CN109988770B - Heavy chain and light chain variable region gene of c-di-AMP synthetase monoclonal antibody, encoded polypeptide and application thereof - Google Patents

Abstract

The invention relates to a heavy chain and light chain variable region gene of a c-di-AMP synthetase monoclonal antibody, a polypeptide coded by the gene and application of the polypeptide. The heavy chain variable region gene has the sequence of SEQ ID NO.1, and the light chain variable region gene has the sequence of SEQ ID NO. 3. The heavy chain variable region gene encoding polypeptide has the sequence of SEQ ID NO.2, and the light chain variable region gene encoding polypeptide has the sequence of SEQ ID NO. 4. The monoclonal antibody DisA-1B8 with the polypeptide coded by the variable region gene has high affinity with Rv3586, can specifically recognize c-di-AMP synthetase in different mycobacteria, but does not recognize the enzyme of other bacteria, and has better specificity. The gene and the polypeptide coded by the gene are applied to the preparation of a reagent, a vaccine and a medicament for infecting mycobacterium tuberculosis and other mycobacteria.

Description

A c-di-AMP synthetase monoclonal antibody heavy chain and light chain variable region genes and Encoded polypeptides and applications thereof

technical field

The invention belongs to the technical field of biomedicine and relates to the related fields of immunology and molecular biology. In particular, it relates to a heavy chain and light chain variable region gene and encoded polypeptide of a monoclonal antibody against c-di-AMP synthetase, and said gene and encoded polypeptide are used as a method for preparing Mycobacterium tuberculosis and other mycobacteria Application of infectious agents, vaccines and drugs.

Background technique

1. Bacterial signal molecule c-di-AMP and its function

Small molecule cyclic nucleotides as the second signal molecule of bacteria is considered to be a milestone discovery in the field of bacteriology research. Bacteria can regulate bacterial growth, nutrition, A series of physiological processes such as uptake, bacterial motility, biofilm formation and pathogenicity (Gomelsky, M., cAMP, c-di-GMP, c-di-AMP and now cGMP: bacteria use themall! Mol Microbiol, 2011.79( 3): p.562-5.).

Cyclic di-adenosine monophosphate (c-di-AMP) is a small molecule nucleotide signaling molecule newly discovered in 2008, which only exists in bacteria and has not been found in eukaryotic cells . A number of studies have reported that c-di-AMP is involved in the regulation of bacterial DNA damage monitoring, bacterial growth, cell wall homeostasis, fatty acid synthesis, ion transport, and bacterial virulence physiological processes. In addition, studies have found that c-di-AMP can also induce host innate immune response, induce host cell autophagy, and activate inflammasome. As a mucosal adjuvant, c-di-AMP can induce high levels of Th1/Th2/Th17 adaptive immune responses in mice co-immunized with antigens, and can directly promote the maturation of antigen-presenting cells, and is considered to be a novel innate immune response. Pathogen-associated molecular patterns (PAMPs) discovered. Therefore, c-di-AMP has become a new target in bacterial drug and vaccine research (Commichau FM, HeidemannJL, Ficner R, Stülke J. Making and breaking of an essential poison: the cyclases and phosphodiesterases that produce and degrade the essential second messenger cyclic di-AMP in bacteria. 2018 Sep 17.pii:JB.00462-18.doi:10.1128/JB.00462-18.).

2. c-di-AMP synthetase

Bacillus subtilis DNA integrity scanning protein (DNA integrity scanning protein, DisA) is the first found Diadenylate cyclase (Diadenylate cyclase, DAC) (Witte G, Hartung S, Buttner K, Hopfner KP: Structural biochemistry of A bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates. MolCell 2008,30(2):167-178.). After the DisA protein is activated, it can catalyze the synthesis of c-di-AMP from two ATP molecules, which interacts with downstream related proteins and regulates the biological processes related to bacteria. The Diadenylate cyclase (DAC) domain contained in DisA is the main enzyme active site of DisA, which is highly conserved in sequence and widely distributed in bacterial and archaeal genomes, such as Staphylococcus aureus, pyogenic Streptococcus, Listeria Pseudomonas, Mycobacterium tuberculosis, etc. (Fahmi T, Port GC, Cho KH. Basel).2017.8(8).pii:E197.doi:10.3390/genes8080197).

