-transfection reagent complex. The transfection reagent can be a conventional liposome transfection reagent (such as Lipo-2000, Lipo-3000) or a polymer transfection reagent (such as PEI), preferably Lipo-3000 or TurboFect TM Transfection Reagent.

[0061] (5) Add the incubated DNA-transfection reagent complex solution to the HEK-293T cells to be transfected and incubate them in a 37° C., 5% CO 2 incubator.

[0062] (6) On the second day after co-transfection (18-20 hours later), add sodium butyrate solution to the culture system to a final concentration of 1-10 mM, preferably 10 mM.

[0063] (7) After culturing for 8-10 hours, the original culture medium was replaced with serum-free DMEM culture medium at 30-40% of the original volume and the culture medium was returned to the incubator for continued cultivation.

[0064] (8) Continue culturing for 15-18 hours, obtain the supernatant of the virus packaging cell culture, centrifuge at 800 rpm for 5 min, and then filter the supernatant through a 0.45 μm filter membrane to obtain a lentiviral solution that can be used for infection.

[0065] Lentiviral infection:

[0066] (1) One day before lentiviral infection, HEK-239T cells in good condition with a confluence of more than 90% were inoculated into T25 culture flasks for subculture at a subculture density of 2 to 8 × 10 4 cells / ml, preferably 5×10 4 cells / ml, the culture volume is 5-10ml, preferably 8ml. The culture medium is DMEM culture medium containing 5-10% FBS, preferably DMEM culture medium containing 10% FBS.

[0067] (2) On the day of virus infection, the cells to be infected must be in good condition and have a confluence of 5-30%, preferably 10%, before they can be used for lentiviral infection experiments.

[0068] (3) Remove the original culture medium from the HEK-293T cells to be infected, and add 2-5 ml of the above-mentioned lentiviral solution to each bottle corresponding to the label, preferably adding 3 ml of the virus solution to each bottle.

[0069] (4) Add polybrene to a final concentration of 2-6 μg / ml, preferably 4 μg / ml, and return to the CO2 incubator for continued culture.

[0070] (5) 4 hours after virus infection, add 3-5 ml (preferably 3 ml) of fresh DMEM complete medium and continue culturing.

[0071] (6) 48 hours after virus infection, cells were diluted and subcultured at a volume ratio of 1:5 to 1:6.

[0072] (7) Four days after virus infection, the cells were observed and passaged. The cells were passaged at a certain density and volume for the second infection experiment. After the second infection with the same protocol, a cell line stably expressing Wnt1 protein (named 293T-Wnt1), a cell line stably expressing Luciferase under TCF / LEF regulation (named 293T-TCFLuc), and a cell line co-expressing Wnt1 and Luciferase containing TCF / LEF transcriptional regulatory elements (named 293T-TCF-Wnt1 / Luc) were obtained.

[0073] This method uses the WNT signaling pathway system. When Wnts protein ligands exist outside the cell matrix and bind correctly to the receptor, the signaling pathway is activated, β-Catenin enters the cell nucleus and binds to the transcription factor TCF / LEF, and activates the expression of downstream genes (luciferase genes). In the osteoporosis pathological model, SOST binds to the Wnt co-receptor LRP5 / 6, blocking the WNT signaling pathway and inhibiting the expression of luciferase. Anti-SOST antibodies effectively bind to SOST, hindering its binding to the co-receptor LRP5 / 6, so that the WNT signaling pathway is restored to an activated state to express luciferase. The expression level of luciferase is positively correlated with the biological activity (titer) of the anti-SOST antibody. The luciferase substrate is added, the chemiluminescence value is measured and a curve is drawn to characterize the biological activity (titer) of the anti-SOST monoclonal antibody. The present invention uses cells (293T-TCFLuc cells) that can express luciferase gene under the regulation of TCF / LEF as effector cells, and cells that stably express Wnt1 protein (293T-Wnt1) as action cells. The two are co-cultured as a detection system to detect the cell activity of anti-SOST antibodies, and can also be used for SOST protein titer detection. The molecular mechanism of the present invention is clear, high in specificity, high in sensitivity, and good in robustness, and is of great significance for anti-SOST antibody drug screening, quality control, and clinical application.

[0074] Compared with the prior art, the present invention has the following advantages and effects:

[0075] (1) The SOST inhibition window reaches about 60 times, which is more than 4 times that of the existing technology.

[0076] (2) Using the co-culture method, the window of anti-SOST antibodies to relieve the inhibition of SOST protein at a certain concentration reaches 9-16 times, which is twice that of the self-constructed co-expression cell line detection method and 5 times that of the prior art CN108866002A.

[0077] (3) High throughput, low cost, and short detection cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 is the fluorescence response value under different inoculation densities and co-culture inoculation ratios.

[0079] Figure 2 This is the inhibitory effect of recombinant human SOST on the WNT signaling pathway under different co-culture ratios.

[0080] Figure 3 This is the curve after optimization of the inhibitory concentration of recombinant human SOST.

[0081] Figure 4The anti-SOST antibody (Romosozumab) relieves the inhibitory effect under different recombinant human SOST protein concentration conditions.

[0082] Figure 5 The inhibitory effect of the recombinant human SOST protein was compared between the detection method of the present invention and the detection method reported in the literature (CN108866002A) (self-constructed cell line co-expression method).

[0083] Figure 6 The inhibition-releasing effect of anti-SOST antibodies was compared using the detection method of the present invention and the detection method reported in the literature (CN108866002A) (self-constructed cell line co-expression method). DETAILED DESCRIPTION

[0084] The following examples are used to further illustrate the present invention, but are not intended to limit the effective scope of the present invention in any way.

[0085] Example 1 Construction of Wnt1 protein, TCF / LEF transcriptional regulatory elements and Luciferase fluorescent protein expression vector

[0086] (1) Construction of Wnt1 protein recombinant expression vector (named plenti-CN-Wnt1)

[0087] Nanjing GenScript was commissioned to synthesize the rmWnt1 protein expression gene sequence (SEQ ID NO: 1) to obtain the recombinant vector pUC57-Wnt1 containing the rmWnt1 protein expression gene. The pUC57-Wnt1 vector and the plenti-CN-Stat6 vector (stored in this laboratory, sequence see SEQ ID NO: 2) were double-digested with NheI and XhoI. The digested products were gel-recovered and purified to obtain the rmWnt1 gene fragment with sticky ends of NheI and XhoI restriction sites and the plenti-CN linearized vector. The above gene fragment and the linearized vector were connected by T4 ligase to obtain the lentiviral recombinant expression vector plenti-CN-Wnt1 (SEQ ID NO: 3) expressing the Wnt1 protein.

[0088] (2) Construction of TCF / LEF transcriptional regulatory elements and Luciferase fluorescent protein recombinant expression vector (named plenti-TCF / LEF-Luciferase)

[0089] Nanjing GenScript was commissioned to synthesize the TCF / LEF transcriptional regulatory gene sequence (SEQ ID NO: 4) to obtain the recombinant vector pUC57-TCF / LEF containing the TCF / LEF gene. The pUC57-TCF / LEF vector and the plenti-S6RE-Luciferase vector (stored in this laboratory, sequence see SEQ ID NO: 5) were double-digested with ClaI and PstI. The digested products were gel-recovered and purified to obtain the TCF / LEF gene fragment with sticky ends of the ClaI and PstI restriction sites and the plenti-Luciferase linearized vector. The above gene fragment and the linearized vector were connected by T4 ligase to obtain the lentiviral recombinant expression vector plenti-TCF / LEF-Luciferase (SEQ ID NO: 6) for the expression of Luciferase fluorescent protein regulated by TCF / LEF.

[0090] Example 2 Construction of 293T-Wnt1, 293T-TCFLuc and 293T-TCF-Wnt1 / Luc cell lines

[0091] Lentiviral packaging:

[0092] After HEK-293T cells (ATCC, Cat#CRL3216) were revived, lentiviral packaging experiments were performed after 2-3 passages.

[0093] (1) One day before lentiviral packaging, HEK-239T cells in good condition with a confluence of more than 90% were inoculated into 6 T25 culture flasks (Corning, Cat#430639) for subculture. The subculture density and culture volume were 3×10 5 cells / ml, 10ml. The culture medium is DMEM culture medium (Gibco, Cat#11965-092) containing 10% FBS. On the day of transfection, the cells were observed under an inverted microscope. The cells were in good condition with a confluence of about 80% and could be used for transfection experiments.

[0094] (2) Add 4.6 μg of target protein expression plasmid, 2.7 μg of psPAX2, and 2.7 μg of pMD2.G (mass ratio of 5:3:3) into serum-free DMEM medium and mix gently; then add 20 μl of transfection reagent TurboFect TMTransfection Reagent (Thermo, Cat# R0531) was gently mixed to a final volume of 1 ml; the reaction was allowed to stand at room temperature for 20 min to form a DNA-transfection reagent complex. The target protein expression plasmids were plenti-CN-Wnt1, plenti-TCF / LEF-luciferase and the positive control plasmid pLVX-IRES-ZsGreen1 (Clontech, Cat# 632187), and a negative control group without DNA transfection was set up. Two parallel experiments were set up for plenti-CN-Wnt1 and plenti-TCF / LEF-luciferase.

