Monoclonal antibody a7 of colp6 of marnifia vesfa and its preparation method and application

Abstract

The application discloses a monoclonal antibody A7 of Marninifia colp6, a light chain variable region of the monoclonal antibody A7 has an amino acid sequence as shown in SEQ ID NO.1, and a heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.2; and the application also discloses a preparation method and application of the monoclonal antibody A7. The light chain variable region gene sequence and the heavy chain variable region gene sequence of the monoclonal antibody A7 of Marninifia colp6 are successfully cloned, the monoclonal antibody A7 with the light chain variable region and the heavy chain variable region can be combined with Marninifia colp6, and the antibody has high purity, high specificity and high detection sensitivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a monoclonal antibody A7 of colp6 of Talaromyces marneffei and a preparation method and application thereof. BACKGROUND

[0002] Talaromyces marneffei (PM) is also known as Talaromyces marneffei, which is a dimorphic fungus and can cause Talaromyces marneffei disease (PSM), which is a rare systemic disease of deep fungal infection. Talaromyces marneffei disease can occur in healthy people, but is more common in immunodeficient or immunosuppressed people. The research results in Vietnam show that PSM ranks the third in the opportunistic infections of AIDS patients. With the increasing number of HIV-infected people, the reports of Talaromyces marneffei disease are also increasing year by year.

[0003] The Talaromyces marneffei glycoprotein reported in the prior art has the same or similar antigenic determinant as the Aspergillus glycoprotein, and immunological detection is prone to cross-reaction, and the glycoprotein is easily cleared by phagocytes after binding with the antibody, but so far a rapid, high-sensitivity and high-specificity method for detecting Talaromyces marneffei antigen has not been developed. SUMMARY

[0004] The present application aims to overcome the deficiencies in the prior art, and provides a monoclonal antibody A7 of colp6 of Talaromyces marneffei and a preparation method and application thereof.

[0005] To achieve the above-mentioned purpose, the technical solutions provided by the present application are as follows:

[0006] A monoclonal antibody A7 of colp6 of Talaromyces marneffei, the protein sequence of the monoclonal antibody A7 contains a light chain variable region and a heavy chain variable region, the light chain variable region has an amino acid sequence as shown in SEQ ID NO. 1, and the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 2; the light chain variable region and the heavy chain variable region are connected by a short peptide, and the short peptide has an amino acid sequence as shown in SEQ ID NO. 3.

[0007] Among them, the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO. 4, and the nucleotide sequence encoding the heavy chain variable region is as shown in SEQ ID NO. 5.

[0008] The preparation method of the above-mentioned monoclonal antibody A7 comprises the following steps:

[0009] The gene of the Colp6 protein of the Marninifia was synthesized, the Colp6 recombinant protein was purified and expressed, the purified Colp6 recombinant protein and the Marninifia were used as immunogens to cross-immunize Balb / c mice, the spleen cells of the immunized mice were fused with mouse myeloma NS-1 cells, the hybridoma cells secreting specific McAb were screened by indirect ELISA, the hybridoma cell strain was used to induce the mice to produce ascites, the antibody was purified by protein G affinity chromatography, and finally the Colp6 protein monoclonal antibody A7 was obtained.

[0010] Further, the amino acid sequence of the Marninifia Colp6 protein is shown as SEQ ID NO. 6.

[0011] Further, the preparation method comprises the following steps:

[0012] Step S1: Synthesis of Colp6 gene

[0013] The amino acid sequence of the Colp6 gene of the Marninifia was obtained from the GenBank sequence database; the codon optimization of the base sequence of the Colp6 gene was performed according to the codon preference of E. coli, and the codon-optimized gene sequence was synthesized;

[0014] Step S2: Expression and purification of Colp6 gene

[0015] The Colp6 gene was linked to the pQE 80L vector to obtain a recombinant vector, the plasmid of the recombinant vector was extracted, the plasmid was transformed into E. coli BL21 (DE3), and expression was induced by IPTG; the bacterial body was collected, the bacterial body was broken by high pressure, the supernatant was collected after centrifugation, and the protein was purified by Ni column affinity chromatography; the purified protein was further purified by Superdex-200 column, and the Colp6 recombinant protein with high purity was obtained, and the Colp6 recombinant protein was collected as an antigen for standby;

