CN101270160B - A monoclonal antibody 1F1 and its application and the hybridoma cell line BCSP31-1F1 secreting the antibody - Google Patents

Abstract

The present invention relates to a monoclonal antibody 1F1 and its application as well as a hybridoma cell line BCSP31-1F1 secreting the antibody, wherein the accession number of the hybridoma cell line is CCTCC C200806. The BCSP31 antigen of Brucella melitensis is expressed in prokaryotes, mice are immunized after purification, and the hybridoma cell line BCSP31-1F1 is obtained by cell fusion and screening. The monoclonal antibody 1F1 is prepared and purified by intraperitoneal inoculation of mice. The obtained monoclonal antibody belongs to the IgG1 subclass and can specifically react with the BCSP31 protein of Brucella melitensis. The heavy chain and light chain variable region genes of the mouse monoclonal antibody against the BCSP31 antigen of the present invention and the encoded polypeptides are constructed and expressed into various forms of small molecule genetic engineering antibodies, such as Fab antibodies, chimeric antibodies, antibody fusion proteins, etc., by genetic engineering methods, to prepare preparations or drugs for diagnosing or treating brucellosis.

Description

A monoclonal antibody 1F1 and its application and the hybridoma cell line BCSP31-1F1 secreting the antibody

technical field

The invention belongs to the field of immune application, relates to related fields such as molecular biology and immunology, and particularly relates to a monoclonal antibody 1F1 against Brucella sheep BCSP31 protein and a hybridoma cell line BCSP31-1F1 secreting said antibody, and the application of said The application of the monoclonal antibody in the preparation of detection reagents for detecting Brucella and diagnosing Brucellosis. The present invention also relates to the heavy chain and light chain variable region genes of the anti-brucella BCSP31 monoclonal antibody and their coded polypeptides, and the application of the genes and polypeptides in the preparation of medicines for diagnosing or treating Brucella and its inflammation .

Background technique

Brucella, also known as Brucella in the past, can infect livestock such as sheep, cattle, and pigs, and can also infect humans, causing brucellosis in humans. Brucella can infect through various routes such as skin, mucous membrane, intestinal tract and respiratory tract, and invade various tissues and organs of the human body. The pathological changes and clinical manifestations of the disease caused by it are diverse, complex and unstable. The clinical diagnosis of the disease is relatively difficult, and there is also a lack of effective preventive agents.

Brucellosis is one of the most widespread zoonotic diseases worldwide. According to the report of the World Health Organization, there are currently more than 160 countries and regions in the world where brucellosis occurs and spreads among humans and animals; about 500,000 people suffer from brucellosis every year in the world. my country's "Law on the Prevention and Control of Infectious Diseases" defines brucellosis as a Class B infectious disease, and is listed as the first of the Class II infectious diseases in the "Regulations for the Implementation of the Regulations on Epidemic Prevention of Livestock and Poultry". At present, there are different degrees of brucellosis epidemics among humans and animals in more than 1,200 cities and counties in 25 provinces, regions, and municipalities directly under the central government, and the threatened population reaches 300 million people. Especially in the past ten years, the epidemic situation of human brucellosis in my country has rebounded significantly, and the magnitude of the rebound has increased year by year, and it has become more and more serious. In addition, brucellosis not only seriously endangers human health, but also has a huge impact on the development of animal husbandry in my country. After cattle, sheep, pigs and other livestock are infected with Brucella, it can cause a large number of livestock infertility, infertility or mass abortion, stillbirth, etc. . According to statistics, in major pastoral areas such as Inner Mongolia, Xinjiang, and Qinghai, livestock abortions and empty pregnants caused by brucellosis each year result in fewer than ten million young animals, and economic losses of more than one billion yuan. In addition, judging from the experience and lessons learned from the occurrence, prevalence and prevention of SARS and avian influenza in recent years, special attention should be paid to the etiological research and diagnosis, prevention and treatment of zoonotic infectious diseases.

