Human growth hormone detection antibody and preparation method thereof

Abstract

The present invention discloses a human growth hormone detection antibody and a preparation method thereof. The amino acid sequence of the detection antibody is one of those shown in SEQ ID NO.1 and SEQ ID NO.2; the preparation steps are: extracting RNA and reverse transcribing, amplifying the obtained cDNA and inserting it into Escherichia coli to construct a human growth hormone primary library; using M13KO7 as an auxiliary phage to invade the human growth hormone primary library to construct a phage primary library; performing three rounds of solid phase panning on the phage primary library, and obtaining the target gene sequence through monoclonal screening, ELISA detection and sequencing; synthesizing the target gene sequence into a prokaryotic expression plasmid, introducing it into Escherichia coli and adding IPTG for expression to obtain a detection antibody. The detection antibody of the present invention has the characteristics of low cost, strong stability and simple subsequent operation, which solves the problem of the need for subsequent animal immunization to prepare antibodies in human growth hormone detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and more specifically, to a human growth hormone detection antibody and a preparation method thereof. Background Art

[0002] Human Growth Hormone (hGH) is a peptide hormone secreted by the anterior pituitary gland of the human body. It is composed of 191 amino acids. Its main functions include promoting growth (stimulating the growth of bones and soft tissues, especially in children and adolescents), metabolic regulation (stimulating protein synthesis, fat decomposition and sugar metabolism), and cell repair (participating in tissue repair and regeneration).

[0003] Growth hormone is widely used, but its detection is difficult and complex. Currently used detection methods include radioimmunoassay (RIA), chemical release immunoassay (CLIA), liquid chromatography-mass spectrometry (LC-MS), and animal immunization. Radioimmunoassay (RIA) uses radiolabeled GH to compete with GH in the sample for binding to antibodies. This method uses radioactive substances, poses safety risks, and is complex and time-consuming. Chemical release immunoassay (CLIA) uses chemiluminescent substances to label antibodies and quantify GH by luminescence intensity. This method has high equipment costs, reliance on imports, and high reagent costs. Liquid chromatography-mass spectrometry (LC-MS) separates GH chromatographically and uses mass spectrometry to detect its molecular weight and fragment ions. This method is expensive and requires specialized technicians, resulting in high costs. Animal immunization involves injecting growth hormone antigens into animals to generate antibodies. These antibodies are then purified and labeled to obtain the corresponding detection antibodies for ELISA experiments. This method has high animal costs and requires re-immunization of the animals each time the antibodies are obtained. This acquisition process is long and involves animal hazards. Therefore, it is necessary to develop a human growth hormone detection antibody that has the characteristics of low cost, strong stability, simple subsequent operation, and can solve the problem of having to prepare serum antibodies through animal immunization in detection. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a human growth hormone detection antibody and a preparation method thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A human growth hormone detection antibody, the amino acid sequence of the detection antibody is one of SEQ ID NO.1 and SEQ ID NO.2.

[0007] A method for preparing a human growth hormone detection antibody comprises the following steps:

[0008] S1. RNA was extracted from the spleen and blood of the striped bamboo shark and reverse transcribed to obtain cDNA. The cDNA was amplified using universal primers to obtain the VNAR gene, which was then inserted into Escherichia coli to construct a primary human growth hormone library.

[0009] S2. Use M13KO7 to assist phage in invading the human growth hormone primary library, display the expressed antibodies on the phage surface, and construct the phage primary library;

[0010] S3. Perform three rounds of solid-phase panning on the primary phage library, and obtain the target gene sequence VNAR through single clone screening, ELISA detection and sequencing.

[0011] S4. The target gene sequence VNAR is synthesized into a prokaryotic expression plasmid, introduced into Escherichia coli and expressed with the addition of IPTG to obtain human growth hormone detection antibodies.

[0012] The present invention is further configured such that, in step S1, the specific steps of extracting RNA from the blood of a striped bamboo shark are as follows: whole blood is collected from the tail vein of a striped bamboo shark, lymphocytes are separated after standing, Trizol reagent is added to the lymphocytes, mixed evenly, and allowed to stand; chloroform is added, the mixture is incubated after shaking, and centrifuged to separate the layers; the upper aqueous phase containing RNA is transferred to a new RNA-free EP tube, isopropanol is added, mixed evenly, allowed to stand, and centrifuged; 75% ethanol is added to wash the precipitate after centrifugation; centrifugation is repeated, the supernatant is removed, the RNA precipitate is dried, RNA-free ddH2O is added to dissolve it, and the precipitate is stored in aliquots.