[0016] Step S3, immunization of mice

[0017] The purified Colp6 recombinant protein and the Marninifia were used as immunogens to immunize 3 female BALB / c mice aged 6-8 weeks, 10 days after conventional immunization, blood was collected from the tail, and the antibody titer produced by the mice was detected by indirect ELISA; the recombinant antigen was injected for the last time through the tail vein 3 days before fusion, and the dose was 60 μg per mouse, and the mice were immunized once;

[0018] Step S4, preparation of NS-1 myeloma cells

[0019] One week before fusion, the NS-1 myeloma cells stored in liquid nitrogen were recovered and cultured in 25 cm 2The cells were subcultured in a cell culture bottle with 15% fetal bovine serum DMEM medium for one week, and the cell concentration was adjusted to 10 6 When the cells were fused, the myeloma cells in logarithmic growth phase were selected, the original medium in the bottle was discarded, and an appropriate amount of serum-free DMEM medium was added. The cells were gently blown off, transferred to a 50 mL centrifuge tube, centrifuged at 400g for 5 minutes, and the cells were washed 3 times. The supernatant was discarded, and the cell pellet was resuspended with serum-free medium and counted for use.

[0020] Step S5, preparation of immune spleen cells

[0021] One of the BALB / c mice with the best immune effect was taken, and the mouse was sacrificed by orbital blood sampling and soaked in 75% alcohol for 5 minutes for disinfection. In the clean bench, the disinfected mouse was fixed, the spleen was removed, and the fat tissue and connective tissue adhered to the cells were removed with scissors. The spleen was washed with serum-free medium and transferred to a 40 μm cell filter screen. The inner core of the syringe was used to gently grind the spleen, and the filter screen was gently washed with serum-free medium. The spleen cell suspension was collected. Centrifugation at 400g for 5 minutes, washing the cells 3 times, discarding the supernatant, and resuspending the cell pellet with serum-free DMEM medium, counting for use.

[0022] Step S6, cell fusion

[0023] The NS-1 myeloma cells and immune spleen cells were mixed in a 50 mL centrifuge tube at a ratio of 1:10, centrifuged at 400g for 10 minutes, the supernatant was aspirated, and the cell pellet was slightly loosened by tapping the bottom of the centrifuge tube. 1 mL of 50% PEG solution pre-warmed to 37°C was slowly added within 1 minute, and the cells were gently stirred with a pipette for 1 minute. Then 10 mL of DMEM medium was added dropwise, 1 mL was added dropwise in the first minute, 1 mL was added in the second minute, 3 mL was added in the third and fourth minutes, and the remaining 5 mL was added after 5 minutes. The cell mixture was incubated in a 37°C water bath for 15 minutes. Then centrifuged at 400g for 5 minutes, remove the supernatant, resuspend the cell pellet with 20 mL of 15% fetal bovine serum DMEM medium, and transfer it to a 75 cm 2 The cell culture bottle was placed in the cell culture incubator for 16-24 hours; the cell suspension after fusion was transferred to a 50 mL centrifuge tube, centrifuged at 400g for 10 minutes; the supernatant was removed, the cell pellet was resuspended with 2.5 mL of 15% fetal bovine serum DMEM medium, and then mixed with 22.5 mL of semi-solid medium and poured into a 3.5 cm diameter flat dish, about 2 mL per flat dish, and cultured at 37°C with 5% CO2; After ten days, white cell colonies can be seen on the surface of the medium in the flat dish under the naked eye.