Brucella BCSP31 protein is a soluble protein present on the outer membrane surface of Brucella with strong immunogenicity ^[4] , and its molecular weight is 31kDa. BCSP31 protein is conserved in 6 species of Brucella. Preliminary experiments have proved that BCSP31 is a virulence factor, and its role may be to directly or indirectly inhibit the proliferation of macrophages in the body, thereby reducing the ability of the immune system to kill infected cells. In addition, gene deletion studies have shown that BSCP31 does not affect bacterial invasiveness and growth rate ^[7] . In recent years, it has been confirmed that BCSP31 has good immunogenicity and is one of the important immune protective antigens, which can induce the body to produce a protective immune response mainly based on B cell immunity ^[6] .

BCSP31 is highly conserved and has high homology in a variety of Brucella that can infect humans. Therefore, BCSP31 has a potentially important role in the detection of Brucellosis. Currently, the main serological detection method for brucellosis is tiger red plate test, but the false positive rate of this method is high. Zeki et al pointed out that with the aid of ELISA detection method, the false positive rate of Tiger Red plate test can be effectively reduced. Therefore, it is particularly feasible to establish an ELISA detection method or a detection kit based on the Brucella BCSP31 protein.

In the 1990s, monoclonal antibodies against Brucella surface antigens A and M (antigens A and M are Brucella bacterium antigens, lipopolysaccharide) and several rough Brucella (not Antigen identification) monoclonal antibody is mainly used in the identification of different types. In addition, the idiotype antibody (polyclonal antibody) was prepared by anti-brucella monoclonal antibody, and the cow was immunized after adding adjuvant. The results showed that the lectin antibody stimulated by it disappeared quickly, and the incomplete antibody could last for more than one year. However, none of the above-mentioned monoclonal antibodies has been practically used in the diagnosis, prevention and treatment of brucellosis. Domestic studies on monoclonal antibodies against BCSP31 have not been reported yet.

Contents of the invention

The invention follows the principle of "giving consideration to both humans and animals", expresses, purifies and identifies the gene BCSP31 related to Brucella infection and immunity; and prepares a monoclonal antibody against the BCSP31 protein by using hybridoma technology. The monoclonal antibody can be applied to the detection of Brucella and the diagnosis and treatment of Brucellosis.

One object of the present invention is to provide a monoclonal antibody 1F1 against Brucella sheep BCSP31 protein and a hybridoma cell line BCSP31-1F1 capable of continuously secreting the monoclonal antibody.

Another object of the present invention is to provide the heavy chain and light chain variable region genes of the monoclonal antibody against BCSP31 antigen and the encoded polypeptides thereof.

Another object of the present invention is to use the monoclonal antibody and its gene or polypeptide in detecting Brucella and diagnosing or treating human or animal brucellosis preparations or medicines.

The present invention is achieved like this:

According to one aspect of the present invention, the monoclonal antibody hybridoma cell line BCSP31-1F1 of the anti-Bruceella sheepis BCSP31 protein has been preserved in the China Center for Type Culture Collection (CCTCC) on March 27, 2008, and the preservation number is CCTCC C200806. Utilize the prokaryotic expression vector of Brucella sheep BCSP31, induce expression, and purify by affinity chromatography to obtain recombinant BCSP31 protein, routinely immunize mice, take immune spleen cells and myeloma cell SP2/0 fusion, and obtain hybridization through ELISA indirect method screening Tumor cell line BCSP31-1F1. After half a year of continuous passage, the hybridoma can still continuously secrete the monoclonal antibody 1F1.

According to another aspect of the present invention, the monoclonal antibody 1F1 secreted by the hybridoma cell line whose deposit number is CCTCC C200806. The above-mentioned hybridoma cells were inoculated into the peritoneal cavity of mice to prepare ascites, and the monoclonal antibody was obtained by purification with n-octanoic acid-ammonium sulfate method. The monoclonal antibody can specifically bind to the Brucella sheep BCSP31 protein.

