CN110172097B

CN110172097B - Monoclonal antibody of mycobacterium tuberculosis CFP-10 protein and application thereof

Info

Publication number: CN110172097B
Application number: CN201910427203.1A
Authority: CN
Inventors: 张贺秋; 冯晓燕; 张佳; 危利; 张玲; 王超男; 车志远; 张磊
Original assignee: Oriental Ocean Beijing Medical Research Institute Co ltd
Current assignee: Oriental Ocean Beijing Medical Research Institute Co ltd
Priority date: 2019-05-22
Filing date: 2019-05-22
Publication date: 2020-11-13
Anticipated expiration: 2039-05-22
Also published as: CN110172097A

Abstract

The invention discloses a monoclonal antibody of mycobacterium tuberculosis-resistant CFP-10 protein and application thereof. In the monoclonal antibody, the amino acid sequence of a light chain CDR1 is shown as SEQ ID NO.1, the amino acid sequence of a light chain CDR2 is shown as SEQ ID NO.2, and the amino acid sequence of a light chain CDR3 is shown as SEQ ID NO. 3; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO. 6. The monoclonal antibody of the anti-mycobacterium tuberculosis CFP-10 protein has good specificity and titer, and can be applied to the detection of the mycobacterium tuberculosis CFP-10 protein.

Description

Monoclonal antibody of mycobacterium tuberculosis CFP-10 protein and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a monoclonal antibody of mycobacterium tuberculosis CFP-10 protein and application thereof.

Background

Tuberculosis is an infectious disease caused by infection of mycobacterium tuberculosis, is mainly transmitted through respiratory tract, leads to systemic multi-system diseases along with continuous deterioration of disease conditions, and belongs to a chronic infectious disease which seriously damages human health. According to the statistical report of the World Health Organization (WHO), about 1040 million new tuberculosis cases are found in 2016 the world, and 8.6 percent new tuberculosis cases in China account for the world, and become the third tuberculosis high-load country in the ranking world. Therefore, the method is very important for the prevention and control of tuberculosis epidemic, early diagnosis and timely standard treatment.

Mycobacterium tuberculosis culture filtrate protein 10(CFP-10) is encoded by RD1 region gene Rv3874, which secretes antigen target protein 6(ESAT-6) in vivo and early to form 1: the 1 heterodimeric complex performs a biological function, thereby provoking a strong specific T cell response. The RD1 region where CFP-10 is located exists only in Mycobacterium tuberculosis and Mycobacterium bovis, and the sequences of the region are deleted in other mycobacteria and BCG, so that the region becomes a specific antigen for tuberculosis diagnosis research. However, effective monoclonal antibodies for immunodetection of CFP-10 antigen are still lacking.

Disclosure of Invention

Therefore, the invention aims to provide a monoclonal antibody against CFP-10 protein prepared by using the fused hybridoma cell strain.

A monoclonal antibody against Mycobacterium tuberculosis CFP-10 protein, which comprises a light chain and a heavy chain, wherein the light chain variable region comprises CDR1, CDR2 and CDR3, the heavy chain variable region comprises CDR1, CDR2 and CDR3, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO. 3; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO. 6.

In the above monoclonal antibody, preferably, the light chain amino acid sequence is shown as SEQ ID No.7, and the heavy chain amino acid sequence is shown as SEQ ID No. 8.

The invention also aims to provide a monoclonal antibody hybridoma cell strain for resisting mycobacterium tuberculosis CFP-10 protein, and the monoclonal antibody hybridoma cell strain secretes the monoclonal antibody.

Preferably, the monoclonal antibody hybridoma cell strain is: the mouse hybridoma cell strain C-10 has the preservation number: CGMCC NO. 17499.

Further, the present invention also aims to provide the following applications:

the monoclonal antibody or the monoclonal antibody hybridoma cell strain is applied to preparation of a product for detecting the CFP-10 protein of the mycobacterium tuberculosis.

The light chain or the heavy chain of the monoclonal antibody is applied to the preparation of a product for detecting the CFP-10 protein of the mycobacterium tuberculosis.