[0024] Step S7, screening of positive hybridoma cells

[0025] The cell mass in the plate is sucked under sterile conditions and placed in the culture solution in a 96-well plate for further culture for 4 days; after 4 days, indirect ELISA detection is performed; at the same time, the serum of a normal mouse before immunization is used as a negative control, and the serum of a mouse after immunization is used as a positive control, and the absorbance value is greater than 2.1 times that of the negative control group to determine the positive; the specific antibody of the cell growth hole is detected, and the hybridoma cell hole with single clone growth and good morphology is cloned again; after at least 3 times, the hybridoma cells in the positive hole are moved to a 24-well culture plate, and when the hybridoma cells in the 24-well culture plate grow well, the hybridoma cells are frozen;

[0026] Step S8, preparation and purification of monoclonal antibody A7

[0027] 0.5 mL of liquid paraffin is injected into the abdominal cavity of an 8-week-old BALB / c mouse; the hybridoma cells are inoculated in a 25 cm 5 culture bottle at a concentration of 1×10 2 cells / ml, and cultured to the best state of cell activity, and the cell number is adjusted to about 1×10 6 cells / ml; the cells are inoculated into the abdominal cavity of a mouse that has been injected with liquid paraffin; the ascites is collected after 7-10 days; the ascites is diluted more than 3 times with 20 mM PBS pH 7.4, and the supernatant is obtained by centrifugation, and purified by HiTrap protein G affinity chromatography to obtain the Colp6 protein monoclonal antibody A7.

[0028] Further, in step S3, the immunization method of the mouse is as follows: 60 μg of antigen is emulsified with an equal volume of Freund's adjuvant by primary emulsification, 3500 times, at 200 times / min; first immunization: 60 μg of antigen is emulsified with 0.2 mL of liquid paraffin, and 0.2 mL of the emulsion is injected subcutaneously into the groin of each mouse with a 2 mL syringe, and the injection time and site are recorded; second immunization: 60 μg of antigen is emulsified with 0.2 mL of liquid paraffin, and 0.2 mL of the emulsion is injected intraperitoneally into each mouse with a 2 mL syringe, and the injection time and site are recorded; third immunization: 60 μg of antigen is emulsified with 0.2 mL of liquid paraffin, and 0.2 mL of the emulsion is injected subcutaneously into the groin of each mouse with a 2 mL syringe, and the injection time and site are recorded.

[0029] Further, in step S7, the method of indirect ELISA detection is as follows: 100 ng of antigen is coated onto an enzyme-labeled plate at 4°C overnight, washed three times, 200 mL of blocking solution is added, incubated at 37°C for 2 h, washed three times, then hybridoma cell culture supernatant is added, incubated at 37°C for 2 h, washed three times with washing solution, then 1:10,000 diluted goat anti-mouse IgG-HRP is added, incubated at 37°C for 1 h, washed three times, then 2 M H2SO4 is added to terminate the reaction after 10 min of color development at 37°C in the dark, and the absorbance value (OD value) is measured at a wavelength of 450 nm by an enzyme-labeled instrument.

[0030] Further, in step S8, the Protein G affinity chromatography column is used for purification, and a new column is first passed through 5 mL of ultrapure water, and then 5 mL of 0.4M PB buffer (pH 7.0) is used to balance the purification column; the antibody is passed through the column, and the process requires slow passing through the column to better bind the antibody protein to the binding site; 10 mL of 0.4M pH 7.0 PB buffer is continued to balance the purification column; 5 mL of 0.1M pH 2.7 glycine-hydrochloric acid buffer is used to remove the antibody on the binding site, and 1M pH 8.0 Tris-HCl is added to neutralize the glycine, so that the pH is kept neutral for the preservation of the antibody; the purified antibody hybridoma cells are subjected to antibody sequence determination.

[0031] The application also protects the use of the monoclonal antibody A7 of the said Rhizomucor Miehei colp6, which is any one of the following (a1) to (a6):

[0032] (a1) identifying Rhizomucor Miehei;

[0033] (a2) preparing a kit for identifying Rhizomucor Miehei;

[0034] (a3) detecting whether the pathogenic microorganism to be tested is Rhizomucor Miehei;

[0035] (a4) preparing a kit for detecting whether the pathogenic microorganism to be tested is Rhizomucor Miehei;

[0036] (a5) detecting whether Rhizomucor Miehei is contained in the sample to be tested;

[0037] (a6) preparing a kit for detecting whether Rhizomucor Miehei is contained in the sample to be tested.