According to another aspect of the present invention, the heavy and light chain variable region genes of the monoclonal antibody 1F1 are obtained from the RNA of the hybridoma BCSP31-1F1 cells, and the heavy chain and light chain of the monoclonal antibody are obtained by RT-PCR. Light chain variable region gene. After sequence determination and BLAST comparative analysis in NCBI, it was confirmed that the nucleotide sequence of the heavy chain variable region of the antibody is shown in <400>1 in the sequence listing, and the encoded amino acid sequence is shown in <400> in the sequence listing 3; the nucleotide sequence of the light chain variable region gene of the antibody is shown in <400>2 in the sequence listing, and the encoded amino acid sequence is shown in <400>4 in the sequence listing.

For the antibody heavy and light chain variable region genes successfully cloned in the present invention, use the professional known antibody gene sequence database (IMGT) and (NCBI) on the Internet respectively to compare the homology of the obtained sequences and their germline gene sources The analysis showed that the obtained gene sequence was indeed derived from the mouse germline gene, which was not completely consistent with the various antibody gene sequences reported so far.

According to still another aspect of the present invention, the present invention also relates to the heavy and light chain variable region genes of the antibody and the antibody and the polypeptides encoded thereof in the preparation of preparations or drugs for Brucella detection and diagnosis and treatment of Brucellosis aspects of application.

Using the monoclonal antibody and recombinant BCSP31 protein of the present invention as core reagents, an ELISA sandwich method is established, which can be used for ELISA detection of brucellosis.

The present invention expresses and purifies BCSP31 protein through gene recombination, and immunizes mice to prepare anti-BCSP 31 specific monoclonal antibody 1F1, which is for the future detection of Brucella and the preparation of diagnostic reagents for Brucella disease, as well as for in-depth research on Brucella The basis of the pathogenic mechanism. The present invention successfully clones the heavy and light chain variable region genes (VH, VL) of the monoclonal antibody by using the RT-PCR method. Based on the above heavy and light chain variable region genes, genetic engineering methods can be used to construct and express various forms of small molecule genetically engineered antibodies, such as ScFv antibodies, Fab antibodies, F(ab) ₂ antibodies, antibody fusion proteins, etc. , for the preparation of a medicament for the diagnosis and treatment of Brucella and Brucellosis.

Description of drawings

Figure 1 is the Western-blot detection results of purified BCSP31 protein.

Figure 2 is the antigen-specific detection results of monoclonal antibodies.

Fig. 3 is the sensitivity detection result of ELISA indirect sandwich method.

Detailed ways

Prokaryotic expression, purification and identification of Brucella sheep BCSP31 protein

BCSP31-pGEX-4T1 is a prokaryotic expression vector for recombinant BCSP31 protein, and its specific construction method can be found in Si Rui et al. (Journal of Zoonoses 2005, 21(11): 961-964). The expression vector was transformed into Escherichia coli DH5α, induced by 10mmol/L IPTG for 4h, collected and resuspended in PBS, ultrasonically lysed, and purified using Amersham’s purification kit, following the instructions. After loading the sample, PBS was used to wash away the impurity proteins, and 1ml of 100U/ml thrombin was slowly poured into the sample port of the GSTrap affinity chromatography column with a syringe, the upper and lower ports were closed, and enzyme digestion was performed at 4°C for 20 hours. Wash with buffer solution, collect the eluate in sections, and measure the protein content by ultraviolet spectrophotometer to 1.5 mg/mL. The results of Western blot detection showed that the obtained BCSP31 protein could specifically react with Brucella positive sera (see Figure 1).