The monoclonal antibody or the monoclonal antibody hybridoma cell strain is applied to the preparation of products for detecting, diagnosing, preventing and treating tuberculosis.

The light chain or heavy chain of the monoclonal antibody is applied to the preparation of products for detecting, diagnosing, preventing and treating tuberculosis.

Meanwhile, the invention also aims to provide a kit for detecting CFP-10 protein, which comprises the monoclonal antibody or the light chain or the heavy chain of the monoclonal antibody.

The kit can be applied to the preparation of products for detecting the CFP-10 protein of the mycobacterium tuberculosis.

The invention provides a monoclonal antibody of an anti-mycobacterium tuberculosis CFP-10 protein and a hybridoma cell strain thereof, wherein the monoclonal antibody of the anti-mycobacterium tuberculosis CFP-10 protein has good specificity and potency, and can be applied to detection of the mycobacterium tuberculosis CFP-10 protein.

Biological material preservation instructions

The monoclonal antibody hybridoma cell strain of the invention: the mouse hybridoma cell strain C-10 is preserved in China general microbiological culture Collection center (CGMCC), the registration numbers of the preservation center are CGMCC No.17499 respectively, and the preservation dates are as follows: 18/04/2019. The addresses of the China general microbiological culture Collection center are as follows: xilu No.1 Hospital No.3, Beijing, Chaoyang, North Chen, zip code 100101.

Drawings

FIG. 1 shows a SDS-PAGE electrophoresis of CFP-10 protein fractions, wherein the indices are: M-Marker; 1-whole bacterium; 2-supernatant fluid; and 3-precipitation.

FIG. 2 shows SDS-PAGE electrophoretic analysis of anti-CFP-10 monoclonal antibody purification, wherein the indices are: M-Marker; 1-purified anti-CFP-10 monoclonal antibody.

FIG. 3 shows the anti-CFP-10 monoclonal antibody subclass identification.

FIG. 4 shows the anti-CFP-10 monoclonal antibody-ELISA immunoassay for CFP-10 protein.

FIG. 5 shows Western Blot for immunodetection of CFP-10 protein by anti-CFP-10 monoclonal antibody.

Detailed Description

In order to explain the technical content, the achieved objects and effects of the technical solution in detail, the following description is given with reference to the specific embodiments.

EXAMPLE 1 preparation of immunogen

The amplification primers were designed for the open reading frame region of CFP-10 using the DNA of Mycobacterium tuberculosis H37Rv strain as a template, as shown in Table 1. PCR amplification is carried out, the fragment is connected to a prokaryotic expression vector pBVIL1 after enzyme digestion, and pBVIL1-CFP-10 recombinant plasmid is constructed.

TABLE 1 amplification primers for CFP-10 fragment

The recombinant expression plasmid with correct sequencing is transformed into HB101 competent cells, a single colony is selected in 3ml LB liquid culture medium containing ampicillin sodium, shaking culture is carried out at 37 ℃ overnight, the next day is inoculated in a fresh LB liquid culture medium and cultured to a logarithmic phase, and then induction is carried out at 42 ℃ overnight. SDS-PAGE gel electrophoresis was performed to analyze the expression of inclusion bodies of the target protein in the pellet, and the results are shown in FIG. 1.

The pBVIL1-CFP-10 bacterial liquid is subjected to amplification culture and fermentation, protein is purified by affinity chromatography through a Ni column, protein peaks are respectively collected by eluting through 25mM Tris-HCl (pH 8.5) solution containing 25mM imidazole and 250mM imidazole, and purified products are analyzed through electrophoresis, and 25mM imidazole eluting protein is taken as immunogen (CFP-10 antigen).

Example 2 hybridoma cell line establishment

Adopting 8-week-old BALB/C male mice, CFP-10 antigen and equivalent Freund's complete adjuvant, and injecting mice (50 μ g/mouse) in the back and abdominal cavity; the same dose was administered 2 and 3 times in the fourth and eighth weeks, and splenocytes were taken 3 days later for fusion.