[0038] The application also protects the use of the monoclonal antibody A7 of the said Rhizomucor Miehei colp6, which is any one of the following (a1) to (a6):

[0039] (b1) identifying Rhizomucor Miehei;

[0040] (b2) detecting whether the pathogenic microorganism to be tested is Rhizomucor Miehei;

[0041] (b3) detecting whether Rhizomucor Miehei is contained in the sample to be tested.

[0042] Compared with the prior art, the application has the following beneficial effects:

[0043] The application successfully clones the light chain variable region gene sequence and the amino acid sequence of the colp6 monoclonal antibody A7 of the genus Marninifia and the monoclonal antibody A7 with the light chain variable region can specifically recognize the genus Marninifia; the monoclonal antibody A7 obtained by the application can be combined with the genus Marninifia colp6, has high purity, strong specificity and high detection sensitivity.

[0044] The accompanying drawings

[0045] Figure 1 The protein electrophoretogram of the Colp6 monoclonal antibody is shown in Fig. 1. Figure 1 In Fig. 1, the molecular weights of the bands from top to bottom in the Maker lane are respectively 116KD, 66.2KD, 45KD, 35KD, 25KD, 18.5KD and 14.5KD.

[0046] Figure 2 The titer determination diagram of the monoclonal antibody A7 is shown in Fig. 2.

[0047] Figure 3 The Western-Blot detection diagram of the Colp6 monoclonal antibody is shown in Fig. 3. DETAILED DESCRIPTION

[0048] The application will be described in detail below with specific embodiments, but it should be understood that the protection scope of the application is not limited by the specific embodiments. The raw materials used in the examples are commercially available unless otherwise specified.

[0049] Example 1: Preparation of the monoclonal antibody of the genus Marninifia colp6

[0050] Step S1: Synthesis of the Colp6 gene

[0051] The amino acid sequence of the Colp6 gene of the genus Marninifia is obtained from the GenBank sequence database, the codon optimization of the base sequence of the Colp6 gene is performed according to the codon preference of the E. coli, and the codon-optimized gene sequence is synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. The Colp6 gene encodes 220 amino acid residues.

[0052] The amino acid sequence of the Colp6 (SEQ ID NO. 6) is shown in Table 1.

[0053] MKFLPSLVVLGLSTQALASSYVDYVTKDQHGLTVYEMVINIINTTTSDFNTRIQSYQGGDLSSILEGCNQVTQIMKLGATILDQQTTKPLTNNEWLSLLSHMEKKGGLEDMLKMAINTLILKKSLILDSGLGSKLGLALYSQQMASIDLGAKFFEKTPPGKVEDFREHWFKNIMWVIGRGVDTFDKSTHHATIPTLPPRGAMATSNSPAIPTFTDAASAN

[0054] Colp6 nucleotide sequence (SEQ ID NO. 7):

[0055] ATGAAGTTCTTACCCTCTCTCGTCGTCCTCGGTCTTTCTACCCAGGCTCTTGCGAGCTCTTACGTCGATTATGTTACTAAGGACCAGCATGGTCTTACTGTTTATGAGATGGTCATAAATATCATTAACACCACTACCTCAGACTTCAATACCCGGATTCAGAGTTACCAGGGTGGTGATCTCAGCAGCATTCTCGAAGGCTGTAATCAAGTTACCCAAATAATGAAGCTTGGGGCTACGATACTCGATCAGCAAACAACGAAACCCCTTACCAATAATGAGTGGCTTAGCCTCCTCTCACATATGGAGAAAAAGGGTGGTTTAGAAGATATGTTGAAAATGGCTATAAATACTCTTATCCTGAAGAAGTCACTTATTCTTGATTCTGGTTTGGGTTCTAAGCTTGGGTTAGCGCTTTACAGTCAGCAGATGGCTTCTATAGACCTCGGTGCTAAGTTCTTTGAGAAGACCCCCCCAGGAAAGGTCGAAGATTTCAGAGAACACTGGTTTAAGAACATCATGTGGGTCATCGGGCGAGGTGTTGATACCTTTGATAAATCTACCCATCACGCGACAATACCTACCTTACCCCCTAGAGGTGCTATGGCGACTAGTAATTCGCCTGCTATCCCTACTTTTACTGATGCTGCTAGTGCTAACTAG