[0013] The present invention is further configured as follows: in step S1, the specific steps of extracting RNA from the spleen of the striped bamboo shark are: grinding the spleen into a powder, taking the spleen powder and adding Trizol reagent, mixing and standing, then adding chloroform, shaking and incubating, centrifuging and stratifying; transferring the upper aqueous phase containing RNA to a new RNA-free EP tube, adding isopropanol, mixing and standing, and centrifuging; adding 75% ethanol to wash the precipitate after centrifugation; centrifuging again, removing the supernatant, drying the RNA precipitate, adding RNA-free ddH2O to dissolve it, and storing it in aliquots.

[0014] The present invention is further configured as follows: step S2 specifically comprises: inoculating the primary library bacterial liquid obtained in step S1 without adding glycerol into 2×YT / Tet / Car liquid culture medium, inoculating two bottles in total, and shaking to the logarithmic phase; adding M13KO7 helper phage to each bottle and then culturing on a shaking table; then adding Kan medium to each bottle and continuing to culture on a shaking table; then adding IPTG medium to each bottle, culturing on a shaking table overnight, and then centrifuging and filtering; transferring the supernatant obtained by filtration to a new centrifuge tube, adding PEG8000-NaCl solution to mix, ice-bathing and centrifuging, discarding the supernatant, and resuspending the phage precipitate with PBS to obtain a phage solution, which is the primary phage library.

[0015] The present invention is further configured such that, in step S3, the first round of panning specifically comprises the following steps: diluting human growth hormone to 5 μg / mL with a PBS solution, coating an ELISA plate with the dilution; discarding the coating solution and washing the plate with a 0.05% PBST solution; blocking the ELISA plate with a 3% BSA blocking solution; discarding the liquid in the plate and washing the plate with a 0.05% PBST solution; taking a portion of the phage obtained in step S2, fixing the volume with a PBS solution, adding a 3% BSA blocking solution to block the phage, fixing the volume with a PBS solution, adding the remaining phage obtained in step S2, slowly shaking, and then discarding the phage mixed liquid in the plate, and washing the plate with a 0.1% PBST solution; adding an eluent for incubation to elute the bound phage; collecting the eluent and fixing the volume with a PBS solution, adjusting the pH to 7.0, to obtain a phage library for the first round of panning;

[0016] The specific steps of the second round of panning are as follows: dilute human growth hormone to 2 μg / mL with PBS solution and use this as the coating solution to coat the ELISA plate; discard the coating solution and wash the plate with 0.05% PBST solution; block the ELISA plate with 3% skim milk powder blocking solution; pour out the liquid in the plate and wash the plate with 0.05% PBST solution; add phage with a titer of 1 / 3 of the phage obtained in the first round of panning, make up the volume with PBS solution, add 3% skim milk powder blocking solution to block the phage, make up the volume again with PBS solution, add the remaining phage obtained in the first round of panning, shake slowly and pour out the phage mixture in the plate, wash the plate with 0.25% PBST solution; add elution solution for incubation to elute the bound phage; collect the eluate and make up the volume with PBS solution, adjust the pH to 7.0, and obtain the phage library for the second round of panning;

[0017] The specific steps of the third round of panning are as follows: human growth hormone is diluted to 1 μg / mL with PBS solution, and this is used as the coating solution to coat the ELISA plate; the coating solution is discarded and the plate is washed with 0.05% PBST solution; the ELISA plate is blocked with 3% BSA blocking solution; the liquid in the plate is poured out and the plate is washed with 0.05% PBST solution; phage with a titer of 1 / 3 of the phage obtained in the second round of panning is added, the volume is fixed with PBS solution, and then 3% BSA blocking solution is added to block the phage, and the volume is fixed with PBS solution again, and the remaining phage obtained in the second round of panning is added, and the phage mixed liquid in the plate is poured out after slow shaking, and the plate is washed with 0.5% PBST solution; the elution solution is added for incubation to elute the bound phage; the eluate is collected and fixed with PBS solution, and the pH is adjusted to 7.0 to obtain the phage library for the third round of panning.