3. c-di-AMP synthetase of Mycobacterium tuberculosis

Tuberculosis (Tuberculosis, TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) infection. According to WHO, in 2016, there were about 10.4 million new TB patients and 1.7 million deaths due to TB in the world. my country is one of the countries with a high burden of TB, and the incidence rate is second only to India and Indonesia. The Ministry of Health of my country has listed TB as one of the major diseases under national key control.

The inventor of this patent application, Bai Yinlan et al., reported for the first time that Rv3586 is a DAC enzyme that synthesizes c-di-AMP in Mtb. Its structure and function are similar to DisA, and it was named Mtb DisA. By analyzing the genome of Mtb, it was found that Rv3586 is the only c-di-AMP synthetase in Mtb. Mtb Rv3586 consists of three domains: N (DAC), M and C (HhH), among which amino acids 1-140 are the N-terminal domain (Domain), which contains the DAC domain (DAC domain), which is c-di-AMP The main active region of the synthetase. Rv3586 has the same DGA and RHR conserved motifs as the DisA protein of Bacillus subtilis. After RHR is mutated, the protein cannot bind ATP and thus loses DAC activity. Both chemically synthesized and biosynthesized c-di-AMP can specifically bind to Rv3586 and is the only substrate for Rv3586 (Bai Y, Yang J, Zhou X, Ding X, Eisele LE, Bai G.2012. Mycobacterium tuberculosis Rv3586 (DacA ) is a diadenylate cyclase that converts ATP or ADPinto c-di-AMP. PLoS One 7:e35206). The previous studies of the research group showed that high titers of Rv3586 antibodies could be detected in the serum of Mtb-infected animals and TB patients, and DisA recombinant protein subunit vaccine immunization could induce a strong humoral immune response in mice (Cao Tianyu, Ji Siyu ,Chu Yangguang,Wang Limei,Kang Jian,Wang Lifei,Xu Zhikai,Bo Yinlan.Expression and purification of Mycobacterium tuberculosis c-di-AMP synthetase and preparation of mouse polyantiserum[J].Chinese Journal of Pathogenic Biology, 2015, (08):681-684+688.). Therefore, Rv3586 is a good target for Mtb new vaccine and drug research.

Searching the genome database found that the c-di-AMP synthetase contained in different bacteria of the genus Mycobacterium had a high homology, such as the attenuated strain of Mtb H37Ra, the only preventive vaccine against tuberculosis BCG (Bacille Calmette-Guérin, BCG), etc. The contained c-di-AMP synthetase is 99% consistent with the sequence of Rv3586. The c-di-AMP synthase MSMEG_6080 contained in the fast-growing, non-pathogenic Mycobacteria smegmatis (Mycobacteria smegmatis, MS) has 84% homology with Rv3586 and contains a conserved motif of DisA. Therefore, c-di-AMP synthetase is highly conserved among bacteria of the genus Mycobacterium, and antibodies that recognize Rv3586 can also recognize other mycobacteria.

At present, there is no report on the research on monoclonal antibody against Mtb c-di-AMP synthetase at home and abroad.

Contents of the invention

The purpose of the present invention is to provide a c-di-AMP synthetase monoclonal heavy chain and light chain variable region gene and its encoded polypeptide, as well as the gene and polypeptide in the preparation of Mycobacterium tuberculosis and other branches Application of reagents, vaccines and medicines for bacillus infection.

The present invention is achieved through the following technical solutions:

The Rv3586 DAC domain gene was amplified from the Mtb H37Rv genome using molecular biology techniques and PCR, cloned into a prokaryotic expression vector, induced to express the target protein, and purified by affinity chromatography to obtain the recombinant Rv3586 DAC domain protein; Mice were routinely immunized with the recombinant protein, the immune spleen cells were fused with myeloma cell SP2/0, and a hybridoma cell line was obtained by ELISA indirect screening, which was named DisA-1B8. The above-mentioned hybridoma cells were inoculated into the peritoneal cavity of mice to prepare ascites, and the obtained monoclonal antibody was purified by medium-pressure liquid chromatography, which can specifically bind to Mtb Rv3586 protein.