[0056] Step S2, expression and purification of Colp6 gene

[0057] The Colp6 gene was linked to the pQE 80L vector to obtain a recombinant vector, the plasmid of the recombinant vector was extracted, the plasmid was transformed into Escherichia coli BL21 (DE3), expression was induced by IPTG; the bacterial body was collected, the bacterial body was broken by high pressure, the supernatant was collected after centrifugation, and the protein was purified by Ni column affinity chromatography; the purified protein was further purified by Superdex-200 column, and high-purity Colp6 recombinant protein was obtained, which was collected as an antigen for standby;

[0058] Step S3, immunization of mice

[0059] The purified Colp6 recombinant protein and the Marnifia fungus were used as immunogens to immunize 3 female BALB / c mice aged 6-8 weeks, tail blood was collected 10 days after conventional immunization, and the antibody titer produced by the mice was detected by indirect ELISA; the recombinant antigen was last injected into the tail vein 3 days before fusion at a dose of 60 μg per mouse, and the mice were immunized once;

[0060] The immunization method of the mice was as follows: 60 μg of antigen per mouse was emulsified with an equal volume of Freund's adjuvant by primary emulsification, 3500 times emulsification, and 200 times / min; the first immunization: 60 μg of antigen was injected into the groin of each mouse subcutaneously with a 2 mL syringe, and the injection time and site were recorded; the second immunization: 60 μg of antigen was injected into the abdomen of each mouse with a 2 mL syringe, and the injection time and site were recorded; the third immunization: 60 μg of antigen was injected into the groin of each mouse subcutaneously with a 2 mL syringe, and the injection time and site were recorded;

[0061] Step S4, preparation of NS-1 myeloma cells

[0062] One week before fusion, the NS-1 myeloma cells stored in liquid nitrogen were recovered and cultured in a 25 cm 2 cell culture bottle with 15% fetal bovine serum DMEM culture solution for one week, and the cell concentration was adjusted to 10 6 cells / mL; at the time of fusion, the logarithmic growth phase myeloma cells were selected, the original bottle culture medium was discarded, an appropriate amount of serum-free DMEM culture solution was added, the cells were gently blown down, transferred into a 50 mL centrifuge tube, centrifuged at 400g for 5 minutes, the cells were washed 3 times, the supernatant was discarded, and the cell pellet was resuspended with serum-free culture solution for standby;

[0063] Step S5, preparation of immune spleen cells

[0064] Take the best immune effect of BALB / c mice, one of the orbital blood, 75% alcohol soak 5 min disinfection; In the clean bench, the good mouse fixed, take out the spleen, scissors to remove the fat tissue and connective tissue of the cells; The spleen with serum-free medium rinse, move into 40 μm cell filter screen, with a syringe core gently grinding, and gently rinse the filter screen with serum-free medium, collection of spleen cell suspension; 400g centrifugation for 5 min, wash the cells 3 times, discard the supernatant, the cell pellet with serum-free DMEM medium suspension, count for use;

[0065] Step S6, cell fusion

[0066] The NS-1 myeloma cells and immune spleen cells were mixed in a 50 mL centrifuge tube at a ratio of 1:10, 400g centrifugation for 10 min, the supernatant was aspirated, and the bottom of the centrifuge tube was tapped to slightly loosen the cell pellet; 1 mL of 50% PEG solution was slowly added within 1 min and the cells were gently stirred with a pipette for 1 min; then 10 mL of DMEM medium was added to the cell mixture, 1 mL was added dropwise in the first minute, 1 mL was added in the second minute, 3 mL was added in the third to fourth minutes, and the remaining 5 mL was added after 5 min; the cell mixture was incubated in a 37°C water bath for 15 min; then 400g centrifugation for 5 min, remove the supernatant, resuspend the cell pellet with 20 mL 15% fetal bovine serum DMEM medium, and transfer to a 75 cm 2 cell culture bottle, and incubate in a cell culture incubator for 16-24 h; the fused cell suspension was transferred to a 50 mL centrifuge tube, 400g centrifugation for 10 min; remove the supernatant, resuspend the cell pellet with 2.5 mL 15% fetal bovine serum DMEM medium, mix with 22.5 mL semi-solid medium, and pour into a 3.5 cm diameter dish, about 2 mL per dish, 37°C, 5% CO2 culture; ten days later, white cell colonies can be seen on the surface of the culture medium in the dish;