[0018] The present invention is further configured such that, in step S3, the specific steps of monoclonal screening are as follows: 88 monoclones are selected from the output plates of the three rounds of panning and placed in a 96-deep-well plate, 2×YT / Car culture medium is added to each well, the plate sealing film is covered, holes are punched in the membrane, and the plate is shaken to the logarithmic phase; the bacterial solution from each well of the deep-well plate is aspirated to a new 96-deep-well plate, 2×YT / Car culture medium is added to the new 96-deep-well plate, the plate sealing film is covered, and the new deep-well plate is labeled as a split plate; 2×YT / Car and IPTG are added to each well of the split-plate deep-well plate, the plate is shaken overnight and then centrifuged, the supernatant is discarded, TEST lysis solution is added to each well, the plate is vortexed to mix, and the plate is lysed on ice, and then cultured overnight.

[0019] The present invention is further configured such that step S4 specifically comprises:

[0020] a. Using the amino acid sequence of the human growth hormone detection antibody shown in SEQ ID NO.1 or SEQ ID NO.2 as a template, construct a VNAR recombinant plasmid, transfect the synthesized plasmid into BL21 competent cells by heat shock method, select single clones on LB plates, culture and freeze to obtain an expression strain;

[0021] b. The obtained expression strain was inoculated into a fermenter containing LB liquid medium for fermentation and cultured until OD600 = 40. The culture temperature was adjusted to 16 ° C, IPTG was added for overnight induction, and the induced bacterial liquid was centrifuged to obtain bacterial cells. The cells were resuspended in Tris solution, broken, centrifuged, and the supernatant was collected and filtered;

[0022] c. Elution and purification are performed on the supernatant obtained in step b to obtain human growth hormone VNAR antibody.

[0023] The present invention is further configured as follows: in step b, the inoculation volume of the expression strain is 1 ml; and IPTG is added to a final concentration of 0.5 mM.

[0024] The present invention is further configured such that step c specifically comprises: removing a GSTrap HP chromatographic column, rinsing it with deionized water, and then equilibrating it with an equilibration buffer; loading a filtered supernatant sample onto the chromatographic column; after loading, washing the chromatographic column with a binding buffer until the A280 absorbance returns to a baseline level; then eluting with an elution buffer, collecting the elution peak, observing the change in A280 absorbance, and collecting the eluate when a clear elution peak appears to obtain a human growth hormone VNAR antibody;

[0025] The equilibration buffer and the binding buffer are both solutions containing 50 mM Tris-HCl and 150 mM NaCl, pH = 7.4; the elution buffer is a solution containing 50 mM Tris-HCl, 150 mM NaCl and 10 mM reduced glutathione, pH = 7.4.

[0026] In summary, the present invention has the following beneficial effects:

[0027] The human growth hormone detection antibody of the present invention has the characteristics of low cost, strong stability and simple subsequent operation. The human growth hormone detection antibody is obtained by constructing a library of Escherichia coli capable of expressing the human growth hormone detection antibody and storing it at -80°C. When used, the Escherichia coli is cultured and an inducer is added to obtain the human growth hormone detection antibody, thereby solving the problem of the conventional animal immunization method for detecting human growth hormone that requires subsequent animal immunization to prepare serum antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the electrophoresis result of spleen RNA and blood RNA of the striped bamboo shark in step 1 of Example 1;

[0029] Figure 2 This is the electrophoresis result of the cDNA gene obtained in step 2 of Example 1;

[0030] Figure 3 This is the electrophoresis result of the VNAR gene obtained by vector amplification in step 2 of Example 1;

[0031] Figure 4 This is a diagram of the primary human growth hormone library in step 2 of Example 1;

[0032] Figure 5 This is a diagram of the phage library obtained from one round of solid phase panning in Example 1;

[0033] Figure 6 This is a diagram of the phage library obtained from the second round of solid phase panning in Example 1;

[0034] Figure 7 This is a diagram of the phage library obtained from three rounds of solid phase panning in Example 1;

[0035] Figure 8 This is the Elisa test result diagram in step 5 of Example 1;

[0036] Figure 9 This is a graph showing the results of the antibody effect detection of the BL21-GST-VNAR1-HIS expression strain in Example 2. DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0038] Example 1 Phage display library construction

[0039] The main process of constructing a phage display library is: RNA extraction and reverse transcription → primary library construction → collection of growth hormone primary library → construction of phage library → three rounds of solid phase panning of phage library → monoclonal Elisa screening → obtaining human growth hormone VNAR.

[0040] 1. RNA extraction and reverse transcription

[0041] Blood was collected from the tail vein of the striped bamboo shark and the spleen of the immunized shark was dissected and extracted. RNA was extracted and reverse transcribed to obtain cDNA.