[0095] (3) Add the DNA-transfection reagent complex solution to the HEK-293T cells to be transfected and incubate in a 37° C., 5% CO 2 incubator.

[0096] (4) On the second day after co-transfection (18-20 hours later), add 100 μl of 1 M sodium butyrate solution to the system to make the final concentration 10 mM.

[0097] (5) After culturing for 8-10 hours, replace the original culture medium with 4 ml of serum-free DMEM medium and return the culture medium to the incubator for continued cultivation.

[0098] (6) Continue culturing for 15-18 hours (about 48 hours after co-transfection), take the supernatant of the virus packaging cell culture, centrifuge at 800 rpm for 5 minutes, and then filter the supernatant through a 0.45 μm filter membrane to obtain a lentiviral solution that can be used for infection.

[0099] Lentiviral infection:

[0100] (1) One day before lentiviral infection, HEK-239T cells in good condition with a confluence of more than 90% were inoculated into 5 T25 culture flasks for subculture. The subculture density and culture volume were 5×10 4 cells / ml, 8ml. The culture medium is DMEM medium containing 10% FBS. On the day of virus infection, the cells were observed under an inverted microscope. The cells were in good condition with a confluence of 10-20%, and could be used for lentivirus infection experiments.

[0101] (2) Remove the original culture medium from the HEK-293T cells to be infected, and add 3 ml of the above-mentioned lentiviral solution to the corresponding marker, such as adding 3 ml of NC virus solution to the NC infection group, adding 3 ml of Green1 virus solution to the pLVX-IRES-ZsGreen1 group, adding 3 ml of Wnt1 virus solution to the plenti-CN-Wnt1 infection group, adding 3 ml of Luciferase virus solution to the plenti-TCF / LEF-luciferase infection group, and adding 2 ml of Wnt1 virus solution and 2 ml of Luciferase virus solution to the Wnt1 / Luciferase co-expression group.

[0102] (3) Add polybrene to a final concentration of 4 μg / ml and return the cells to the CO2 incubator for continued culture.

[0103] (4) 4 hours after virus infection, add 4 ml of fresh DMEM complete medium and continue culturing.

[0104] (5) 48 hours after virus infection, cells were diluted and subcultured at a volume ratio of 1:5 to 1:6.

[0105] (6) Four days after virus infection, cells were observed and processed. Under a fluorescence microscope, the positive infection group showed strong green fluorescence, and the cell fluorescence expression rate (the percentage of cells expressing GFP) was more than 95%. The cells in the plenti-CN-Wnt1 infection group, the plenti-TCF / LEF-luciferase infection group, and the Wnt1 / Luciferase co-expression group were 5×10 4 cells / ml, and the cell density and volume of 8ml were passaged for the second infection experiment. After the second infection with the same scheme, a cell line stably expressing Wnt1 protein (named 293T-Wnt1), a cell line expressing Luciferase under the regulation of TCF / LEF (named 293T-TCFLuc), and a cell line co-expressing Wnt1 and Luciferase (named 293T-TCF-Wnt1 / Luc) were obtained.

[0106] Example 3 Co-culture of 293T-Wnt1 cells and 293T-TCFLuc cells and reporter gene detection experiment

[0107] 293T-Wnt1 cells and 293T-TCFLuc cells were taken 4 days after secondary infection, and after being processed according to the cell passaging method, the cell suspension was centrifuged at 800 rpm for 5 min, the supernatant was removed, and 2 ml of DMEM complete medium was added to resuspend the cells. 50 μl of the cell suspension was taken for counting. The counting results are shown in Table 1 below:

[0108] Table 1 293T-Wnt1 cell and 293T-TCFLuc cell counting results

[0109] Cell Name <![CDATA[Viable cell density (×10 6 cells / ml)]]> Cell viability (%) Diameter(μm) 293T-Wnt1 2.4 96 14.5 293T-TCFLuc 3.6 97 13.9

[0110] Under the conditions of 293T-TCFLuc cell seeding density of 10000 cells / well, 20000 cells / well and 30000 cells / well, the mixing ratio of 293T-TCFLuc and 293T-Wnt1 was set to 40:1, 30:1, 20:1, 10:1, 5:1, 2.5:1, 1:1, 1:2, and 100 μl of cell dilution / well was inoculated into a white transparent bottom 96-well plate (Corning, Cat#3610), and the plate was returned to the CO2 incubator for incubation for 18-20 hours, and then the plate was stained with Bright-Glo TM The fluorescence response value was detected by Luciferase Assay System luciferase detection kit (Promega, Cat#2620). The test results are attached. Figure 1 .

[0111] Results: Under the condition of constant 293T-TCFluc seeding density, the expression level of Luciferase increased with the increase of 293T-Wnt1 cell density; under the condition of constant 293T-TCFLuc to 293T-Wnt ratio, the expression level of Luciferase increased with the increase of 293T-TCFLuc seeding density. Probably due to the error of cell seeding operation, when the 293T-TCFLuc cell seeding density was 10000 cells / well and 20000 cells / well. When the two cell seeding ratios were ≤2.5, the expression of Luciferase was not linear. Considering the response level of Wnt signaling pathway (expression level of Luciferase), simplicity and accuracy of experimental operation, two parameter conditions were selected for further optimization: 30000 cells / well for 293T-TCFLuc seeding density and 10 and 5 for 293T-TCFLuc to 293T-Wnt seeding ratio.

[0112] Example 4 Detection of the inhibitory effect of rhSOST protein under co-culture conditions

[0113] (1) Preparation of rhSOST protein diluent

[0114] Take the recombinant human SOST protein (rhSOST) stock solution (Cat#HST-H5245, Acro), and dilute it to 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5.0, 10, 20, 40, 80 μg / ml with complete medium containing 10% FBS in DMEM (Gibco, Cat#11965-092). Incubate at room temperature for use.

[0115] (2) Treatment of 293T-TCFLuc and 293T-Wnt1 cells and inoculation in 96-well plates

[0116] 293T-Wnt1 cells and 293T-TCFLuc cells with good growth status and cell confluence of more than 90% were taken, and after being processed according to the cell passaging method, the cell suspension was centrifuged at 800 rpm for 5 minutes, the supernatant was removed, and 2 ml of 10% FBS DMEM complete medium was added to resuspend, and 50 μl of the cell suspension was counted. The counting results are shown in Table 2 below:

[0117] Table 2 293T-Wnt1 cell and 293T-TCFLuc cell counting results

[0118] Cell Name <![CDATA[Viable cell density (×10 6 cells / ml)]]> Cell viability (%) Diameter(μm) 293T-Wnt1 1.8 97 15.2 293T-TCFLuc 2.3 96 14.5

[0119] Under the condition of 293T-TCFLuc cell seeding density of 30000 cells / well, the mixing ratio of 293T-TCFLuc and 293T-Wnt1 was set to 10:1, 5:1, and 50 μl of cell dilution / well was inoculated into a white transparent bottom 96-well plate (Corning, Cat#3610). The rhSOST dilution was added according to the preset seeding layout, and the plate was returned to the CO2 incubator for incubation for 18-20 hours. Then, the plate was stained with Bright-Glo TM The fluorescence response value was detected by Luciferase Assay System luciferase detection kit (Promega, Cat#2620). The inhibitory effect curve of rhSOST is shown in the attached Figure 2 The detection results are shown in Table 3. In this embodiment, the detection window is calculated by dividing the highest response value in the inhibition curve by the background value.

[0120] Table 3 The inhibitory effect of rhSOST when the co-culture ratio of 293T-TCFLuc and 293T-Wnt1 cell lines was 5:1 and 10:1 respectively

[0121] Co-culture ratio IC50(μg / ml) Detection window 5:1 0.289 72 times 10:1 0.208 42 times

[0122] Result analysis: When the 293T-TCFLuc cell seeding density was 30,000 cells / well and the mixing ratio of 293T-TCFLuc and 293T-Wnt1 was 5:1, a higher signal-to-noise ratio (72 times) was obtained, and the IC50 values ​​of rhSOST were close.

[0123] Example 5 Optimization of rhSOST protein inhibitory effect detection concentration

[0124] In order to make the fitting curve of the rhSOST inhibitory effect show obvious upper and lower platforms, the detection concentration dilution multiple of the rhSOST solution was further optimized under the conditions of 293T-TCFLuc cell seeding density of 30,000 cells / well and a mixing ratio of 293T-TCFLuc and 293T-Wnt1 of 5:1.

[0125] (1) Preparation of rhSOST protein diluent

[0126] Take the recombinant human SOST protein (rhSOST) stock solution (Cat#HST-H5245, Acro), and dilute it to 0.008, 0.020, 0.051, 0.128, 0.32, 0.8, 2.0, 5.0, 10, 20, 40, 80 μg / ml with DMEM complete medium containing 10% FBS. Incubate at room temperature for use.