[0067] Step S7, positive hybridoma cell screening

[0068] Under sterile conditions, the cell mass in the dish was aspirated and placed in 96-well plate culture medium, and continued to culture for 4 days; after 4 days, indirect ELISA detection was performed; at the same time, pre-immune mouse serum was used as negative control and post-immune mouse serum as positive control, then the absorbance value was greater than 2.1 times that of the negative control group to determine positive; the cell growth hole was detected to have specific antibodies, and the hybridoma cell hole was single clone growth and good in morphology, and was cloned again; after at least 3 times, the positive hole hybridoma cells were moved to a 24-well culture plate, and when the hybridoma cells in the 24-well plate grew well, the hybridoma cells were frozen;

[0069] The method of the indirect ELISA detection is: coating the antigen to the enzyme-labeled plate at 100 ng / well, 4℃ overnight, washing three times, adding 200 mL blocking solution, incubating at 37℃ for 2 h, washing three times, then adding the hybridoma cell culture supernatant, incubating at 37℃ for 2 h, washing three times with washing solution, then adding 1:10000 diluted goat anti-mouse IgG-HRP, incubating at 37℃ for 1 h, washing three times, then developing color at 37℃ for 10 min in the dark, adding 2M H2SO4 to terminate the reaction after sufficient color development, and determining the absorbance value (OD value) at a wavelength of 450 nm by using an enzyme-labeled instrument;

[0070] Step S8, preparation and purification of monoclonal antibody

[0071] Injecting 0.5 mL of liquid paraffin into the abdominal cavity of an 8-week-old BALB / c mouse; inoculating hybridoma cells at a concentration of 1×10 5 cells / ml in a 25 cm 2 culture bottle, and culturing until the cell activity is optimal, and then adjusting the cell number to about 1×10 6 cells / ml for inoculation into the abdominal cavity of the mouse that has been injected with liquid paraffin; collecting the ascites after 7-10 days; diluting the ascites more than 3 times with 20 mM PBS pH 7.4, centrifuging to obtain the supernatant, and purifying by HiTrap protein G affinity chromatography; the method of protein G affinity chromatography purification is as follows: first equilibrating the new column with 5 mL of ultrapure water, and then with 5 mL of 0.4M PB buffer (pH 7.0) to equilibrate the purification column; slowly passing the antibody through the column to better bind the antibody protein to the binding site; continuing to equilibrate the purification column with 10 mL of 0.4M PB buffer (pH 7.0); eluting the antibody from the binding site with 5 mL of 0.1M glycine-hydrochloric acid buffer (pH 2.7), and neutralizing the glycine with 1M Tris-HCl (pH 8.0) to maintain a neutral pH suitable for antibody storage. Two strains (A5, A7) of purified Colp6 monoclonal antibodies were subjected to protein electrophoresis, and the loading amount was 1 μL. The results showed that the A5 and A7 antibodies had high purity, and the light chain and heavy chain of the antibody could be clearly seen (see Figure 1 ).

[0072] Step S9, sequence determination of the monoclonal antibody

[0073] The hybridoma cells of A5 and A7 were sent to Nanjing Kingsriver Biotech Co., Ltd. for antibody sequence determination. The method is as follows: total RNA was isolated from the hybridoma cells according to the technical manual of RNeasy Plus Micro Kit. Then according to the technical manual of SMARTScribe Reverse Transcriptase, total RNA was reverse transcribed into cDNA using isotype-specific antisense primer or universal primer. The antibody fragments of heavy chain and light chain were amplified according to the standard operating procedure of GenScript cDNA rapid amplification (RACE). The amplified antibody fragments were cloned into standard cloning vectors respectively. Colony PCR was performed to screen clones with correct size of inserted fragments.