Hybridoma preparation

1. Preparation of immune splenocytes

Take the above-mentioned purified BCSP31 protein and fully mix Freund's complete adjuvant, and carry out multi-point subcutaneous immunization on the back at 100 μg/mouse; after 2 to 3 weeks, take the purified BCSP 31 protein and incomplete Freund's complete adjuvant Mix well, and carry out multi-point subcutaneous immunization on the back at 100 μg per mouse; 2 to 3 weeks later, take the purified BCSP31 protein and inoculate it intraperitoneally at 100 μg per mouse, and immunize three times in total. Two weeks after the last immunization, the antibody titer in the mouse serum was determined by ELISA method. High-titer mice were boosted with intraperitoneal immunization once as the source of spleen cells.

2. Cell Fusion

On the day of fusion, collect 3×10 ⁷ cells/ml of mouse myeloma cells (Sp2/0) in the logarithmic growth phase, in good growth state, and with a live cell count higher than 95%, and wash 2 times with incomplete culture medium (each 1000rpm centrifugal 5min). The splenocytes of the above immunized mice were ground and made into a suspension, washed twice with incomplete culture medium as above, and the number of cells was adjusted to 2×10 ⁸ cells/ml. Myeloma cells and splenocytes were mixed together at a ratio of 1.5:10, washed once with incomplete culture medium in a 50ml plastic centrifuge tube (centrifuged at 1200rpm for 8min). Discard the supernatant and aspirate the residual liquid. Flick the bottom of the centrifuge tube to loosen the precipitate, slowly add 0.7ml PEG at room temperature within 60 seconds, then suck into the pipette and let it stand for 30 seconds, then blow it out slowly within 30 seconds. Add 25ml of incomplete 1640 medium within 5min, centrifuge at 1000rpm for 8min, and discard the supernatant. Resuspend the pellet in 10ml of HAT medium, add 2 drops/well to a 96-well plate covered with feeder cells. Place in a 5% CO ₂ incubator at 37°C to continue culturing.

3. Selection culture and screening of positive clones

Culture the fused cells with HAT medium, change the medium once after 4 to 5 days of culture, suck out 1/2 of the liquid in the 96-well plate, add 1×HAT complete culture medium, 100 μl/well, and place in a 5% CO2 incubator at 37°C Continue to culture and observe the growth status of cell clones every day. After maintaining the culture with HAT selection medium for 1 week, switch to HT medium and maintain the culture for another 3-4 days.

When the hybridoma cells cover 1/10 of the bottom of the well (about day 8-10), detect the culture supernatant by indirect ELISA: coat BCSP31 protein at a concentration of 5 μg/ml, 100 μl/well, overnight at 4°C, wash Then add 3% BSA to block, react at 37°C for 2h, add cell culture supernatant after washing, react at 37°C for 1h, add 1:3000 HRP-labeled goat anti-mouse IgG diluted with diluent after washing, and react at 37°C for 1h , after fully washing, add the substrate solution (OPD), develop color at room temperature for 30min, stop the reaction with 2M H2SO4 and measure the OD value at 492nm. Sp2/0 cell culture supernatant and immune mouse serum were used as negative and positive controls, respectively. Cells in positive wells were cloned multiple times by limiting dilution method to establish hybridoma cell lines. A hybridoma cell line was obtained and continuously cultured for half a year. The cell line can still secrete monoclonal antibody stably. The cell line was named: BCSP31-1F1.