[0127] (2) Treatment of 293T-TCFLuc and 293T-Wnt1 cells and inoculation in 96-well plates

[0128] 293T-Wnt1 cells and 293T-TCFLuc cells with good growth status and cell confluence of more than 90% were taken, and after being processed according to the cell passaging method, the cell suspension was centrifuged at 800 rpm for 5 minutes, the supernatant was removed, and 2 ml of 10% FBS DMEM complete medium was added to resuspend, and 50 μl of the cell suspension was counted. The counting results are shown in Table 4 below:

[0129] Table 4 293T-Wnt1 cell and 293T-TCFLuc cell counting results

[0130] Cell Name <![CDATA[Live cell density (×10 6 cells / ml)]]> Cell viability (%) Diameter(μm) 293T-Wnt1 1.4 98 14.5 293T-TCFLuc 1.8 97 15.3

[0131] The 293T-TCFLuc cell seeding density was set to 30,000 cells / well, and the mixing ratio of 293T-TCFLuc and 293T-Wnt1 was 5:1. 50 μl of cell dilution / well was inoculated into a white transparent bottom 96-well plate. The rhSOST dilution was added according to the preset inoculation layout. After returning to the CO2 incubator for 18-20 hours, the plate was incubated with Bright-Glo TMLuciferase Assay System luciferase detection kit detects the fluorescence response value. The rhSOST inhibition effect curve is attached. Figure 3 The test results are shown in Table 5. After optimization, the response values ​​are evenly distributed on the high platform, the oblique line and the low platform, making the protein titer detection more accurate. The calculation method of the detection window in this embodiment is the highest response value in the inhibition curve divided by the background value.

[0132] Table 5 The inhibitory effect of rhSOST after concentration gradient optimization when the co-culture ratio of 293T-TCFLuc and 293T-Wnt1 cell lines was 5:1

[0133] Co-culture ratio IC50(μg / ml) Detection window 5:1 0.44 60 times

[0134] Example 6 Selection of rhSOST concentration for anti-SOST antibody activity detection

[0135] According to the inhibition curve of rhSOST, three rhSOST concentrations with more obvious inhibition on Wnt1 signaling pathway, 1.25μg / ml, 2.5μg / ml, and 5.0μg / ml, were selected to test the activity of Romosozumab injection (Amgen).

[0136] (1) Preparation of rhSOST and Romosozumab Injection

[0137] Take Romosozumab injection, dilute it to 1 mg / ml with DMEM complete medium, and then dilute it to 0.625, 1.25, 2.5, 5.0, 10, 20, 40, 60, 80, 120, 160, 240 μg / ml;

[0138] The recombinant human SOST protein (rhSOST) stock solution (Cat#HST-H5245, Acro) was taken and diluted to 5, 10, and 20 μg / ml respectively with DMEM complete culture medium containing 10% FBS.

[0139] 90 μl of rhSOST dilutions of different concentrations were mixed with 90 μl of Romosozumab dilutions of different concentration gradients, and incubated at room temperature for 30-40 min.

[0140] (2) Treatment of 293T-TCFLuc and 293T-Wnt1 cells and inoculation in 96-well plates

[0141] 293T-Wnt1 cells and 293T-TCFLuc cells with good growth status and cell confluence of more than 90% were taken, and after being processed according to the cell passaging method, the cell suspension was centrifuged at 800 rpm for 5 minutes, the supernatant was removed, and 2 ml of 10% FBS DMEM complete medium was added to resuspend, and 50 μl of the cell suspension was counted. The counting results are shown in Table 6 below:

[0142] Table 6 293T-Wnt1 cell and 293T-TCFLuc cell counting results

[0143] Cell Name <![CDATA[Live cell density (×10 6 cells / ml)]]> Cell viability (%) Diameter(μm) 293T-Wnt1 1.2 98 14.2 293T-TCFLuc 2.2 97 14.6

[0144] Under the condition of 293T-TCFLuc cell seeding density of 30,000 cells / well, the mixing ratio of 293T-TCFLuc and 293T-Wnt1 was set to 5:1, and 50 μl of cell dilution / well was inoculated into a white transparent bottom 96-well plate. According to the preset inoculation layout, 50 μl / well of rhSOST and Romosozumab mixture was added, and the plate was returned to the CO2 incubator for incubation for 18-20 hours, and then Bright-Glo TM The fluorescence response value was detected by Luciferase Assay System luciferase detection kit (Promega, Cat#2620). The inhibitory effect curve of rhSOST is shown in the attached Figure 4 The detection results are shown in Table 7. In this embodiment, the detection window is calculated by dividing the highest response value in the inhibition release curve by the background value.

[0145] Table 7 Romosozumab bioactivity test results when the co-culture ratio of 293T-TCFLuc and 293T-Wnt1 cell lines was 5:1 and the rhSOST concentrations were 1.25μg / ml, 2.5μg / ml, and 5.0μg / ml, respectively

[0146] rhSOST concentration (μg / ml) EC50(μg / ml) Detection window 1.25 2.04 5 times 2.5 4.71 11 times 5.0 8.96 18 times

[0147] Result analysis: With the increase of rhSOST concentration, the EC50 value and signal-to-noise ratio of Romosozumab increased accordingly. Considering the antibody dosage and the response value distribution of the curve, 2.5 μg / ml was selected as the optimal rhSOST concentration for detecting antibody activity.

[0148] Example 7 Comparison of the inhibitory effects of rhSOST detected by co-culture and co-expression methods

[0149] The detection scheme of the present invention is compared with the detection scheme of Wnt1 and Luciferase co-expressing cells in the prior art.

[0150] (1) Preparation of rhSOST solution

[0151] Take the recombinant human SOST protein (rhSOST) stock solution (Cat#HST-H5245, Acro), and dilute it to 0.0256, 0.064, 0.16, 0.4, 1.0, 2.5, 5.0, 10, 20, 40 μg / ml with DMEM complete medium containing 10% FBS. Incubate at room temperature for use.

[0152] (2) Treatment of 293T-TCFLuc, 293T-Wnt1 and 293T-TCF-Wnt1 / Luc cells and inoculation in 96-well plates

[0153] Cells with good growth status and cell confluence of more than 90% were taken, and after being processed according to the cell passaging method, the cell suspension was centrifuged at 800 rpm for 5 minutes, the supernatant was removed, and 2 ml of 10% FBS DMEM complete medium was added to resuspend, and 50 μl of the cell suspension was counted. The counting results are shown in Table 8 below:

[0154] Table 8 Cell counting results

[0155]

[0156]

[0157] Co-culture cell seeding: Under the condition of 293T-TCFLuc cell seeding density of 30,000 cells / well, the mixing ratio of 293T-TCFLuc and 293T-Wnt1 was set to 5:1, that is, DMEM complete medium was used to prepare 0.6×10 6 cells / ml 293T-TCFLuc cells and 0.12×10 6 cells / ml 293T-Wnt1 cell suspension was inoculated into a white transparent bottom 96-well plate at 50 μl / well;

[0158] Co-expression cell seeding: 293T-TCF-Wnt1 / Luc seeding density was 20,000 cells / well, i.e., 293T-TCF-Wnt1 / Luc cells were diluted to 0.4×10 in DMEM complete medium. 6 cells / ml;

[0159] According to the preset inoculation layout, add 50 μl / well of rhSOST solution with different concentration gradients, put it back into the CO2 incubator and incubate for 18-20 hours. TMThe fluorescence response value was detected by Luciferase Assay System luciferase detection kit (Promega, Cat#2620). The inhibitory effect curve of rhSOST is shown in the attached Figure 5 The test results are shown in Table 9. In this embodiment, the detection window is calculated by dividing the highest response value in the inhibition curve by the background value.

[0160] Table 9 Inhibitory effect of rhSOST on Wnt signaling pathway under co-culture and co-expression conditions

[0161]

[0162] Result analysis:

[0163] The co-culture detection method has better data fit at low concentrations, and the rhSOST inhibition window is more than 4 times that of the co-expression detection method.

[0164] Example 8 Comparison of the Anti-SOST Antibody Cell Activity Detection by Co-culture and Co-expression Methods

[0165] The detection scheme of the present invention is compared with the detection scheme of Wnt1 and Luciferase co-expressing cells in the prior art.

[0166] (1) Preparation of rhSOST and Romosozumab Injection

[0167] Take Romosozumab injection, dilute it to 1 mg / ml with DMEM complete medium containing 10% FBS, and then dilute it to 0.625, 1.25, 2.5, 5.0, 10, 20, 40, 60, 80, 120, 160, 240 μg / ml;

[0168] The recombinant human SOST protein (rhSOST) stock solution (Cat#HST-H5245, Acro) was taken and diluted to 10 μg / ml with DMEM complete medium containing 10% FBS.