The heavy and light chain variable region genes of cell lines secreting anti-c-di-AMP synthetase Mtb Rv3586 monoclonal antibody of the present invention are obtained by RT-PCR method by extracting the RNA of the above-mentioned DisA-1B8 hybridoma cells Heavy and light chain variable region genes of monoclonal antibodies. 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 has the sequence of SEQ ID NO.1, and the encoded amino acid sequence has the sequence of SEQ ID NO.2; The nucleotide sequence of the light chain variable region gene of the antibody has the sequence of SEQ ID NO.3, and the encoded amino acid sequence has the sequence of SEQ ID NO.4.

For the monoclonal antibody heavy and light chain variable region genes of the successfully cloned anti-c-di-AMP synthetase Mtb Rv3586 of the present invention, respectively carry out homology comparison and other with known antibody gene sequence database (IMGT and (NCBI) The analysis of the source of the germline gene showed that the obtained gene sequence was from the mouse germline gene, which was not completely consistent with the various antibody gene sequences reported so far.

The invention also relates to the application of the heavy and light chain variable region genes of the antibody and the encoded polypeptides in the preparation of reagents, vaccines and medicines for Mycobacterium tuberculosis and other Mycobacterium infections.

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-mentioned heavy and light chain variable region genes, it 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 tuberculosis Preparation of diagnostic reagents, drugs or vaccines for mycobacterial infection. Since the c-di-AMP synthetase gene is highly conserved in mycobacteria, the heavy and light chain variable region genes of the monoclonal antibody obtained in this study also lay the foundation for the development of reagents, vaccines and drugs for other mycobacterial infections .

Compared with the prior art, the present invention has the following beneficial technical effects:

1. The heavy and light chain variable region genes and amino acid sequences of the monoclonal antibody against c-di-AMP synthetase Mtb Rv3586 successfully cloned in the present invention. Sequence analysis has confirmed the uniqueness of the antibody sequence.

2. The monoclonal antibody with the above heavy and light chain variable region genes and amino acid sequences can specifically recognize Rv3586 and carry out immune reaction, and can recognize DAC domain proteins in different mycobacteria, with good specificity.

3. Analyze the heavy chain and light chain variable region genes of the monoclonal antibody DisA-1B8, and obtain the CDR region of the variable region, which provides support for laboratory Mtb and other mycobacteria detection and clinical detection.

Description of drawings

Fig. 1 The results of detecting mAb antibody subclasses by indirect ELISA method.

Figure 2 is a diagram of the detection results of mAb antibody affinity detection by indirect ELISA method.

Figure 3 Western-blot detection results of mAb recognition of Rv3586.

Figure 4 Western-blot detection of mAb recognition results for different bacterial DAC domain proteins.

Detailed ways

The present invention immunizes female BALB/c mice with prokaryotically expressed and purified DAC domain protein of Mtb Rv3586, prepares a group of mouse monoclonal antibodies against c-di-AMP synthetase, and screens out stable and secreted high-affinity antibodies The hybridoma cell line of the anti-c-di-AMP synthetase monoclonal antibody, the total RNA of the cell line was extracted, and the gene sequence of the monoclonal antibody was obtained by RT-PCR method; the sequence of the variable region of the monoclonal antibody gene was confirmed by sequencing , and determined the uniqueness of the corresponding protein sequence and its complementarity determining regions (CDR) sequence through analysis; the heavy and light chain variable region genes of the monoclonal antibody and their encoded polypeptides were used to isolate tuberculosis Provide technical support for the development of reagents, vaccines and drugs for Mycobacterium and other mycobacterial infections. Hereinafter, the heavy and light chain variable region genes of the monoclonal antibody and the preparation method of the encoded polypeptide and the uniqueness of the antibody sequence will be described in detail, which is an explanation of the present invention rather than a limitation.

The present invention is specifically implemented according to the following steps:

1 Preparation of mouse anti-c-di-AMP synthetase high-affinity antibody

1.1 Preparation and purification of monoclonal antibodies

According to the monoclonal antibody preparation method (practical monoclonal antibody technology, edited by Xu Zhikai, P9-P11), immunize female BALB/c mice (purchased from the Experimental Animal Center of Air Force Military Medical University) with the purified Mtb DAC domain protein, and subcutaneously immunize for the first time Antigen 50 μg/rat + Freund’s incomplete adjuvant; two weeks apart, two subcutaneous immunizations; the third subcutaneous immunization antigen 25 μg/rat; one week after the completion of the third immunization, blood was collected from the tail vein to test the immune effect. The immunized mice were boosted with 25 μg of antigen per mouse. Three days after the completion, the mice were sacrificed, and at the same time, the spleen lymphocyte suspension of the immunized mice was prepared and counted.