[0074] The sequencing results of monoclonal antibody A7 are as follows:

[0075] The amino acid sequence of the light chain variable region of monoclonal antibody A7 is (SEQ ID NO. 1): DIVMTQSPLSLPVSLGDQASIFCRSSQSLVHSNGKIYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVGAEDLGVYFCSQSTHVPLTFGAGTKLELKR

[0076] The amino acid sequence of the heavy chain variable region of monoclonal antibody A7 is (SEQ ID NO. 2): EVKLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSDSNTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARAAYYGSSFGYWGQGTLVTVSA

[0077] The short peptide connecting between the light chain variable region and the heavy chain variable region has the amino acid sequence (SEQ ID NO. 3):

[0078] GGGGSGGGGSGGGG

[0079] Example 2: Determination of the titer of monoclonal antibody A7

[0080] Colp6 protein was coated at 100 ng / well, and the monoclonal antibody A7 was diluted at 1 :2000, 1 :4000, 1 :8000, 1 :16000, 1 :320000, 1 :640000, 1 :1280000, 1 :2560000, 1 :512000, 1 :1024000, 1 :4096000 (the original antibody concentration was 1 mg / mL). The A450nm value was determined by indirect ELISA method. The maximum dilution of the monoclonal antibody reacting with the immunized protein target antigen was the titer, and the determination hole reading value greater than 2.1 was positive. The specific operation steps were as follows: coating antigen, 4°C overnight coating. 3% BSA blocking solution, 37°C blocking for 2h; using Colp6 monoclonal antibody as the primary antibody, 37°C incubation for 1h; HRP-goat anti-mouse IgG as the secondary antibody, 37°C incubation for 1h. TMB color developing solution color development for 5min; 2mM sulfuric acid termination. The results showed that the titer of monoclonal antibody A7 was more than 2 million (see Figure 2). Figure 2 )

[0081] Example 3 Specificity identification of monoclonal antibody A7

[0082] Western-Blot was used to detect the specificity of the antibody using Colp6 protein as the antigen. The specific experimental steps were as follows: Colp6 protein loading amount was 1 μg, electrophoresis, and electrotransfer to PVDF membrane, the membrane was taken out, TBS membrane washing, 5minx3.3% BSA 4°C blocking overnight. The purified monoclonal antibody was added at a ratio of 1:50000 (the antibody was used at a concentration of 0.02 μg / ml), and incubated for 1h. The membrane was washed with TBST for 5minx3. The HRP-labeled goat anti-mouse IgG antibody (secondary antibody) was added at a dilution of 1:10000, and incubated for 1h. The membrane was washed with TBST for 5minx3. The ECL color developing solution was prepared at a ratio of A / B liquid 1:1, and exposed on the luminescent instrument. The Western-blotting results showed that the monoclonal antibody A7 could bind to Colp6 protein (see Figure 3). Figure 3 ).

[0083] The foregoing description of specific exemplary embodiments of the application is intended to be illustrative only and is not intended to limit the application to the precise forms described. Many modifications and variations are possible in light of the above teachings without departing from the spirit or essential characteristics of the application. The exemplary embodiments were chosen and described in order to explain the principles of the application and its practical application to thereby enable others skilled in the art to best utilize the application and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims

1. A monoclonal antibody A7 of colp6 of Marninifia vesfae, characterized in that, The protein sequence of the monoclonal antibody A7 contains a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 1 and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 2; the light chain variable region and the heavy chain variable region are connected by a short peptide having an amino acid sequence as shown in SEQ ID NO. 3; wherein the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO. 4, and the nucleotide sequence encoding the heavy chain variable region is as shown in SEQ ID NO.

5.

2. A detection reagent or kit containing the monoclonal antibody A7 of claim 1.

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