[0169] 200 μl of rhSOST dilutions of different concentrations were mixed with 200 μl of Romosozumab dilutions of different concentration gradients and incubated at room temperature for 40 min.

[0170] (2) Treatment of 293T-TCFLuc, 293T-Wnt1 and 293T-TCF-Wnt1 / Luc cells and inoculation in 96-well plates

[0171] Cells with good growth status and cell confluence of more than 90% were taken, and after being processed according to the cell passaging method, the cell suspension was centrifuged at 800 rpm for 5 minutes, the supernatant was removed, and 2 ml of 10% FBS DMEM complete medium was added to resuspend, and 50 μl of the cell suspension was counted. The counting results are shown in Table 10 below:

[0172] Table 10 Cell counting results

[0173]

[0174] Co-culture cell seeding: Under the condition of 293T-TCFLuc cell seeding density of 30,000 cells / well, the mixing ratio of 293T-TCFLuc and 293T-Wnt1 was set to 5:1, that is, DMEM complete medium containing 10% FBS was used to prepare 0.6×10 6 cells / ml 293T-TCFLuc cells and 0.12×10 6 cells / ml 293T-Wnt1 cell suspension was inoculated into a white transparent bottom 96-well plate at 50 μl / well;

[0175] Co-expression cell seeding: 293T-TCF-Wnt1 / Luc seeding density was 20,000 cells / well, i.e., 293T-TCF-Wnt1 / Luc cells were diluted to 0.4×10 in DMEM complete medium containing 10% FBS. 6 cells / ml (reference CN108866002A);

[0176] According to the preset inoculation layout, add 50 μl / well of rhSOST with different concentration gradients and the anti-SOST antibody mixture to be tested, put it back into the CO2 incubator and incubate for 18-20 hours. TM The fluorescence response value was detected by Luciferase Assay System luciferase detection kit (Promega, Cat#2620). The anti-SOST antibody inhibition relief effect curve is attached. Figure 6 The detection results are shown in Table 11. In this embodiment, the detection window is calculated by dividing the highest response value in the inhibition curve by the background value.

[0177] Table 11 The inhibitory effects of anti-SOST antibodies under co-culture and co-expression conditions

[0178]