The mouse myeloma cells SP2/0 in the logarithmic growth phase were collected and counted, and the myeloma cells and spleen lymphocytes were fused at a ratio of 5:1. The fused cell suspension was added to a 96-well plate containing feeder cells (peritoneal macrophages of normal female BALB/c mice), and cultured at 37° C. and 5% CO ₂ . After the cell clones appeared, the cell supernatant was taken for indirect ELISA detection, and positive clones were selected. Cells containing positive clones were cloned by limiting dilution until a hybridoma cell line capable of stably secreting antibodies was obtained, which was named DisA-1B8. The Ig subclass of the antibody secreted by the hybridoma cell line was determined, and the results showed that the monoclonal antibody was IgG1 subclass, kappa light chain (Figure 1).

1.2 Monoclonal antibody titer and affinity determination

The relative affinity of monoclonal antibody DisA-1B8 was detected by indirect ELISA. The coating antigen is purified DisA protein, the sample to be tested is a purified monoclonal antibody in gradient dilution, the detection antibody is HRP-goat anti-mouse IgG, the chromogenic substrate is TMB, and the chromogenic stop solution is 2M H ₂ SO ₄ . ELISA results showed that the purified monoclonal antibody DisA-1B8 had a titer of 1:25 600 and an affinity of 3.9×10 ^-5 (Figure 2).

1.3 Recognition of Rv3586 protein by monoclonal antibody

Load protein Marker, DisA protein, DAC domain protein (DisA domain) and Mycobacterium smegmatis DisA (MS DisA) purified protein samples in sequence, and perform SDS-PAGE; after electrophoresis, transfer to membrane and block; the primary antibody is purified Antibody DisA-1B8 (1:20 000); the secondary antibody was HRP-goat anti-mouse IgG (1:5 000); ECL luminescence method was used to detect the recognition of monoclonal antibodies to different antigens. Western blot results showed that the monoclonal antibody could specifically recognize Mtb DisA protein, Mtb DAC domain protein and MS DisA purified protein recombinantly expressed in Escherichia coli, indicating that it had good specificity (Figure 3).

1.4 Recognition of monoclonal antibodies to other bacterial c-di-AMP synthetases

The bacterial proteins of Staphylococcus aureus (Staphylococcus aureus, SA), Mycobacterium smegmatis, BCG, Mtb attenuated strain (H37Ra) and Mtb standard strain (H37Rv) were prepared respectively. The protein marker and each protein sample to be tested were loaded in turn, and the purified DisA protein was used as a positive control for SDS-PAGE; after electrophoresis, the membrane was transferred and blocked; the primary antibody was a purified monoclonal antibody (1:20 000); The secondary antibody was HRP-goat anti-mouse IgG (1:5000); the protein expression in different bacteria was observed by ECL luminescence. Western blot results showed that the monoclonal antibody could recognize the c-di-AMP synthetase of BCG, H37Ra and H37Rv, but not the enzyme in MS and SA, so it had genus specificity (Figure 4).

2 Cloning of heavy chain and light chain variable region genes of monoclonal antibodies

2.1 Amplification of Monoclonal Antibody Heavy and Light Chain Variable Region Sequences

The above-mentioned hybridoma cells secreting monoclonal antibodies were completely cultured with RPMI1640 containing 10% fetal bovine serum in a 37°C, 5% CO ₂ incubator until logarithmic growth phase. Total RNA of hybridoma cells was extracted by lysis method using TRIzol (Invitrogen Company), and cDNA was synthesized by reverse transcription kit (TaKaRa Company) after quantification. The cDNA obtained by reverse transcription was used as a PCR amplification template, and PCR amplification kit (TaKaRa Company) was used for amplification. The primers for the variable region of the heavy chain were VHF (upstream primer) and VHR (downstream primer), and the light chain could be The variable region primers were VL F (upstream primer) and VL R (downstream primer) for amplification to obtain VH and VL gene fragments of the monoclonal antibody DisA-1B8, respectively.