[0042] The specific steps for extracting RNA from the blood of striped bamboo sharks are as follows: 12 mL of whole blood was taken from the tail vein of the striped bamboo shark, and the blood was allowed to stand at room temperature for 2 hours to separate the PBMC cells (lymphocytes), and 1 mL of Trizol reagent (RNA extraction reagent) was added to the lymphocytes, and the mixture was mixed thoroughly by pipetting up and down with a pipette, and the mixture was allowed to stand at room temperature for 5-10 minutes; chloroform was added at a ratio of 200 μl chloroform per 1 mL of Trizol reagent, the tube was covered, and the tube was placed in an oscillator for vigorous shaking for 15 seconds, and then incubated at room temperature for 5-10 minutes; after the incubation was completed, the sample was centrifuged at 4°C and 12000×g for 15 minutes to separate the mixture into a red phenol-chloroform lower layer and middle layer and a colorless upper aqueous phase; the upper aqueous phase containing RNA was transferred to a new RNA-free EP tube, and isopropanol was added at a ratio of 0.5 mL of isopropanol per 1 mL of upper aqueous phase, mixed thoroughly, and allowed to stand on ice for 10 minutes. n; then centrifuge at 4°C and 12,000 × g for 10 min (RNA precipitates on the sides and bottom of the tube after centrifugation); add 1 mL of 75% ethanol (prepared with DEPC water) to wash the precipitate, and invert the EP tube to clean the isopropanol remaining on the tube wall; then centrifuge at 4°C and 12,000 × g for 5 min. Use a small pipette tip to remove the supernatant, let the RNA precipitate dry in an ultra-clean bench for about 5 min, add 20-50 μl of RNA-free ddH2O (double-distilled water) to dissolve the RNA precipitate, and store in aliquots.

[0043] The specific steps for extracting RNA from the spleen of the striped bamboo shark are as follows: grind the spleen into powder, take 100 mg of spleen powder, add 1 mL of Trizol reagent, pipette up and down to mix thoroughly, and let it stand at room temperature for 5-10 minutes; add 200 μl of chloroform, cover the tube, place it in a shaker and shake vigorously for 15 seconds, and then incubate it at room temperature for 5-10 minutes; after the incubation is completed, centrifuge the sample at 4°C and 12000×g for 15 minutes; transfer the upper layer containing RNA to a new RNA-free EP tube, add isopropanol at a ratio of 0.5 mL of isopropanol per 1 mL of upper layer, mix thoroughly and let it stand on ice for 10 minutes; then centrifuge it at 4°C and 12000×g for 10 minutes; add 1 mL Wash the precipitate with 75% ethanol (prepared with DEPC water) and invert the EP tube to clean the isopropanol remaining on the tube wall; then centrifuge at 12000×g for 5 minutes at 4°C. Use a small pipette to remove the supernatant and air-dry the RNA precipitate in an ultra-clean bench for about 5 minutes. Add 20-50μl RNA-free ddH2O (double-distilled water) to dissolve the RNA precipitate and store in aliquots.

[0044] The above methods were used to extract RNA from the blood and spleen of the striped bamboo shark and reverse transcription experiments were carried out. Two groups of blood and spleen RNA extraction and reverse transcription were performed, respectively, and recorded as spleen group 1#, spleen group 2#, blood group 1#, and blood group 2#. The electrophoresis results of the extracted RNA are shown in Figure 2. Figure 1 shown.

[0045] 2. Construction and collection of human growth hormone primary library

[0046] 2.1 The cDNA obtained by reverse transcription of spleen RNA and PBMC RNA was mixed, and the VNAR gene was amplified using universal primers (IgNAR1 / 2 / 3 / 4). The VNAR gene was inserted into Escherichia coli and transformed by electroporation to obtain a primary library.

[0047] 2.2 Collection of human growth hormone primary library: 50 clones were randomly selected from the human growth hormone primary library for monoclonal sequencing, and the sequencing results were collected to ensure that the VNAR rate was above 70%.