Purification of monoclonal antibodies

The hybridoma cells were mixed evenly with serum-free medium, and the number of cells was adjusted to 1-2×10 ⁶ cells/ml, and 0.5 ml was injected intraperitoneally into each BALB/c mouse. About 10-15 days after hybridoma cells were inoculated, the abdomen of the mice was obviously enlarged. Ascites was extracted, centrifuged at 3000rpm for 15min, and the supernatant was collected. Ascitic fluid was purified by octanoic acid-saturated ammonium sulfate method. Dilute the ascites with 4 times the volume of acetic acid buffer, adjust the pH to 4.5 with 1mol/L sodium hydroxide; add n-octanoic acid in an amount of 25 μl per ml of the diluted sample, stir while adding, and add slowly. After standing for 30min, centrifuge at 10000×g for 30min. Add 10-fold concentrated PBS (9 samples plus 1 part of 10×PBS) to adjust the pH to 7.4. The supernatant was cooled to 4°C. Add 45% saturated ammonium sulfate to precipitate the supernatant, stir for 30 min, and centrifuge at 5000×g for 15 min. Dissolve the precipitate with a small amount of PBS, and dialyze overnight with 50-100 times the volume of PBS at 4°C. The concentration of mAb was determined to be 2.0 mg/mL. SDS-PAGE electrophoresis identified as a purified monoclonal antibody with a purity of 97%. The monoclonal antibody can be used to prepare reagents for detecting Brucella antibodies.

Antigen-specific test results of monoclonal antibodies

Take the above-mentioned purified BCSP31 protein for SDS-PAGE electrophoresis, transfer to NC membrane or PVDF membrane, react with the above-mentioned purified monoclonal antibody at a concentration of 1-50ug/mL, and use 1:1000-1:10000 diluted HRP to label Rabbit anti-mouse IgG detection, DAB color development. The results showed that the monoclonal antibody could specifically react with BCSP31 protein (see Figure 2).

Determination of monoclonal antibody subclasses

The mouse monoclonal antibody subtype kit from Sigma Company was used to detect the subtype of the prepared monoclonal antibody. The subclass of the above-mentioned monoclonal antibody is IgG1.

Application of Monoclonal Antibody in Diagnosis of Sheep Brucellosis

1. Detection of Brucella antibody by ELISA indirect sandwich method

Coat the purified monoclonal antibody (1-10 μg/ml) on the ELISA plate, 100 μl/well, and incubate overnight at 4°C. The ELISA plate was washed with PBS/T for 3 min×3 times, and then the purified BCSP31 protein (10-50 μg/ml) was added, 100 μl/well, and reacted at 37° C. for 1 h. After washing, goat serum to be tested (1:100) was added, 100 μl/well, and reacted at 37° C. for 1 h. After washing with PBS/T, add 1:2000-50000 diluted HRP-labeled rabbit anti-goat (goat or sheep) IgG (Zhongshan Bioengineering Co., Ltd.), 100 μl/well, and react at 37° C. for 1 h. After the ELISA plate was washed with PBS/T for 3 min×3 times, it was washed with o-phenylenediamine (O-phenyl-enediamine, OPD) or tetramethylbenzidine (3,3′,5,5′-tetramethyl-benzidine, TMB) For substrate color development, 100 μl/well, react at 37°C for 15 minutes. Stop the reaction with 25 μl/well of stop solution (2 M H ₂ SO 4 ) and measure the OD value at 492 nm. Recombinant BCSP31 protein sheep immune serum or Brucella patient serum was used as positive control, and normal sheep or human serum was used as negative control. Judgment result: P/N value = sample OD value/negative control OD value, the P/N value of the positive control must be greater than or equal to 2.1, the P/N value of the sample to be tested is greater than or equal to 2.1, it is judged as a positive result, and less than 2.1 is a negative result Or suspicious, need to re-test once, still below 2.1 is considered a negative result.

The HRP-labeled rabbit anti-goat IgG involved in the ELISA indirect sandwich method is selected based on the species source of the serum to be tested. For example, when detecting goat-derived serum, HRP-labeled rabbit anti-goat IgG is required; when detecting sheep-derived serum, HRP-labeled rabbit anti-sheep IgG is required. If it is necessary to detect other species infected sera, such as deer, pigs and other animals, HRP-labeled anti-IgG antibodies of the corresponding species can be used. Thus, the method can be widely used in the diagnosis of brucellosis.