[0179] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention. Sequence Listing <110> Federal Biotech (Zhuhai Hengqin) Co., Ltd. <120> A method for detecting the activity of anti-sclerostin antibodies based on a dual-cell system <130> LAC220310P <141> 2022-05-24 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1120 <212> DNA <213> Artificial Sequence <400> 1 gctagcatgg ggctctgggc gctgttgcct ggctgggttt ctgctacgct gctgctggcg 60 ctggccgctc tgcccgcagc cctggctgcc aacagcagtg gccgatggtg gggtattgtg 120 aacgtagcct cctccacgaa cctgcttaca gactccaaga gtctgcaact ggtactggag 180 cccagtctgc agctgttgag ccgcaaacag cggcgtctga tacgccaaaa tccggggatt 240 ctgcacagcg tgagtggggg gctgcagagt gccgtgcgcg agtgcaagtg gcagttccgg 300 aatcgccgct ggaactgtcc cactgctcca gggccccacc tcttcggcaa gatcgtcaac 360 cgaggctgtc gagaaacggc gtttatcttc gctatcacct ccgccggggt cacccattcg 420 gtggcgcgct cctgctcaga aggttccatc gaatcctgca cgtgtgacta ccggcggcgc 480 ggccccgggg gccccgactg gcactggggg ggctgcagcg acaacattga cttcggccgc 540 ctcttcggcc gggagttcgt ggactccggg gagaaggggc gggacctgcg cttcctcatg 600 aaccttcaca acaacgaggc aggccgtacg accgtattct ccgagatgcg ccaggagtgc 660 aagtgccacg ggatgtccgg ctcatgcacg gtgcgcacgt gctggatgcg gctgcccacg 720 ctgcgcgccg tgggcgatgt gctgcgcgac cgcttcgacg gcgcctcgcg cgtcctgtac 780 ggcaaccgcg gcagcaaccg cgcttcgcgg gcggagctgc tgcgcctgga gccggaagac 840 ccggcccaca aaccgccctc cccccacgac ctcgtctact tcgagaaatc gcccaacttc 900 tgcacgtaca gcggacgcct gggcacagca ggcacggcag ggcgcgcctg taacagctcg 960 tcgcccgcgc tggacggctg cgagctgctc tgctgcggca ggggccaccg cacgcgcacg 1020 cagcgcgtca ccgagcgctg caactgcacc ttccactggt gctgccacgt cagctgccgc 1080 aactgcacgc acacgcgcgt actgcacgag tgtctgtgac 1120 <210> 2 <211> 10425 <212> DNA <213> Artificial Sequence <400> 2 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct 240 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 300 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 360 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc 420 ccgaacaggg acttgaaagc gaaagggaaa ccagagctct ctcgacgcag gactcggctt 480 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600 attagatcgc gatgggaaaaatcggtta aggccagggg gaagaaaaaataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag aaaatactg ggacagctac aaccatccct tcagacagga 780 tcagagaac ttagatcatt atataca gtagcaaccc tctattgtgt gcatcaagg 840 frequently frequently frequently aaaaaagt 900 agaccaccg cacagcaagc ggccgctgat cttcagacct ggaggagg atatgaggga 960 attgaatt taggtagc 1020 acccaccaag gcaagagaa gagtggtgca gagaaaaa agagcagtgg gataggagc 1080 ttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag gcgcacagc atctgttgca accacagtc tggggcatca agcagctcca 1260 ggcaagaatc ctggctgtgg aagatacct aaaggatcaa cagctcctgg ggatttgggg 1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg atgctagtt ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag tgggacag aattacaa 1440 ttacacaagc ttatacact ccttaattga agaatcgcaaaccagcaag aaagaatga 1500 acaagaatta ttggattag aataatgggc aagtttgtgg aattggtttta acatacaaa 1560 ttggctgtgg tatataaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620 agttttgct gtactttcta tagtgaatag agttaggcag ggattattcac cattatcgtt 1680 1740 1740 1740 tggagagaga gagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaagaa tagtagacat 1860 atagcaaca gaatacaa ctaagaatt aaaaaaaaaaaaat tcaaattttt 1920 atcgatgtac gggccagata tacgcgttga cattgatt tgactagtta ttaatagtaa 1980 tcaattacgg gtcattagt tcatagccca tatggagt tccgcgttac attackacg 2040 gtaaatggcc cgcctggctg accgcccaac gaccccgcc cattgacgtc aataatgacg 2100 tatgttccca tagtaacgcc atagggact ttccattgac gtcaatgggt ggactattta 2160 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 2220 gacgtcaatg acggtaaatg gccgcctgg cattatgccc agtacatgac cttatgggac 2280 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 2340 tggcagtaca tcaatgggcg tggatagcgg ttgactcac ggggatttcc aagtctccac 2400 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 2460 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 2520 ataagcagag ctctctggct aactgcagcg tctcaagctt cagaattctc agatccgcta 2580 gcgctaccgg tcgccaccat gtctctgtgg ggtctggtct ccaagatgcc cccagaaaaa 2640 gtgcagcggc tctatgtgga ctttccccaa cacctgcggc atcttctggg tgactggctg 2700 gagagccagc cctgggagtt cctggtcggc tccgacgcct tctgctgcaa cttggctagt 2760 gccctacttt cagacactgt ccagcacctt caggcctcgg tggggagagca gggggagggg 2820 agcaccatct tgcaacacat cagcaccctt gagagcatat atcagaggga ccccctgaag ctggtggcca ctttcagaca aatacttcaa ggagagaaaa aagctgttat ggaacagttc cgccacttgc caatgccttt ccactggaag caggaagaac tcaagtttaa gacaggcttg cggaggctgc agcaccgagt aggggagatc caccttctcc gagaagccct gcagaagggg 3060 gctgaggctg gccaagtgtc tctgcacagc ttgatagaaa ctcctgctaa tgggactggg 3120 ccaagtgagg ccctggccat gctactgcag ggagaccactg ggagctaga ggcagccaaa gccctagtgc tgaagagaat ccagatttgg aaacggcagc agcagctggc agggaatggc gcaccgtttg aggagagcct ggccccactc caggagaggt gtgaaagcct ggtggacatt 3300 tattcccagc tacagcagga ggtagggggcg gctggtgggg agcttgagcc caagacccgg 3360. 3420. gcatcgctga ctggccggct ggatgaagtc ctgagaaccc tcgtcaccag ttgcttcctg gtggagaagc agcccccca ggtactgaag actcagacca agttccaggc tggagttcga 3480. ttcctgttgg gcttgaggtt cctgggggcc ccagccaagc ctccgctggt cagggccgac 3540 atggtgacag agaagcaggc gcgggagctg agtgtgcctc agggtcctgg ggctggagca 3600 gaaagcactg gagaaatcat caacaacact gtgcccttgg agaacagcat tcctgggaac 3660 tgctgctctg ccctgttcaa gaacctgctt ctcaagaaga tcaagcggtg tgagcggaag 3720 ggcactgagt ctgtcacaga ggagaagtgc gctgtgctct tctctgccag cttcacactt 3780 ggccccggca aactccccat ccagctccag gccctgtctc tgcccctggt ggtcatcgtc 3840 catggcaacc aagacaacaa tgccaaagcc actatcctgt gggacaatgc cttctctgag 3900 atggaccgcg tgccctttgt ggtggctgag cgggtgccct gggagaagat gtgtgaaact 3960 ctgaacctga agttcatggc tgaggtgggg accaaccggg ggctgctccc agagcacttc 4020 ctcttcctgg cccagaagat cttcaatgac aacagcctca gtatggaggc cttccagcac 4080 cgttctgtgt cctggtcgca gttcaacaag gagatcctgc tgggccgtgg cttcaccttt 4140 tggcagtggt ttgatggtgt cctggacctc accaaacgct gtctccggag ctactggtct 4200 gaccggctga tcattggctt catcagcaaa cagtacgtta ctagccttct tctcaatgag 4260 cccgacggaa cctttctcct ccgcttcagc gactcagaga ttgggggcat caccattgcc 4320 catgtcatcc ggggccagga tggctctcca cagatagaga acatccagcc attctctgcc 4380 aaagacctgt ccattcgctc actgggggac cgaatccggg atcttgctca gctcaaaaat 4440 ctctatccca agaagcccaa ggatgaggct ttccggagcc actacaagcc tgaacagatg 4500 ggtaaggatg gcaggggtta tgtcccagct accatcaaga tgaccgtgga aagggaccaa 4560 ccacttccta ccccagagct ccagatgcct accatggtgc cttcttatga ccttggaatg 4620 gcccctgatt cctccatgag catgcagctt ggcccagata tggtgcccca ggtgtaccca 4680 ccacactctc actccatccc cccgtatcaa ggcctctccc cagaagaatc agtcaacgtg 4740 ttgtcagcct tccaggagcc tcacctgcag atgcccccca gcctgggcca gatgagcctg 4800 ccctttgacc agcctcaccc ccagggcctg ctgccgtgcc agcctcagga gcatgctgtg 4860 tccagccctg accccctgct ctgctcagat gtgaccatgg tggaagacag ctgcctgagc 4920 cagccagtga cagcgtttcc tcagggcact tggattggtg aagacatatt ccctcctctg 4980 ctgcctccca ctgaacagga cctcactaag cttctcctgg aggggcaagg ggagtcgggg 5040 ggagggtcct tgggggcaca gcccctcctg cagccctccc actatgggca atctgggatc 5100 tcaatgtccc acatggacct aagggccaac cccagttggt gactcgaggg ggttggggtt 5160 gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc 5220 cgggaaacgc agcggcgccg accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt 5280 cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct 5340 aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg 5400 tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg 5460 atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg 5520 ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg 5580 ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat 5640 caccgacctc tctccccagg gggatcctga ggatcgtttc gcatgattga acaagatgga 5700 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 5760 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 5820 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg 5880 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 5940 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 6000 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 6060 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 6120 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 6180 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg 6240 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 6300 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 6360 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 6420 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg 6480 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat 6540 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct 6600 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag 6660 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 6720 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc 6780 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct 6840 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg 6900 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg 6960 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 7020 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgga 7080 attaattcga gctcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct 7260 ggctaactag ggacccact gcttaagcct caataagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca 7380 gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaattcaga gagtgagagg aacttgttta ttgcagctta taatggttac 7560. aataagca atagcatcac aaatttcaca aataagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc 7620. 7680. 7680. 7680. 7680. 7680. 7680. 7680. gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 7740 agtagtgagg aggctttttt ggaggcctag gcttttgcgg gcccaaattc gtaatcatgg 7800. tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 7860 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 7920 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 7980 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 8040 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 8100 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 8160 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 8220 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 8280 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 8340 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 8400 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 8460 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 8520 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 8580 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 8640 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 8700 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 8760 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 8820 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 8880 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 8940 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 9000 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 9060 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 9120 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 9180 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 9240 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 9300 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 9360 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 9420 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 9480 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 9540 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 9600 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 9660 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 9720 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 9780 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 9840 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 9900 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 9960 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 10020 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 10080 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt 10140 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg 10200 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc 10260 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta 10320 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg 10380 ttttcccagt cacgacgttg taaaacgacg gccagtgcca agctg 10425 <210> 3 <211> 8978 <212> DNA <213> Artificial Sequence <400> 3 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120 cgatcgtgcc ttattagga ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct 240 ctggttagac cagatctgag cctggggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgt gcccgtctgt tgtgtgactc 360 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc 420 480. ccgaacaggg acttgaaagc gaaagggaa ccgagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcggggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataatta aaacatatag tatgggcaag caggggagcta gaacgattcg cagttaatcc tggcctgtta 780. aaccatcag aaggctgtag acaatactg ggacagctac aaccatccct tcagacagga 840. tcagaagaac ttagatcatt father gtagcaaccc tctattgtgt gcatcaaagg frequently frequently frequently aaaaaagt 900 agaccaccg cacagcaagc ggccgctgat cttcagacct ggaggagg atatgaggga 960 attgaatt taggtagc 1020 acccaccaag gcaagagaa gagtggtgca gagaaaaa agagcagtgg gataggagc 1080 ttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag gcgcacagc atctgttgca accacagtc tggggcatca agcagctcca 1260 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg atgctagtt ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag tgggacag aattacaa 1440 ttacacaagc ttatacact ccttaattga agaatcgcaaaccagcaag aaagaatga 1500 acaagaatta ttggattag aataatgggc aagtttgtgg aattggtttta acatacaaa 1560 ttggctgtgg tatataaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620 agttttgct gtactttcta tagtgaatag agttaggcag ggattattcac cattatcgtt 1680 1740 1740 1740 tggagagaga gagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaagaa tagtagacat 1860 atagcaaca gaatacaa ctaagaatt aaaaaaaaaaaaat tcaaattttt 1920 atcgatgtac gggccagata tacgcgttga cattgatt tgactagtta ttaatagtaa 1980 tcaattacgg gtcattagt tcatagccca tatggagt tccgcgttac attackacg 2040 gtaaatggcc cgcctggctg acccccac gaccccgcc cattgacgtc aaatgacg 2100 tatgttccca tagtaacgcc atagggact ttccattgac gtcaatgggt ggacttta 2160 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 2220 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 2280 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 2340 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 2400 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 2460 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 2520 ataagcagag ctctctggct aactgcagcg tctcaagctt cagaattctc agatccgcta 2580 gcatggggct ctgggcgctg ttgcctggct gggtttctgc tacgctgctg ctggcgctgg 2640 ccgctctgcc cgcagccctg gctgccaaca gcagtggccg atggtggggt attgtgaacg 2700 tagcctcctc cacgaacctg cttacagact ccaagagtct gcaactggta ctggagccca 2760 gtctgcagct gttgagccgc aaacagcggc gtctgatacg ccaaaatccg gggattctgc 2820 acagcgtgag tggggggctg cagagtgccg tgcgcgagtg caagtggcag ttccggaatc 2880 gccgctggaa ctgtcccact gctccagggc cccacctctt cggcaagatc gtcaaccgag 2940 gctgtcgaga aacggcgttt atcttcgcta tcacctccgc cggggtcacc cattcggtgg 3000 cgcgctcctg ctcagaaggt tccatcgaat cctgcacgtg tgactaccgg cggcgcggcc 3060 ccgggggccc cgactggcac tgggggggct gcagcgacaa cattgacttc ggccgcctct 3120 tcggccggga gttcgtggac tccggggaga aggggcggga cctgcgcttc ctcatgaacc 3180 ttcacaacaa cgaggcaggc cgtacgaccg tattctccga gatgcgccag gagtgcaagt 3240 gccacgggat gtccggctca tgcacggtgc gcacgtgctg gatgcggctg cccacgctgc 3300 gcgccgtggg cgatgtgctg cgcgaccgct tcgacggcgc ctcgcgcgtc ctgtacggca 3360 accgcggcag caaccgcgct tcgcgggcgg agctgctgcg cctggagccg gaagacccgg 3420 cccacaaacc gccctccccc cacgacctcg tctacttcga gaaatcgccc aacttctgca 3480 cgtacagcgg acgcctgggc acagcaggca cggcagggcg cgcctgtaac agctcgtcgc 3540 ccgcgctgga cggctgcgag ctgctctgct gcggcagggg ccaccgcacg cgcacgcagc 3600 gcgtcaccga gcgctgcaac tgcaccttcc actggtgctg ccacgtcagc tgccgcaact 3660 gcacgcacac gcgcgtactg cacgagtgtc tgtgactcga gggggttggg gttgcgcctt 3720 ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa 3780 cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg 3840 atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg 3900 gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact 3960 agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct 4020 gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg 4080 cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca 4140 ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac 4200 ctctctcccc agggggatcc tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 4260 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 4320 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 4380 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt 4440 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 4500 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 4560 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 4620 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 4680 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 4740 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg 4800 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 4860 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 4920 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 4980 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcggtcgaca 5040 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc 5100 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta 5160 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt 5220 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg 5280 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta 5340 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt 5400 tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg 5460 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca 5520 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc 5580 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct ggaattaatt 5640 cgagctcggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt 5700 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt 5760 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac 5820 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg 5880 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc ttattattca gtatttata cttgcaaaga aatgaatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat 6240 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg 6300. aggaggcttt tttggaggcc tagcttttg cgggcccaaa ttcgtaatca tggtcatagc 6360. tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 6600. tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 6660 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 6720 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 6780 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 6840 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 6900 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 6960 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 7020 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 7080 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 7140 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaggacag 7200 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 7260 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 7320 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 7380 agtgggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 7440 7500 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 7560 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggaggct 7620 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 7680 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 7740 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 7800 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 7860 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 7920 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 7980 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 8040 tagatgctt ttctgtgact ggtgagtact caccaagtc attctgagaa tagtgtatgc 8100 ggcgaccgag ttgcttgc ccggcgtcaa tacggtaa taccgcgcca catagcagaa 8160 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaactca aggatcttac 8220 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caacgatct tcagcatctt 8280 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaatgcc gcaaaaaagg 8340 gaataagggc wakacggaaa tgttgaatac tcatactctt cctttttca tattattgaa 8400 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 8460 aacaaatagg gttccgcgc acatttcccc gaaagtgcc acctgacgtc taagaaacca 8520 ttattatcat gatacacc tataaaaata ggcgtatcac gaggccctt cgtctcgcgc 8580 gtttcggtga tgacggtgaa acctctgac acatgcagct cccggacg gtcacagctt 8640 gtctgtaagc ggatgccgggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg 8700 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata 8760 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc 8820 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc 8880 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc 8940 agtcacgacg ttgtaaaacg acggccagtg ccaagctg 8978 <210> 4 <211> 252 <212> DNA <213> Artificial Sequence <400> 4 gatctggccg gtacctgagc tcagatcaaa gggtttaaga tcaaagggct taagatcaaa 60 gggtataaga tcaaagggcc taagatcaaa gggactaaga tcaaagggtt taagatcaaa 120 gggcttaaga tcaaagggcc tactcgagga tatcaagatc tggcctcggc ggccaagctt 180 cgaagacact agagggtata taatggaagc tcgacttcca gcttggcaat ccggtactgt 240 tggtacactg ca 252 <210> 5 <211> 8984 <212> DNA <213> Artificial Sequence <400> 5 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60 acatgcctta caaggagaga aaagcaccg tgcatgccga ttggtggaag taaggtggta 120 cgatcgtgcc ttaggaa ggcacagac gggtctgaca tggattgac gaaccactga 180 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct 240 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 300 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 360 tggtaactag agatccctca gaccttta gtcagtgtgg aaatctcta gcagtggcgc 420 ccgaacagggg acttgaaagc gaaagggaaa ccagagctct ctcgacgcag gactcggctt 480 gctgaagcgc gcacggcaag agggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600 attagatcgc gatgggaaaaatcggtta aggccagggg gaagaaaaaataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag aaaatactg ggacagctac aaccatccct tcagacagga 780 tcagagaac ttagatcatt atataca gtagcaaccc tctattgtgt gcatcaagg 840 frequently frequently frequently aaaaaagt 900 agaccaccg cacagcaagc ggccgctgat cttcagacct ggaggagg atatgaggga 960 attgaatt taggtagc 1020 acccaccaag gcaagagaa gagtggtgca gagaaaaa agagcagtgg gataggagc 1080 ttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag gcgcacagc atctgttgca accacagtc tggggcatca agcagctcca 1260 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg atgctagtt ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag tgggacag aattacaa 1440 ttacacaagc ttatacact ccttaattga agaatcgcaaaccagcaag aaagaatga 1500 acaagaatta ttggattag aataatgggc aagtttgtgg aattggtttta acatacaaa 1560 ttggctgtgg tatataaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620 agttttgct gtactttcta tagtgaatag agttaggcag ggattattcac cattatcgtt 1680 1740 1740 1740 tggagagaga gagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaagaa tagtagacat 1860 atagcaaca gaatacaa ctaagaatt aaaaaaaaaaaaat tcaaattttt 1920 atcgatact ggccgtacc cgctgttgct caatcgactt cccagaca gcgctgttgc 1980 tcaatcgact tcccagaac agcagggatc tgcctcggc ggccaagctc cgctgttgct 2040 caatcgactt cccagaca gcgctgttgc tcaatcgact tcccagaac agcaggtc 2100 tggcctcggc ggccaagctt agacactaga gggtatatata tggaagctcg acttccagct 2160 tggcaatccg gtactgttgg tacactgcag cgtctcaagc ttcagaattc agcttggcat 2220 tccggtactg ttggtaaagc caccatggaa gacgccaaaa acataaagaa aggcccggcg 2280 ccattctatc cgctggaaga tggaaccgct ggagagcaac tgcataaggc tatgaagaga 2340 tacgccctgg ttcctggaac aattgctttt acagatgcac atatcgaggt ggacatcact 2400 tacgctgagt acttcgaaat gtccgttcgg ttggcagaag ctatgaaacg atatgggctg 2460 aatacaaatc acagaatcgt cgtatgcagt gaaaactctc ttcaattctt tatgccggtg 2520 ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga acgacattta taatgaacgt 2580 gaattgctca acagtatggg catttcgcag cctaccgtgg tgttcgtttc caaaaagggg 2640 ttgcaaaaaa ttttgaacgt gcaaaaaaag ctcccaatca tccaaaaaat tattatcatg 2700 gattctaaaa cggattacca gggatttcag tcgatgtaca cgttcgtcac atctcatcta 2760 cctcccggtt ttaatgaata cgattttgtg ccagagtcct tcgataggga caagacaatt 2820 gcactgatca tgaactcctc tggatctact ggtctgccta aaggtgtcgc tctgcctcat 2880 agaactgcct gcgtgagatt ctcgcatgcc agagatccta tttttggcaa tcaaatcatt 2940 ccggatactg cgattttaag tgttgttcca ttccatcacg gttttggaat gtttactaca 3000 ctcggatatt tgatatgtgg atttcgagtc gtcttaatgt atagatttga agaagagctg 3060 tttctgagga gccttcagga ttacaagatt caaagtgcgc tgctggtgcc aaccctattc 3120 tccttcttcg ccaaaagcac tctgattgac aaatacgatt tatctaattt acacgaaatt 3180 gcttctggtg gcgctcccct ctctaaggaa gtcggggaag cggttgccaa gaggttccat 3240 ctgccaggta tcaggcaagg atatgggctc actgagacta catcagctat tctgattaca 3300 cccgaggggg atgataaacc gggcgcggtc ggtaaagttg ttccattttt tgaagcgaag 3360 gttgtggatc tggataccgg gaaaacgctg ggcgttaatc aaagaggcga actgtgtgtg 3420 agaggtccta tgattatgtc cggttatgta aacaatccgg aagcgaccaa cgccttgatt 3480 gacaaggatg gatggctaca ttctggagac atagcttact gggacgaaga cgaacacttc 3540 ttcatcgttg accgcctgaa gtctctgatt aagtacaaag gctatcaggt ggctcccgct 3600 gaattggaat ccatcttgct ccaacacccc aacatcttcg acgcaggtgt cgcaggtctt 3660 cccgacgatg acgccggtga acttcccgcc gccgttgttg ttttggagca cggaaagacg 3720 atgacggaaa aagagatcgt ggattacgtc gccagtcaag taacaaccgc gaaaaagttg 3780 cgcggaggag ttgtgtttgt ggacgaagta ccgaaaggtc ttaccggaaa actcgacgca 3840 agaaaaatca gagagatcct cataaaggcc aagaagggcg gaaagatcgc cgtgtaattc 3900 tctcgagggg gttggggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg 3960 gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct 4020 tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga 4080 cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt 4140 gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc cagggagcaa 4200 tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg 4260 agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc 4320 tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg 4380 gctccctcgt tgaccgaatc accgacctct ctccccaggg ggatccaccg gagcttacca 4440 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac 4500 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc 4560 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca 4620 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga 4680 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt 4740 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc 4800 ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca 4860 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg 4920 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg 4980 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgag 5040 tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg 5100 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt 5160 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg 5220 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc 5280 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc 5340 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc 5400 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc 5460 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg 5520 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc 5580 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctggaa 5640 ttaattcgag ctcggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc 5700 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc 5760 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg 5820 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag 5940 tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagga acttgtttat tgcagcttat aatggttaca grandmother tagcatcaca grandmother grandmother tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct 6180. aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 6240 actattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccaga gtagtgagga ggcttttttg gaggcctagg cttttgcggg cccaaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 6480. gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 6600 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 6660 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 6720 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 6780 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 6840 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 6900 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 6960 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 7020 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 7080 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 7140 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 7200 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 7260 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg ttttttgtt tgcaagcagc 7320 agattacgcg cagaaaaaaa ggatctcaag aagatcctttt gatctttct acggggtctg 7380 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 7440 tcttcaccta gatcctttta attaaaaat gaagttttaa atcaatctaa agtatatatg 7500 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 7560 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 7620 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 7680 cagattatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 7740 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 7800 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 7860 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 7920 ccatgttgtg caaaaaagcg gttagctcct tcggtcctc gatcgttgtc agaagtaagt 7980 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 8040 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 8100 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 8160 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 8220 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 8280 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 8340 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 8400 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 8460 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 8520 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 8580 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 8640 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 8700 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 8760 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 8820 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 8880 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 8940 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctg 8984 <210> 6 <211> 8970 <212> DNA <213> Artificial Sequence <400> 6 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct 240 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 300 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 360 tggtaactag agatccctca gaccttta gtcagtgtgg aaatctcta gcagtggcgc 420 ccgaacagggg acttgaaagc gaaagggaaa ccagagctct ctcgacgcag gactcggctt 480 gctgaagcgc gcacggcaag agggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600 attagatcgc gatgggaaaaatcggtta aggccagggg gaagaaaaaataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag aaaatactg ggacagctac aaccatccct tcagacagga 780 tcagagaac ttagatcatt atataca gtagcaaccc tctattgtgt gcatcaagg 840 frequently frequently frequently aaaaaagt 900 agaccaccg cacagcaagc ggccgctgat cttcagacct ggaggagg atatgaggga 960 attgaatt taggtagc 1020 acccaccaag gcaagagaa gagtggtgca gagaaaaa agagcagtgg gataggagc 1080 ttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgag gcgcacagc atctgttgca accacagtc tggggcatca agcagctcca 1260 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320 ttgctctgga aaactcattt gcaccactgc tgtgccttgg atgctagtt ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggag tgggacag aattacaa 1440 ttacacaagc ttatacact ccttaattga agaatcgcaaaccagcaag aaagaatga 1500 acaagaatta ttggattag aataatgggc aagtttgtgg aattggtttta acatacaaa 1560 ttggctgtgg tatataaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620 agttttgct gtactttcta tagtgaatag agttaggcag ggattattcac cattatcgtt 1680 1740 1740 1740 tggagagaga gacagagaca gatccattcg attagtgac ggatctcgac ggtatcggtt aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaaga tagtagcat aatagcaaca gacatacaa ctaaagatt acaaaaacaa attack tcaaaatttt atcgatctgg ccggtacctg agctcagatc aaagggttta agatcaaagg gcttaagatc aaagggtata agatcaaagg gcctaagatc aaagggacta agatcaaagg gtttaagatc aaagggctta agatcaaagg gcctactcga ggatatcaag atctggcctc ggcggccaag cttcgaagac actagagggt fatheragcgactt ccagcttggc aatccggtac tgttggtaca ctgcagcgtc tcaagcttca gattcagct tggcattccg gtactgttgg taaagccacc atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatccgct ggaagatgga accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt gcttttacag atgcacatat cgaggtggac atcacttacg ctgagtactt cgaaatgtcc gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt 2520 tatcggagtt gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag 2580 tatgggcatt tcgcagccta ccgtggtgtt cgtttccaaa aaggggttgc aaaaaatttt 2640 gaacgtgcaa aaaaagctcc caatcatcca aaaaattatt atcatggatt ctaaaacgga 2700 ttaccaggga tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa 2760 tgaatacgat tttgtgccag agtccttcga tagggacaag acaattgcac tgatcatgaa 2820 ctcctctgga tctactggtc tgcctaaagg tgtcgctctg cctcatagaa ctgcctgcgt 2880 gagattctcg catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat 2940 tttaagtgtt gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat 3000 atgtggattt cgagtcgtct taatgtatag atttgaagaa gagctgtttc tgaggagcct 3060 tcaggattac aagattcaaa gtgcgctgct ggtgccaacc ctattctcct tcttcgccaa 3120 aagcactctg attgacaaat acgatttatc taatttacac gaaattgctt ctggtggcgc 3180 tcccctctct aaggaagtcg gggaagcggt tgccaagagg ttccatctgc caggtatcag 3240 gcaaggatat gggctcactg agactacatc agctattctg attackcccg agggggatga taaccgggc gcggtcggta aagttgttcc attttttga gcgaaggttg tggatctgga 3360. taccgggaaa acgctgggcg ttaatcaaag aggcgaactg tgtgtgagag gtcctatgat 3480. tatgtccggt tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct ggagacatag cttactggga cgaagacgaa cacttcttca tcgttgaccg cctgaagtct ctgattaagt acaaaggcta tcaggtggct cccgctgaat tggaatccat cttgctccaa caccccaaca tcttcgacgc aggtgtcgca ggtcttcccg acgatgacgc cggtgaactt cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga 3720 gatcgtggat tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac gagtaccga aaggtcttac cggaaaactc gacgcaaga aaatcagaga gatcctcata aaggccaaga agggcggaa gatcgccgtg taattctctc gaggggttg gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt 3960 ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc 4020 agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg 4080 cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc 4140 gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga 4200 ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg 4260 gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg 4320 cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac 4380 cgaatcaccg acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag 4440 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc 4500 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg 4560 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg 4620 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg 4680 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg 4740 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg 4800 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc 4860 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc 4920 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc 4980 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgagtcga caatcaacct 5040 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg 5100 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc 5160 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt 5220 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc 5280 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg 5340 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact 5400 gacaattccg tggtgttgtc ggggaagctg acgtcctttc catggctgct cgcctgtgtt 5460 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg 5520 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc 5580 cctcagacga gtcggatctc cctttgggcc gcctccccgc ctggaattaa ttcgagctcg 5640 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa 5700 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt 5760 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac 5820 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg 5880 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct 5940 agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata 6000 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc 6060 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 6120 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc 6180 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta 6240 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct 6300 tttttggagg cctaggcttt tgcgggccca aattcgtaat catggtcata gctgtttcct 6360 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 6420 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 6480 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 6540 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 6600 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 6660 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 6720 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 6780 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gacttaag ataccaggcg 6840 ttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 6900 ctgtccgcct ttctccctc gggaagcgtg gcgctctc atagctcacg ctgtaggtat 6960 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgacc ccccgttcag 7020 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 7080 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 7140 gctacagagt tctgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 7200 atctgcgctc tgctgaagcc agttaccttc ggaaaagag ttggtagctc ttgatccggc 7260 aaacaaacca ccgctggtag cggtggtttt ttgtttgca agcagcagat tacgcgcaga 7320 aaaaaaggat ctcaagaga tccttgatc tttctacgg ggtctgacgc tcagtggac 7380 gaaaactcac gttaaggat ttggtcatg agatttca aaaggatctt cacctagatc 7440 cttttaaattt aaaaatgaag ttttaatca atctaagta tatatgagta aacttggtct 7500 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 7560 tccatagttg cctgactccc cgtcgtgtag atatacga tacgggagg cttaccatct 7620 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 7680 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 7740 7800 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 7860 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 7920 aaagcggtta gctccttgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 7980 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 8040 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 8100 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 8160 gtgctcatca ttggaaaacg ttcttcgggg cgaaaaactct caaggatctt accgctgttg 8220 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 8280 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 8340 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 8400 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taacaaata 8460 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 8520 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt 8580 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa 8640 gcggatgccg gggcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg 8700 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt 8760 gaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg 8820 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg 8880 aaaggggat gtgctgcaag gcgattaagt tgggttaacgc cagggttttc ccagtcacga 8940 cgttgtaaaa cgacggccag tgccaagctg 8970