[0048] The cDNA obtained after reverse transcription from spleen group 1# in step 1 was mixed with the cDNA obtained after reverse transcription from blood group 1#. The cDNA obtained after reverse transcription from spleen group 2# in step 1 was mixed with the cDNA obtained after reverse transcription from blood group 2#. The two mixed cDNAs were then mixed and the human growth hormone primary library was constructed and collected according to the methods in 2.1 and 2.2. During the experiment, the two mixed cDNAs were tested by electrophoresis. The results are shown in the figure. Figure 2 ( Figure 2Human growth hormone 1# represents the electrophoresis result of the mixture of cDNA obtained by reverse transcription of spleen 1# group and cDNA obtained by reverse transcription of blood 1# group. Figure 2 Human growth hormone 2# represents the electrophoresis result of the mixture of cDNA obtained by reverse transcription of spleen group 2# and cDNA obtained by reverse transcription of blood group 2#); during the experiment, the amplified VNAR gene was also subjected to electrophoresis test (four test experiments were set up), and the test results are shown in Figure 3 The human growth hormone primary library obtained by this step is shown in Figure 2. Figure 4 As shown (the culture medium in the figure is set as follows: coating volume 10 μl, storage capacity 3.2x10 9 The dilution multiples of the 6 culture media from left to right and from top to bottom are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 ).

[0049] 3. Construction of primary phage library

[0050] Build it as follows:

[0051] 3.1 Inoculate 2 mL of the human growth hormone primary library into 200 mL of 2×YT / Tet / Car liquid medium, inoculating two bottles in total. Shake at 37°C and 220 rpm until the culture reaches the logarithmic phase.

[0052] 3.2 Add 200 μL of M13KO7 helper phage to each bottle and culture at 37°C and 80 rpm in a shaking incubator for 0.5 h.

[0053] 3.3 Add 200 μL of Kan medium to each bottle and culture in a shaking incubator at 37°C and 220 rpm for 0.5 h.

[0054] 3.4 After that, add 100 μL IPTG medium to each bottle and culture overnight at 30°C and 220 rpm in a shaking incubator.

[0055] 3. Centrifuge the bacterial solution overnight at 8000 rpm and 4°C for 40 min.

[0056] 3.6 Filter the supernatant through a 0.22 μm filter membrane into a new centrifuge tube. Add PEG8000-Na Cl solution at 1 / 5 of the supernatant volume, mix thoroughly, and place on ice for 1 hour.

[0057] Centrifuge at 78,000 rpm and 4°C for 30 min, discard the supernatant, and invert the tube onto absorbent paper to remove any residual liquid.

[0058] 3.8 Resuspend the phage pellet in 1 mL of PBS to obtain the phage solution, which is the primary phage library.

[0059] 3.9 Detect the titer of the obtained phage primary library.

[0060] 4. Three rounds of solid phase panning of phage library:

[0061] 4.1 The first round of selection:

[0062] 4.1.1 Dilute human growth hormone to 5 μg / mL in 0.01 M PBS to prepare coating solution. Inject the coating solution into a 96-well ELISA plate at 100 μl / well and coat overnight at 4°C.

[0063] 4.1.2 Discard the coating solution in the Elisa plate and wash three times with 200 μL 0.05% PBST solution.

[0064] 4.1.3 Prepare 3% BSA blocking solution, add 200 μL of blocking solution to each well, and block at 37°C for 1 hour.

[0065] 4.1.4 Pour off the liquid in the plate and wash five times with 200 μL 0.05% PBST.

[0066] 4.1.5 Take 100 μL of the phage collected in step 3 and dilute to 600 μL with 0.01 M PBS solution; add 600 μL of 3% BSA blocking solution to block the phage, block at room temperature for 1 hour, and then dilute to 5 mL with 0.01 M PBS solution.

[0067] 4.1.6 Add the remaining phage collected in step 3 and shake slowly at 25℃ for 1 hour.

[0068] 4.1.7 Discard the phage mixture from the plate and wash six times with 0.1% PBST solution. Add 100 μL of eluent (add 0.75 g of Glycine and 0.05 g of BSA, dissolve in 45 mL of ddH2O, add 670 μL of 6 M HCl solution, and adjust the volume to 50 mL with ddH2O. The pH value is now 2.2. Filter sterilize and store at 4°C to obtain eluent) to each well. Incubate at room temperature for 5 min to elute the bound phage.

[0069] 4.1.8 Collect the eluate and dilute to 5 mL with 0.01 M PBS solution. Immediately adjust the pH to 7.0 with neutralizing solution (dissolve 1.21 g Tris in 45 mL ddH2O, add 225 μL 6 M HCl solution, and dilute to 50 mL with ddH2O, pH 9.1. Filter and sterilize, and store at 4°C to obtain the neutralizing solution) to obtain the phage library for the first round of panning.

[0070] 4.2 Second round of selection:

[0071] 4.2.1 Dilute human growth hormone to 2 μg / mL with 0.01 M PBS solution to prepare coating solution. Inject the coating solution into a 96-well ELISA plate at 100 μl / well and coat overnight at 4°C.