SP20 myeloma cells were revived to be in logarithmic growth phase. Taking immune BALB/c mouse, removing eyeball to collect blood and supply positive control serum, killing mouse at the same time of cervical dislocation, disinfecting body surface with 75% alcohol for 3-5min, taking spleen, and preparing spleen cell suspension.

Taking the spleen cells and myeloma cells according to the ratio of 5: 1, mixing uniformly in serum-free DMEM medium, centrifuging at 1500rpm for 5 minutes, fully absorbing supernatant, lightly shaking the bottom of a centrifuge tube, vibrating to disperse cells, adding 1ml of preheated 50% PEG fused cells within 45-60s, gently shaking while adding, standing for 90 seconds after adding, adding serum-free DMEM medium to stop fusion (1 ml in the first minute, 2ml in the second minute and 8ml in the third minute), standing for 10min at 37 ℃, centrifuging at 1500rpm for 5 minutes, suspending precipitates with HAT medium, subpackaging in 96-well feeder cell-containing cell plates at 37 ℃ and 5% CO₂Cultured in a cell culture box.

After 5 days of culture in a cell incubator, the HAT culture medium is used for replacing liquid once, the HT culture medium is used for replacing liquid on the 10 th day, when 10% -50% of the bottom of a hole is covered by the fused cell, positive clone is screened by adopting an indirect ELISA method, and clone strain is preserved and named as mouse hybridoma cell strain C-10.

EXAMPLE 3 monoclonal antibody preparation and subtype analysis thereof

The C-10 hybridoma cells were recovered and cultured in 1640 medium containing 10% fetal bovine serum. Each BALB/c mouse was injected intraperitoneally with 0.5ml of liquid paraffin. After 10 days, the cells were harvested and resuspended in 10ml of physiological saline, 0.5ml per mouse was injected intraperitoneally (cell density approximately 1X 10)⁷Pieces/ml). After 2 weeks, ascites were collected.

1) Purification of monoclonal antibodies

The results of antibody Purification using the Melon Gel Monoclonal IgG Purification Kit from Thermo are shown in FIG. 2. The purified antibody is subpackaged and stored at-20 ℃.

2) Subclass identification

The monoclonal antibody is identified as a Mouse IgG1 subtype by adopting the Pierce packed Isotyping Kit-Mouse Kit antibody subtype identification, as shown in figure 3.

EXAMPLE 4 sequencing of monoclonal antibody variable regions

Culturing a mouse hybridoma cell strain C-10, extracting total RNA of the hybridoma cell by a Trizol method, carrying out reverse Transcription of cDNA by a High Capacity cDNA reverse Transcription Kit of Thermo Fisher, designing and synthesizing a heavy-light chain primer of the antibody (Beijing Tianyihui Biotechnology limited company, Beijing) according to a mouse monoclonal antibody primer sequence in a book compiled by Shenzou, carrying out PCR amplification (an amplification program is preheating at 95 ℃ for 5min, carrying out 30 cycles (30 seconds at 95 ℃, 35 seconds at 60 ℃ and 45 seconds at 72 ℃) and finally extending for 10min at 72 ℃), connecting a PMD18-T vector, expressing Escherichia coli HB109, and selecting positive clones for sequencing.

Fab light chain amino acid sequence: 112 amino acids as shown in SEQ ID NO.7 and below:

DVLMTQTPLALPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLNISRVEAEDLGVYYCFQGSHVPWTFGGGSKLEIK。

wherein, the CDR region information is as follows:

CDR 1: 27-37aa SEQ ID NO.1 is QSIVHSNGNTY;

CDR 2: 55-57aa SEQ ID NO.2 is KVS;

CDR 3: 94-102aa SEQ ID NO.3 is FQGSHVPWT.

Fab heavy chain amino acid sequence: 118 amino acids as shown in SEQ ID NO.8 and as follows:

VQLQQSGAELARPGASVKLSCKASGYIFTSYWIQWLKQRPGQGPEWIGAIYPGDGDTSYTQKFKGKATLTADRSSSTAYMQLSSLASEDSAVYYCARHFFGNSYFAYWGQGTLVTVSA。

wherein, the CDR region information is as follows:

CDR 1: 25-32aa SEQ ID NO.4 is GYIFTSYW;

CDR 2: 50-57aa SEQ ID NO.5 is IYPGDGDT;

CDR 3: 96-107aa SEQ ID NO.6 is ARHFFGNSYFAY.