2. The sensitivity of the above ELISA indirect sandwich method

The above-mentioned ELISA method was used to detect the serially diluted Brucella sheep serum, and the dilution ratio of the serum was from the stock solution to 10 ^-7 . The results showed that the sensitivity of this method was 5×10 ^-4 (Fig. 3).

3. Application of monoclonal antibody in detecting Brucella sheep

The monoclonal antibody described in this patent can be used for the detection of Brucella: the monoclonal antibody described in this patent can be directly used for the detection of Brucella. Through the isolation and culture of the sample, the obtained bacteria are reacted with the monoclonal antibody, and then reacted with the enzyme-labeled secondary antibody or the fluorescent-labeled secondary antibody. The secondary antibody here is consistent with the above-mentioned HRP-labeled antibody, and the antibody can be labeled as HRP , AP or FITC. Can be used for the identification of Brucella.

The monoclonal antibody described in this patent can even be directly labeled with FITC, HRP, AP, etc., and directly used for the detection of Brucella or its antigen BCSP31.

Amplification, Cloning and Sequence Determination of Monoclonal Antibody Heavy Chain and Light Chain Variable Region Genes

Total RNA of hybridoma cell 1F1 was extracted with Trizol (Gibco). cDNA was synthesized using the cDNA First Strand Synthesis Kit from Promega, USA. Amplification was performed using specific heavy chain variable region (VH) primers (Table 1) and light chain variable region (VL) primers (Table 2). The PCR amplification conditions were: 94°C for 5 min, 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and after 30 cycles, 72°C for 10 min. Cloned into the T easy Vector vector of Promega Company of the United States, the DNA sequence was determined in Aoke Biotechnology Co., Ltd. Wherein the heavy chain variable region gene of the monoclonal antibody has a sequence listed in Sequence List <400>1, and the light chain variable region gene has a sequence listed in Sequence List <400>3.

Amplification primers: Synthesized according to Luo Wen et al. (Construction and Sequence Analysis of Murine Monoclonal Antibody 3G1 Single-chain Antibody Gene. Journal of Xi'an Union University. 2003; 6(2): 25-8).

Table 1: Heavy chain H variable region primers

*The degenerate codes in the brackets in the primer sequence

*The degenerate codes in the brackets in the primer sequence

Sequence analysis of the heavy and light chain variable region genes of the cloned anti-BCSP31 antigen monoclonal antibody is as follows

The following two databases are used respectively:

http://imgt.cines.fr

http://www.ncbi.nlm.nih.gov/blast

The homology comparison of the obtained sequence with other antibody genes that have been reported at present, and the source of germline genes are analyzed, and the results are as follows:

(1) Germline gene source of 1F1 heavy chain <400>1:

V-GENE: BN000872 IGHV1-62-2*01, or AC163348 IGHV1-71*01

D-GENE: J00434 IGHD1-1*01

J-GENE: V00762 IGHJ1*01, or X63164 IGHJ1*03

Analysis by FR-IMGT and CDR-IMGT shows:

CDR1: ggc tac att ttc act gag tat att

CDR2: ttt tcc cct gga agt ggt agt a ta

CDR3: gca aga cac gaa gcc tac ggt agt acc ctt tac tgg tac ttc gatgtc

Homology comparison results in NCBI show:

RID: XJEJ7N2R016

Database: All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, environmental samples or phase 0, 1 or 2 HTGS sequences)

6,540,228 sequences; 23,165,110,747 total lettersQuery＝Length＝384

Database: All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, environmental samples or phase 0, 1 or 2 HTGS sequences)

2,655,401 sequences; 12,045,272,748 total lettersgi|84487350|dbj|AB159968.1|Mus musculus mRNA for immunoglobulinH-chain V-region, partial cds, clone: 0S_b-01

Length＝357

Score＝558 bits(618), Expect＝6e-156

Identities=344/368 (93%), Gaps=12/368 (3%)

Strand=Plus/Plus

(2) Germline gene source of 1F1 light chain <400>3:

V-GENE: Y15976 IGKV6-15*01

J-GENE: V00777 IGKJ4*01

Analysis by FR-IMGT and CDR-IMGT shows:

CDR1: cag aat gtg ggt act tat

CDR2: tcg gca tcc

CDR3: cag caa tat aac agc tat cca ttc acg

Homology comparison results in NCBI show:

RID: XJAVVTUD014

Query＝Length＝4 50

Database: All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, environmental samples or phase 0, 1 or 2 HTGS sequences)

6,540,228 sequences; 23,165,110,747 total lettersgi|5853231|gb|AF178624.1|AF178624 Mus musculus 23-7 immunoglobulin light chain variable region

mRNA, partial cds

Length＝321

Score=513bits(568), Expect=3e-142

Identities=304/317 (95%), Gaps=0/317 (0%)

Strand=Plus/Plus

The above analysis results show that the obtained monoclonal antibody 1F1 gene sequence is indeed derived from the mouse germline gene, and is not completely consistent with various other known antibody genes reported at present, and belongs to a new functional BCSP31-targeting gene sequence. antibody gene.

Application of Heavy and Light Chain Variable Region Genes of Anti-BCSP31 Antigen Monoclonal Antibody and Polypeptide Products Encoded

It has been reported in the literature that the BCSP31 protein is a Brucella cell surface protein, which is highly conserved. It is a Brucella immunodominant antigen and protective antigen, which can stimulate the body to produce a protective immune response.

The heavy and light chain variable region genes of the monoclonal antibody of the present invention can be remodeled and expressed into various forms of protein drugs, which can be directly used in the diagnosis and treatment of anti-SARS-Coy inflammation. For example: small molecule antibodies. There are mainly Fab antibodies composed of VH-CH1 and VL-C1, single-chain antibodies formed by linking VH gene and VL gene with a polypeptide (GLy4Ser) ₃ linker, and Fv fragments formed by non-covalent bonding of VH and VL Antibodies, single-domain antibodies composed of one functional domain of VH or VL, the smallest recognition unit composed of a single CDR, etc.; multivalent miniature antibodies. There are mainly diabody, (ScFv) ₂ , Flex minibody, LD minibody, F(ab') ₂ , F(ab') ₃ , (ScFv) ₄ and so on. Due to the characteristics of multivalent antigen binding sites, high affinity, moderate molecular size, ability to penetrate tissues, and slow clearance rate in the kidney, it has high clinical application value; bispecific antibody. It is a type of antibody with dual specificity and dual function, also known as bifunctional antibody; recombinant antibody fusion protein. A recombinant protein with specific biological activity directed to the target site formed by linking gene fragments such as Fab or Fv with non-antibody toxins or enzymes and other protein genes; recombinant immunotoxin. It is produced by recombining genes encoding antibodies and toxins, which is characterized by high efficiency, low non-specific toxicity, and good stability in vivo; human-derived full antibodies mainly include complete antibodies composed of VH-CH and VL-C1.

Sequence Listing

<110> Fourth Military Medical University of Chinese People's Liberation Army

<120>A monoclonal antibody 1F1 and its application and hybridoma cell line secreting the antibody

BCSP31-1F1

<160>4

<210>1

<211>384

<212>DNA

<213> mouse

<220>

<221>V-region

<222>(7)...(376)

<400>1

atggccgagg cccagctgca gcagtctgga gctgagctgg tgaaacccgg ggcatcagtg 60

aagctgtcct gcaaggcttc tggctacatt ttcactgagt atattataca ctggataaag 120

cagaggtctg gacagggtct tgagtggatt gggtggtttt cccctggaag tggtagtata 180

aagtacaatg agaaatttaa ggacaaggcc aattgactg cggacaaatc ctccagcaca 240

gtctatatgg agcttagtag attgacatct gaagactctg cggtctattt ctgtgcaaga 300

cacgaagcct acggtagtac cctttactgg tacttcgatg tctggggcca agggaccacg 360

gtcaccgtct cctcaaatca ctag 384

<210>2

<211>123

<212>PRT

<213> mouse

<220>

<221>V-segment

<222>(1)...(123)