[0072] 4.2.2 Discard the coating solution in the Elisa plate and wash three times with 200 μL 0.05% PBST solution.

[0073] 4.2.3 Prepare 3% skim milk powder blocking solution, add 200 μL of blocking solution to each well, and block at 37°C for 1 hour.

[0074] 4.2.4 Pour off the liquid in the plate and wash five times with 200 μL 0.05% PBST.

[0075] 4.2.5 Add phage with a titer of 1 / 3 of that obtained in the first round of panning and dilute to 600 μL with 0.01 M PBS solution; block the phage with 600 μL of 3% skim milk powder blocking solution at room temperature for 1 hour, then dilute to 5 mL with 0.01 M PBS solution.

[0076] 4.2.6 Add the remaining phage from the first round of panning and shake slowly at 25°C for 45 minutes.

[0077] 4.2.7 Pour off the phage mixture in the plate, wash eight times with 0.25% PBST solution, add 100 μL of elution buffer to each well, incubate at room temperature for 5 minutes, and elute the bound phage.

[0078] 4.2.8 Collect the eluate and dilute to 5 mL. Immediately adjust the pH to 7.0 with neutralizing solution to obtain the phage library for the second round of panning.

[0079] 4.3 The third round of selection:

[0080] 4.3.1 Dilute human growth hormone to 1 μg / mL with 0.01M PBS solution to prepare coating solution. Inject the coating solution into a 96-well ELISA plate at 100 μl / well and coat overnight at 4°C.

[0081] 4.3.2 Discard the coating solution in the Elisa plate and wash three times with 200 μL 0.05% PBST solution.

[0082] 4.3.3 Add 200 μL of 3% BSA blocking solution to each well and block at 37°C for 1 hour.

[0083] 4.3.4 Pour off the liquid in the plate and wash five times with 200 μL 0.05% PBST.

[0084] 4.3.5 Add phage with a titer of 1 / 3 of that obtained in the second round and dilute to 600 μL with 0.01 M PBS solution; block the phage with 600 μL of 3% BSA blocking solution and block at room temperature for 1 hour. Then dilute to 5 mL with 0.01 M PBS solution.

[0085] 4.3.6 Add the remaining phage from the second round of panning and shake slowly at 25°C for 30 minutes.

[0086] 4.3.7 Pour off the phage mixture in the plate, wash eight times with 0.5% PBST solution, add 100 μL of elution buffer to each well, incubate at room temperature for 5 minutes, and elute the bound phage.

[0087] 4.3.8 Collect the eluate and dilute to 5 mL. Immediately adjust the pH to 7.0 with neutralizing solution to obtain the phage library for the third round of panning.

[0088] 4.4 Inoculate MC1061F' bacterial suspension (Escherichia coli) into 30 mL of 2×YT / Tet liquid medium and culture at 37°C and 220 rpm until the logarithmic phase.

[0089] 4.5 Add the phage obtained from three rounds of panning to 30 mL of bacterial culture cultured to the logarithmic phase, and incubate at 37°C and 150 rpm for 30 min for infection.

[0090] 4.6 Take 100 μL of the infected bacterial solution and perform a 10-fold gradient dilution, then spread it on a 2×YT / Tet / Car / 2% glucose plate for subsequent single colony selection.

[0091] The phages obtained from the three rounds of solid phase panning of the primary phage library obtained in step 3 are shown in Figure 5-Figure 7 .

[0092] 5. Monoclonal screening, ELISA detection, and sequencing

[0093] 5.1 After three rounds of panning, pick 88 clones from the output plate of the third round of panning and place them in a 96-well deep-well plate (column 12 is not used). Add 600 μL of 2×YT / Car medium to each well, cover with a sealing film, poke holes in the film, label the deep-well plate, and shake at 37°C and 220 rpm until the logarithmic phase.

[0094] 5.2 Pipette 100uL of bacterial solution from each well of the deep-well plate into a new 96-well deep-well plate, then add 100uL of 2×YT / Car medium to the new 96-well deep-well plate, cover with a sealing film, and label the new deep-well plate. This is a split plate.

[0095] 5.3 Add 100 μL of 2×YT / Car and 0.6 μL of IPTG to each well of the split-plate deep-well plate and shake overnight at 30°C and 220 rpm.

[0096] 5.4 The next day, centrifuge the deep-well plate at 4000 rpm for 20 minutes, discard the supernatant, add 300 μL of LTEST lysis buffer to each well, vortex to mix, and lyse on ice for 1 hour, shaking every 15 minutes during lysis. Then, incubate at 4°C overnight.