EXAMPLE 5 immunodetection of monoclonal antibodies against CFP-10 antigen

1. Enzyme linked immunosorbent assay

Diluting CFP-10 antigen with carbonate coating buffer solution to 2.5 μ g/ml, coating each well with 100 μ l, and standing at 4 deg.C overnight; washing the plate with washing solution 2 times at 200. mu.l/well; adding 100 mul/hole sealing liquid, sealing for 6 hours at room temperature; washing the plate with washing solution 5 times at 200. mu.l/well; by usingDiluting CFP-10 monoclonal antibody 10, 100, 1000, 10000, 20000, 40000, 80000, 160000, 320000, 640000 and 1280000 times by using antibody diluent, loading 100 mul/well and setting blank wells (100 mul antibody diluent); incubating at 37 ℃ for 30 min; washing the plate with washing solution 5 times at 200. mu.l/well; incubating goat anti-mouse secondary antibody marked by HRP for 20min at 37 ℃; washing the plate with washing solution 5 times at 200. mu.l/well; adding a freshly prepared substrate solution, incubating at 100. mu.l/well for 10 minutes at 37 ℃; add 50. mu.l/well 2M H₂SO₄Terminating the reaction, and measuring the light absorption value of each hole by adopting a microplate reader with dual wavelength of 450nm/630nm, wherein the result is shown in figure 4, the prepared monoclonal antibody can identify the CFP-10 antigen expressed by recombination, and the titer is higher than 1: 640000.

2. western Blot immunoblotting

The CFP-10 antigen is loaded, subjected to 240U constant-pressure SDS-PAGE electrophoresis, and then subjected to 200mA constant-current membrane transfer for 1.5 hours; cutting a film with a proper size, and washing the film for 5min by TBS; blocking with blocking solution (TBST, 0.05% skimmed milk powder) for 1 hr; diluting the CFP-10 monoclonal antibody with a confining liquid at 1:200, and incubating overnight at 4 ℃; washing the membrane for 3 times (5 min each time) with TBST; diluting a horseradish peroxidase-labeled goat anti-mouse secondary antibody by using a confining liquid 1:300, and incubating for 1 hour; washing the membrane for 3 times (5 min each time) with TBST; developing, mixing ECL luminescence solution A and solution B in a ratio of 1:1, and exposing by a BIO-RAD gel imager. As a result, as shown in FIG. 5, the prepared monoclonal antibody recognized CFP-10 antigen.

Sequence listing

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Claims

1. A monoclonal antibody against Mycobacterium tuberculosis CFP-10 protein, which comprises a light chain and a heavy chain, wherein the light chain variable region comprises CDR1, CDR2 and CDR3, the heavy chain variable region comprises CDR1, CDR2 and CDR3, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO. 3; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO. 6.

2. The monoclonal antibody of claim 1, wherein the light chain amino acid sequence is set forth in SEQ ID No.7 and the heavy chain amino acid sequence is set forth in SEQ ID No. 8.

3. A monoclonal antibody hybridoma cell strain for resisting mycobacterium tuberculosis CFP-10 protein is as follows: the mouse hybridoma cell strain C-10 has the preservation number: CGMCC NO. 17499.

4. The monoclonal antibody of claim 1 or 2 or the monoclonal antibody hybridoma cell line of claim 3, for use in the preparation of a product for detecting mycobacterium tuberculosis CFP-10 protein.

5. Use of the monoclonal antibody of any one of claims 1-2 or the monoclonal antibody hybridoma cell line of claim 3 in the preparation of a product for detecting, diagnosing, preventing and treating tuberculosis.

6. A kit for detecting CFP-10 protein, which is characterized in that: comprising the monoclonal antibody of any one of claims 1-2.

7. The use of the kit for detecting CFP-10 protein according to claim 6 in the preparation of a product for detecting CFP-10 protein of Mycobacterium tuberculosis.