<400>2

Glu Ala Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly

1 5 10 15

Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr

20 25 30

Glu Tyr Ile Ile His Trp Ile Lys Gln Arg Ser Gly Gln Gly Leu

35 40 45

Glu Trp Ile Gly Trp Phe Ser Pro Gly Ser Gly Ser Ile Lys Tyr

50 55 60

Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser

65 70 75

Ser Ser Thr Val Tyr Met Glu Leu Ser Arg Leu Thr Ser Glu Asp

80 85 90

Ser Ala Val Tyr Phe Cys Ala Arg His Glu Ala Tyr Gly Ser Thr

95 100 105

Leu Tyr Trp Tyr Phe Asp Val Trp Gly Glu Gly Thr Thr Val Thr

110 115 120

Val Ser Ser

123

<210>3

<211>450

<212>DNA

<213> mouse

<220>

<221>V-region

<222>(130)...(450)

<400>3

atgaccatga ttacgccaag ctatttaggt gacactatag aatactcaag ctatgcatcc 60

aacgcgttgg gagctctccc atatggtcga cctgcaggcg gccgcgaatt cactagtgat 120

tggctgcagg atattcagct gacacaagct caaaaattca tgtccacatc agtaggagac 180

agggtcagcg tcacctgcaa ggccagtcag aatgtgggta cttatgtagc ctggtatcaa 240

cagaaaccag ggcaatctcc taaatcactg atttactcgg catcctaccg gtacagtgga 300

gtccctgatc gcttcacagg cagtggatct gggacagatt tcactctcac catcagcaat 360

gtacagtctg aagacttggc agagtatttc tgtcagcaat ataacagcta tccattcacg 420

ttcggctcgg ggaccaagct ggagatctaa 450

<210>4

<211>106

<212>PRT

<213> mouse

<220>

<221>V_segment

<222>(1)...(106)

<400>4

Asp Ile Gln Leu Thr Gln Ala Gln Lys Phe Met Ser Thr Ser Val

1 5 10 15

Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly

20 25 30

Thr Tyr Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys

35 40 45

Ser Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp

50 55 60

Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75

Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln

80 85 90

Tyr Asn Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu

95 100 105

Ile

106

Claims (7)

1. the proteic mouse monoclonal antibody 1F1 of anti-sheep Bacillus brucellae BCSP31 is to be the hybridoma cell line secretion generation of CCTCC C200806 by deposit number.

2. can secrete the hybridoma cell line BCSP31-1F1 of monoclonal antibody according to claim 1 for one kind, its deposit number is: CCTCC C200806.

3. by the described a kind of monoclonal antibody 1F1 of claim 1, it is characterized in that: its heavy chain variable region gene has the sequence of sequence table < 400>1, and chain variable region gene has the sequence of sequence table < 400>3.

4. by the described monoclonal antibody 1F1 of claim 3, it is characterized in that: its heavy chain variable region gene encoded polypeptides has the sequence of sequence table < 400>2, and its chain variable region gene encoded polypeptides has < 400>4 sequence.

5. be used for detecting the application of Bacillus brucellae and diagnosis human or animal gibraltar fever detection reagent in preparation by the described monoclonal antibody 1F1 of claim 1.

6. be used for detecting the application of Bacillus brucellae and diagnosis or treatment human or animal's gibraltar fever reagent and medicine in preparation by the heavy chain of the described monoclonal antibody 1F1 of claim 3 and chain variable region gene.

7. be used for detecting the application of Bacillus brucellae and diagnosis or treatment human or animal's gibraltar fever reagent and medicine in preparation by the heavy chain of the described monoclonal antibody 1F1 of claim 4 and chain variable region gene encoded polypeptides.

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