[0097] 5.5 Coat the ELISA plate and label the sample names on the plate (in the ELISA plate, the wells are numbered A1-A12, B1-B12, C1-C12, D1-D12, E1-E12, F1-F12, G1-G12, and H1-H12, with A12, B12, C12, D12, E12, F12, G12, and H12 being control wells, and the rest being post-induction clone wells);

[0098] 5.6 Carry out ELISA test according to the following process (test results are shown in Figure 8 );

[0099]

[0100] 5.7 The target clone solution (i.e. Figure 8 The cloned bacterial solution corresponding to 1-13 in the sample was aspirated into an EP tube, the EP tube cap was labeled and sent to Hangzhou Youkang Sequencing Company for sequencing. The sequencing primer was "p3Rd v-SeqF".

[0101] The amino acid sequences of the partial VNAR genes obtained by sequencing are shown in SEQ ID NO.1-SEQ ID NO.7:

[0102] SEQ ID NO.1( Figure 8 The sequence of the clone solution corresponding to 12 is obtained):

[0103] VEQTPTTTTKEAGESLTINCVLKGSSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAGYWIGYCSGIVGIDAAYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQI

[0104] SEQ ID NO.2( Figure 8 (Sequence obtained from the clone solution corresponding to 4)

[0105] VEQTPTTTTKEAGESLTINCVLKGSSYALCDTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAWPLVGGYCSVGYLYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0106] SEQ ID NO.3( Figure 8 The sequence of the clone solution corresponding to 13 is obtained):

[0107] VEQTPTTTTKEAGESLTINCVLKGSSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAGYWIGYCSGIVGIDAAYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0108] SEQ ID NO.4( Figure 8 (Sequence obtained from the clone solution corresponding to 2):

[0109] VEQTPTTTTKEAGESLTINCVLKGNSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAGVWIGYCSGIVGIDAAYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0110] SEQ ID NO.5( Figure 8 (Sequence determined from the clone solution corresponding to 8):

[0111] VEQTPTTTTKEAGESLTINCVLKGSSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAGGWIGYCSDIIGIDAAYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0112] SEQ ID NO.6( Figure 8 The sequence of the clone solution corresponding to 11 is obtained):

[0113] VEQTPTTTTKEAGESLTINCVLKGSSYALCDTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYPALYCSRWDSARDYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0114] SEQ ID NO.7( Figure 8 (Sequence obtained from the clone solution corresponding to 6):

[0115] VEQTPTTTTKEAGESLTINCVLKGSNYALCDTYWYFTKKGATKKENLSNGGRYAETVNKASKSFSLRISDLRIEDSGTYHCKGSYNWERLLAHTYYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0116] Example 2 Construction of VNAR recombinant plasmid, fermentation purification and detection of antibody expression

[0117] 1. Construction of VNAR recombinant plasmid

[0118] 1.1 Two VNAR sequences (SEQ ID NO. 1 and SEQ ID NO. 2) were selected and named Sequence 1 and Sequence 2, respectively. A GST tag was added to the N-terminus of the amino acid sequence of Sequence 1 and a 6xHIS tag was added to the C-terminus to form the amino acid sequence shown in SEQ ID NO. 8; a GST tag was added to the N-terminus of the amino acid sequence of Sequence 2 to form the amino acid sequence shown in SEQ ID NO. 9.

[0119] SEQ ID NO.8:

[0120] MKYLLPTAAAGLLLLAAQPAMAGGSGGSHHHHHGGGSGGGSMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALD VVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKGGGVEQTPTTTTKEAGESLTINCVLKGSSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAGYWIGYCSGIVGIDAAYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQI

[0121] SEQ ID NO.9:

[0122] MKYLLPTAAAGLLLLAAQPAMAGGSGGSMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYM DPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKGGGVEQTPTTTTKEAGESLTINCVLKGSSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAWPLVGGYCSVGYLYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0123] 1.2 The two sequences were converted into DNA sequences and codon optimized. The two target genes were double-digested with NdeⅠ and EcoRⅠ restriction sites and integrated into the pET22b(+) plasmid respectively. The plasmids pET22b(+)GST-VNAR1-HIS and pET22b(+)-GST-VNAR2 were synthesized by outsourcing.

[0124] 1.3 The two synthesized plasmids were transfected into BL21 competent cells by heat shock method, and single clones were selected by LB plate (ampicillin resistance) and cultured and frozen to obtain BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 expression strains, respectively.