The PCR reaction system was 50 μL, and the amplification program was: 95°C for 1 min; 95°C for 5 s, 58°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 5 min. The primer sequences are (degenerate primers in brackets):

2.2 Cloning of monoclonal antibody heavy chain and light chain variable region sequences

The PCR product was subjected to 1% agarose gel electrophoresis, and the PCR cleaning and recovery kit (Axygen Company) was used to reclaim the target gene fragment, and the target gene recovery fragment was respectively combined with the pMD19-T Vector (TaKaRa Company) using a DNA ligation kit (TaKaRa Company). ) vector connection, the connection product was transformed into E.coli DH5α competent cells, spread on LB plates containing Amp antibiotics, and cultured overnight at 37°C.

Pick the clones on the LB plate containing Amp antibiotics as PCR templates, use primers VHF, VHR corresponding to the heavy chain and primers VL F, VL R corresponding to the light chain as primers, and identify and screen positive E.coli DH5α transformants by PCR Expand the culture, extract the plasmid with a small amount of plasmid extraction kit (Axygen company), and send it to Sangon Bioengineering (Shanghai) Co., Ltd. for gene sequencing. The gene sequence of the heavy chain variable region obtained by sequencing is shown in SEQ ID NO.1, and the gene sequence of the light chain variable region is shown in SEQ ID NO.3.

3 Homology analysis of heavy chain and light chain variable region sequences of monoclonal antibodies

3.1 Homology analysis of the nucleotide sequence of the variable region of the monoclonal antibody

After the sequence of the variable region was correctly sequenced, the NCBI (GenBank+EMBL+DDBJ+PDB) database (http://www.ncbi.nlm.nih. gov/blast) and the IMGT database (http://www.imgt.org) for nucleotide sequence homology analysis, compared the obtained sequence with other antibody genes that have been reported, and analyzed Its germline gene source.

Sequence comparison results showed that the light chain variable region gene sequence of the monoclonal antibody DisA-1B8 had the highest homology with the mouse Ig light chain variable region gene sequence numbered GenBank: S61689.1, reaching 313/321 (98 %). The heavy chain variable region gene sequence of monoclonal antibody DisA-1B8 has the highest homology with the mouse Ig heavy chain variable region gene sequence numbered GenBank: AY648645.1, which is 292/298 (98%). The result is displayed as follows:

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

V-GENE: Musmus IGHV1S137*01

J-GENE: Musmus IGHJ2*01

D-GENE: Musmus IGHD2-10*02F

Analysis by FR-IMGT and CDR-IMGT shows:

CDR1: ggctacacattcactgattatgct

CDR2: attagtacttactatggtgatgct

CDR3: gcaagaggggatggtaattacctctttgcttac

Homology comparison results in NCBI show:

RID: 0CU6NS9T015

Query Length: 354

Database Name:All non-redundant GenBank+EMBL+DDBJ+PDB sequences(noEST,STS,GSS or HTGS sequences)

Sequence ID: AY648645.1 Mus musculus clone SPLH23 immunoglobulin heavychain variable region mRNA, partial cds

Length: 414

Score: 518 bits (280)

Expect: 6e-143

Identities: 292/298 (98%)

Gaps: 0/298 (0%)

Strand: Plus/Plus

(2) Germline gene source of monoclonal antibody light chain <400>3:

V-GENE: Musmus IGKV12-44*01

J-GENE: Musmus IGKJ4*01

Analysis by FR-IMGT and CDR-IMGT shows:

CDR1: gagaatattacagttat

CDR2: aatgcaaaa

CDR3: caacatcatttatggtactccattcacg

Homology comparison results in NCBI show:

RID:0CJD9YNX014(Expires on 12-05 16:32pm)

Query Length: 321

Database Name:All non-redundant GenBank+EMBL+DDBJ+PDB sequences(noEST,STS,GSS or HTGS sequences)

Sequence ID: S61689.1 anti-ferritin immunoglobulin light chain, mRNA Partial

Length: 490

Score: 549 bits (297)

Expect: 2e-152

Identities: 313/321 (98%)

Gaps: 0/321 (0%)

Strand: Plus/Plus

Sequence homology analysis showed that the nucleotide sequences encoding the heavy and light chain variable regions of the monoclonal antibody DisA-1B8 were derived from mouse germline genes, but were not completely consistent with the various antibody gene sequences reported so far , indicating that the present invention has uniqueness in the gene sequence.