Claims

1. A method for detecting the cell activity of an anti-SOST antibody or SOST, characterized in that: The method comprises: co-culturing a cell line stably expressing Wnt1 protein and a cell line expressing a reporter gene under the regulation of TCF / LEF elements to detect the cell activity of anti-SOST antibodies or SOST; Wherein, the reporter gene is selected from Luciferase protein; The cell line expressing Wnt1 protein is 293T-Wnt1; the cell line expressing Luciferase protein is 293T-TCFLuc, and the 293T-TCFLuc and 293T-Wnt1 are co-cultured; The sequence of the TCF / LEF regulatory element is SEQ ID NO: 4; the amino acid sequence of the Wnt1 protein is the amino acid sequence after translation of SEQ ID NO:

1.

2. The method according to claim 1, characterized in that In the method, Wnt1 protein is expressed by the lentiviral expression vector plenti-CN-Wnt1, which is the nucleotide sequence shown in SEQ ID NO: 3; TCF / LEF element and Luciferase protein are expressed by the lentiviral expression vector plenti-TCF / LEF-Luciferase, which is the nucleotide sequence shown in SEQ ID NO:

6.

3. The method according to claim 1, characterized in that: The cell line is selected from 293 cell subtypes or other human adherent cells.

4. The method according to claim 1, characterized in that: The method includes: the 293T-TCFLuc cell inoculation density is 10,000 to 30,000 cells / well.

5. The method according to claim 4, characterized in that The mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 10~0.5:

1.

6. The method according to any one of claims 1 to 5, characterized in that: The method comprises: (1) Preparation of recombinant human SOST protein and anti-SOST antibody solution to be tested, or preparation of SOST protein solution to be tested; (2) Treatment of 293T-TCFLuc and 293T-Wnt1 cells and inoculation in 96-well plates; (3) Add the mixture of the recombinant human SOST protein and the anti-SOST antibody solution to be tested or the SOST protein solution to be tested in step (1) to the 96-well plate inoculated in step (2), and return the plate to the CO2 incubator for incubation; (4) Detect the fluorescence response value of step (3) using a luciferase assay kit, and calculate the EC50 value of the anti-SOST antibody to be tested or the IC50 value of the SOST protein to be tested.

7. The method according to claim 6, characterized in that The step (1) comprises: Preparation of recombinant human SOST protein and anti-SOST antibody solution: (1-1) Take the anti-SOST antibody solution to be tested, dilute it to 1 mg / ml with DMEM complete medium containing FBS, and then dilute it stepwise to 0.625, 1.25, 2.5, 5.0, 10, 20, 40, 60, 80, 120, 160, 240 μg / ml; (1-2) Take the recombinant human SOST protein stock solution and dilute it to 5-20 μg / ml with DMEM complete medium containing FBS; (1-3) Mix a certain volume of rhSOST dilution with an equal volume of anti-SOST antibody solution of different concentration gradients and incubate at room temperature for 30-60 minutes, or Preparation of SOST protein solution to be tested: The recombinant human SOST protein stock solution was diluted to 0.008, 0.020, 0.051, 0.128, 0.32, 0.8, 2.0, 5.0, 10, 20, 40, 80 μg / ml with DMEM complete medium containing FBS, and incubated at room temperature for use.

8. The method according to claim 6, characterized in that The step (2) comprises: (2-1) Take 293T-Wnt1 cells and 293T-TCFLuc cells with good growth status and cell confluence of more than 90%, treat them according to the cell passaging method, centrifuge the cell suspension, remove the supernatant, add DMEM complete medium containing FBS to resuspend, and take the cell suspension to count; (2-2) Dilute and mix the cells according to the conditions that the 293T-TCFLuc cell seeding density is 10,000 to 30,000 cells / well, the mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 10 to 5:1, and the actual required cell amount; after the two cell lines are diluted and mixed according to the above ratio, 50 μl of cell dilution solution / well is inoculated into a white transparent bottom 96-well plate.

9. The method according to claim 6, characterized in that The step (3) comprises: adding the mixed solution of rhSOST and the anti-SOST antibody to be tested or the recombinant human SOST protein solution to be tested prepared and incubated in step (1) at 50ul / well into the 96-well plate inoculated with cells in step (2), and returning the plate to a 5% CO2 incubator for incubation for 18-20h.

10. The method according to claim 6, characterized in that The step (4) comprises: using a luciferase detection kit to detect the fluorescence response value of the 96-well plate after the incubation in step (3), using SoftMax or other data analysis software to process the raw data, and calculating the EC50 value of the anti-SOST antibody to be tested or the IC50 value of the recombinant human SOST protein to be tested.

11. The method according to claim 6, characterized in that The method comprises: (1) Preparation of recombinant human SOST protein and anti-SOST antibody solution: Take the anti-SOST antibody solution to be tested, dilute it to 1 mg / ml with DMEM complete medium containing FBS, and then dilute it stepwise to 0.625, 1.25, 2.5, 5.0, 10, 20, 40, 60, 80, 120, 160, 240 μg / ml; Take the recombinant human SOST protein stock solution and dilute it to 10 μg / ml with DMEM complete medium containing FBS; Take a certain volume of rhSOST dilution and mix it with an equal volume of anti-SOST antibody solution of different concentration gradients, and incubate at room temperature for 40 minutes, or Preparation of SOST protein solution to be tested: Take the recombinant human SOST protein stock solution and dilute it to 0.008, 0.020, 0.051, 0.128, 0.32, 0.8, 2.0, 5.0, 10, 20, 40, 80 μg / ml with DMEM complete medium containing FBS, and incubate at room temperature for use; (2) Treatment of 293T-TCFLuc and 293T-Wnt1 cells and inoculation of 96-well plates: Take 293T-Wnt1 cells and 293T-TCFLuc cells with good growth status and cell confluence of more than 90%, treat them according to the cell passaging method, centrifuge the cell suspension, remove the supernatant, add an appropriate amount of DMEM complete medium containing FBS to resuspend, and take the cell suspension to count; The cells were diluted and mixed according to the conditions that the 293T-TCFLuc cell seeding density was 30,000 cells / well, the mixing ratio of 293T-TCFLuc and 293T-Wnt1 was 5:1, and the actual required cell amount; after the two cell lines were diluted and mixed according to the above ratio, 50 μl of cell dilution solution / well was inoculated into a white transparent bottom 96-well plate; (3) Add 50ul / well of the rhSOST and anti-SOST antibody mixture or SOST protein solution prepared and incubated in step (1) into the 96-well plate seeded with cells in step (2), and return to the 5% CO2 incubator for incubation for 18-20h; (4) Using a luciferase assay kit to detect the fluorescence response value of the 96-well plate after the incubation in step (3), using SoftMax or other data analysis software to process the raw data, and calculating the EC50 value of the anti-SOST antibody to be tested or the IC50 value of the recombinant human SOST protein to be tested.

12. The method according to claim 6, characterized in that The anti-SOST antibodies to be tested include lomosozumab.

13. A cell line 293T-Wnt1 stably expressing Wnt1 protein according to any of the preceding claims, characterized in that: The cell line is constructed by lentivirus infection method, and the vector expressing Wnt1 protein is a recombinant lentivirus expression vector plenti-CN-Wnt1, which has a nucleotide sequence shown in SEQ ID NO:

3.

14. A cell line 293T-TCFLuc expressing Luciferase under the control of TCF / LEF elements in the method according to any of the preceding claims, characterized in that: The cell line is constructed by lentivirus infection method, and the vector expressing TCF / LEF regulatory expression element and Luciferase protein is a recombinant lentivirus expression vector plenti-TCF / LEF-Luciferase, which has a nucleotide sequence shown in SEQ ID NO:

6.

15. Use of the cell line according to claim 13 or 14 in detection of β-Catein / Wnt signaling pathway.

16. The use according to claim 15, characterized in that The cell line 293T-Wnt1 stably expressing Wnt1 protein according to claim 13 and the cell line 293T-TCFLuc expressing Luciferase protein under the regulation of TCF / LEF elements according to claim 14 are co-cultured to detect the cell activity of anti-SOST antibodies or SOST.

17. The use according to claim 16, characterized in that The 293T-TCFLuc cell seeding density is 10,000 to 30,000 cells / well.

18. The use according to claim 17, characterized in that The mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 10~0.5:

1.

19. The method according to claim 3, characterized in that The cell line is selected from HEK-293T or HEK-293.

20. The method according to claim 19, characterized in that The cell line selected is HEK-293T.

21. The method according to claim 4, characterized in that The method includes: the 293T-TCFLuc cell inoculation density is 30,000 cells / well.

22. The method according to claim 5, characterized in that The mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 10~5:

1.

23. The method according to claim 22, characterized in that The mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 5:

1.

24. The method according to claim 7, characterized in that The step (1) comprises: Preparation of recombinant human SOST protein and anti-SOST antibody solution: (1-2) Take the recombinant human SOST protein stock solution and dilute it to 10 μg / ml using DMEM complete medium containing FBS.

25. The method according to claim 7, characterized in that The step (1) comprises: (1-3) Take a certain volume of rhSOST dilution and mix it with an equal volume of anti-SOST antibody solution with different concentration gradients, and incubate at room temperature for 40 minutes.

26. The method according to claim 8, characterized in that The step (2) comprises: (2-2) Based on the 293T-TCFLuc cell seeding density of 30,000 cells / well, the mixing ratio of 293T-TCFLuc and 293T-Wnt1 was 5:

1.

27. Use of the cell line according to claim 15 in detecting the cell activity of anti-SOST antibodies or SOST.

28. The use according to claim 17, characterized in that: The 293T-TCFLuc cell seeding density is 30,000 cells / well.

29. The use according to claim 18, characterized in that The mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 10~5:

1.

30. The use according to claim 29, characterized in that The mixing ratio of 293T-TCFLuc and 293T-Wnt1 is 5:1.

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