[0125] 2. Fermentation

[0126] The BL21-GST-VNAR1-HIS expression strain and the BL21-GST-VNAR1 expression strain were fermented according to the following method to obtain two supernatants respectively:

[0127] 2.1 The expression strain was pre-cultured in a shake flask and inoculated into a 5 L fermentor containing LB liquid medium (the inoculation volume of the control expression strain was 1 ml). After culturing to OD600 = 40, the culture temperature was adjusted to 16 ° C and IPTG was added with a final concentration of 0.5 mM for overnight induction for 20 h.

[0128] 2.2 Centrifuge the induced bacterial solution at 8000g for 10 minutes to obtain the bacterial cells.

[0129] 2.3 Resuspend the cells in 20 mM Tris (pH 8.0) at a mass ratio of 1:10 based on wet cell weight. Disrupt the cells twice at 1000 bar. Centrifuge the solution at 8000 g for 10 minutes, collect the supernatant, and filter through a 0.45 μm capsule.

[0130] 3. Purification of supernatant after cell wall breaking

[0131] The two supernatants obtained from the fermentation step were purified according to the following methods to obtain BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 expression strain antibodies:

[0132] 3.1 Solution preparation:

[0133] Binding buffer: 50 mM Tris-HCl, 150 mM NaCl, pH = 7.4.

[0134] Elution buffer: 50 mM Tris-HCl, 150 mM NaCl, 10 mM reduced glutathione, pH = 7.4.

[0135] Equilibration buffer: Same as binding buffer.

[0136] 3.2 Column preparation

[0137] A. Remove the GSTrap HP column and flush it with deionized water at a flow rate of 5 ml / min for 10 minutes to completely remove the ethanol.

[0138] B. Equilibrate the column with equilibration buffer at 5 ml / min until the baseline is stable.

[0139] 3.3 Load the supernatant sample obtained by purification in step 2 onto the chromatographic column with a loading volume of 500 ml and a loading flow rate of 5 ml / min.

[0140] 3.4 After loading, wash the column with binding buffer at 5 ml / min until the A280 absorbance returns to the baseline level.

[0141] 3.5 Elute with elution buffer at 2 ml / min, collect the elution peak, observe the change in A280 absorbance, and collect the eluate when a clear elution peak appears.

[0142] 3.6 After elution, rinse the column with binding buffer at 5 ml / min for 10 min, then rinse with 20% ethanol at 5 ml / min for 10 min, and store the column in 20% ethanol.

[0143] 3.7 Replace the two eluates into 20 mM PB pH 7.5 solution using ultrafiltration tubes, measure the protein concentration using the BCA method, and dilute to 1 mg / ml with 20 mM PB pH 7.5 solution.

[0144] 4. Detection of the effect of antibodies expressed in BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 strains

[0145] The effects of antibodies expressed in BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 were tested according to the following methods:

[0146] 4.1 Dilute the antibody to 50 μg / ml with coating solution and add 100 μl / well of the diluted solution to the ELISA plate overnight. The next day, wash the plate and block for 1-2 hours.

[0147] 4.2 Use diluent to add 100 μl of human growth hormone to each well at the following concentrations: 20 ng, 10 ng, 5 ng, 2.5 ng, 1.25 ng, 0.625 ng, and 0 ng. Incubate at 37°C for 1 hour.

[0148] 4.3 Remove the ELISA plate, wash with washing solution, add 100 μl of 1:1000 diluted antibody to each well, and incubate for 1 hour;

[0149] 4.4 Add HRP-labeled mouse anti-HIS monoclonal antibody (purchased from Sangon Biotech) and TMB for color development, stop the reaction, and read the results.

[0150] The results of the antibody effect test of BL21-GST-VNAR1-HIS expression strain are shown in Table 1.

[0151] Table 1

[0152]

[0153] Example 3 Construction and storage of BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 expression strains

[0154] 3.1 Preparation of 40% glycerol: Draw 12 ml of glycerol into a syringe, stir thoroughly and dissolve it in 18 ml of water for injection. Sterilize it by high pressure sterilization (121°C, 40 minutes) and set aside.

[0155] 3.2 Mix the BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 expression strains with 40% glycerol at a volume ratio of 1:1, dispense into cell cryopreservation tubes at 1.0 ml / tube, and store at -80°C or below.

[0156] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A human growth hormone detection antibody, characterized in that The amino acid sequence of the detection antibody is shown in SEQ ID NO.1.

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