3.2 Homology Analysis of Amino Acid Sequences of Variable Regions of Monoclonal Antibodies

The amino acid sequence of the heavy chain variable region of the monoclonal antibody DisA-1B8 is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4. Amino acid sequence homology analysis (Blastp) was performed in the non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF protein database. The analysis results showed that the light chain amino acid sequence of the monoclonal antibody DisA-1B8 had the highest homology with the mouse Ig light chain variable region protein numbered 1F6L_L, reaching 105/107 (98%). The heavy chain amino acid sequence of the monoclonal antibody DisA-1B8 has the highest homology of 107/119 (90%) with the mouse Ig heavy chain variable region protein numbered BAN13750.1. The results of homologous comparison of heavy chain and light chain amino acids are as follows:

(1) Alignment results of monoclonal antibody heavy chain amino acid sequences <400>2:

RID: 0T7095MW014

Query ID:lcl|Query_305570

Query Length: 118

Database Name:All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF excluding environmental samples from WGS projects

Sequence ID: BAN13750.1 immunoglobulin heavy chain, partial [Musmusculus]

Length: 120

Score:216bits(550)

Expect: 1e-70

Identities: 107/119 (90%)

Positives: 110/119 (92%)

Gaps: 3/119 (2%)

(2) Monoclonal antibody light chain amino acid sequence <400>4 homology comparison results:

RID: 0T62E9NP015

Query ID:lcl|Query_87287

Query Length: 107

Database Name:All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF excluding environmental samples from WGS projects

Sequence ID: 1F6L_L Chain L, variable light chain dimer of anti-ferritin antibody

Length: 114

Score: 219bits(557)

Expect: 4e-72

Identities: 105/107 (98%)

Positives: 106/107 (99%)

Gaps: 0/107 (0%)

Homology analysis shows that the amino acid sequence of the heavy and light chain variable regions of the monoclonal antibody DisA-1B8 is a mouse-derived protein. Although it has homology with other protein amino acid sequences, no amino acid completely identical to the present invention has been found sequence, indicating that the present invention also has uniqueness in amino acid sequence.

3.3 Determine the CDR area

The heavy chain and light chain variable region sequences of monoclonal antibody DisA-1B8 obtained by sequencing were analyzed on the VBASE2 website (http://www.vbase2.org/vbase2.php) to obtain the CDR region.

The three complementarity determining region (CDR) sequences of the heavy chain variable region of the monoclonal antibody DisA-1B8, as shown in the part of SEQ ID NO.2<222>, are specifically:

CDR1: Gly-Tyr-Thr-Phe-Thr-Asp-Tyr-Ala

CDR2: Ile-Ser-Thr-Tyr-Tyr-Gly-Asp-Ala

CDR3: Ala-Arg-Gly-Asp-Gly-Asn-Tyr-Leu-Phe-Ala-Tyr

The three complementarity determining region (CDR) sequences of the light chain variable region of the monoclonal antibody DisA-1B8, as shown in SEQ ID NO.4<222>, specifically:

CDR1: Glu-Asn-Ile-Tyr-Ser-Tyr

CDR2: Asn-Ala-Lys

CDR3: Gln-His-His-Tyr-Gly-Thr-Pro-Phe-Thr

Application of Anti-Mtb c-di-AMP Synthetase Monoclonal Antibody DisA-1B8 Heavy and Light Chain Variable Region Gene and Its Coded Polypeptide Product

Genome analysis shows that the c-di-AMP synthetase contained in different bacteria of the Mycobacterium genus has high homology, and the homology of this enzyme in most Mtb strains is higher than 90%, such as H37Ra, c-di-AMP of BCG - The amino acid sequence of AMP synthetase has 100% and 99% similarity with Rv3586, respectively, while the enzyme in fast-growing, non-pathogenic MS has 84% homology with it. The monoclonal antibody DisA-1B8 comprising the heavy chain and light chain variable region sequences of the present invention can recognize c-di-AMP synthetase in Mtb different virulence strains H37Rv and H37Ra, BCG vaccine strain and fast-growing MS. Since c-di-AMP synthetase is highly conserved among bacteria of the genus Mycobacterium, it is speculated that the monoclonal antibody comprising the heavy chain and light chain variable region sequences of the present invention can also recognize other mycobacteria other than this study, but It does not recognize c-di-AMP synthase from other bacteria, so it has genus specificity and can be used in the differential diagnosis of bacterial infections. Previous studies have also shown that Rv3586 has strong antigenicity and can stimulate the body to produce a higher immune response. Therefore, the heavy chain and light chain variable region sequences of the present invention can also be used in the development of Mycobacterium tuberculosis vaccines.

The heavy chain and light chain variable region sequences of the anti-c-di-AMP synthetase monoclonal antibody successfully obtained in the present invention can also be used to construct and express small molecule genetically engineered antibodies or drugs in various forms, such as: small Molecular antibodies, mainly Fab antibodies, single chain antibodies, Fv fragment antibodies, single domain antibodies and the smallest recognition unit composed of a single CDR, etc., are used in the development of diagnostic reagents, drugs or vaccines for Mycobacterium tuberculosis infection. Since the c-di-AMP synthetase gene is highly conserved in mycobacteria, the heavy and light chain variable region genes of the monoclonal antibody obtained in this study also lay the foundation for the development of diagnostic reagents, vaccines and drugs for other mycobacterial infections. A certain basis.

sequence listing

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

<120> A c-di-AMP synthetase monoclonal antibody heavy chain and light chain variable region gene and encoded polypeptide and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 354

<212>DNA

<213> Artificial sequence (unknown)

<400> 1

caggcccagc tgcagcagtc aggggctgag ctggtgaggc ctggggtctc agtgaagatt 60

tcctgcaagg tttctggcta cacattcact gattatgcta tgcactgggt gaagcagagt 120

catgcaaaga gtctagagtg gattggagtt attagtactt actatggtga tgctagctac 180

aaccagaagt tcaagggcaa ggccacaatg actgtagaca aatcctccag cacactctat 240

atggaacttg ccagactgac atctgaggat tctgccatct attackgtgc aagaggggat 300

ggtaattacc tctttgctta ctggggccaa gggaccacgg tcaccgtctc ctca 354

<210> 2

<211> 118

<212> PRT

<213> Artificial sequence (unknown)

<400> 2

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Tyr Gly Asp Ala Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Leu Tyr

65 70 75 80

Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Gly Asn Tyr Leu Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 3

<211> 321

<212>DNA

<213> Artificial sequence (unknown)

<400> 3

gacattcaga tgacccagtc tccagcctcc ttatctgcat ctgtgggaga aactgtcacc 60

atcacatgtc gagcaagtga gaatattac agttatttag catggtatca gcagaaacag 120

ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagaagg tgtgccatca 180

aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240

gaagattttg ggagatatta ctgtcaacat catttatggta ctccattcac gttcggctcg 300

gggaccaagg tggagatcaa a 321

<210> 4

<211> 107

<212> PRT

<213> Artificial sequence (unknown)

<400> 4

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

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

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Arg Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys

100 105

Claims (4)

1. A heavy chain and light chain variable region gene of a c-di-AMP synthetase monoclonal antibody, characterized in that: its heavy chain variable region gene has the sequence of SEQ ID NO.1, and the light chain variable region gene The gene has the sequence of SEQ ID NO.3. 2. The polypeptide encoded by the heavy chain and light chain variable region genes of the monoclonal antibody according to claim 1 is characterized in that: the polypeptide encoded by its heavy chain variable region gene has the sequence of SEQ ID NO.2, and its light chain variable region gene encoding polypeptide has the sequence of SEQ ID NO.2. The polypeptide encoded by the chain variable region gene has the sequence of SEQID NO 4. 3. the application of the heavy chain and the light chain variable region gene of the monoclonal antibody as claimed in claim 1 as the preparation of reagents, vaccines and medicines for mycobacterium tuberculosis and other mycobacterial infections. 4. The application of the polypeptide encoded by the heavy chain and light chain variable region genes of the monoclonal antibody as claimed in claim 2 as the preparation of reagents, vaccines and medicines for mycobacterium tuberculosis and other mycobacterial infections.

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