CN110240637A - Mycobacterium bovis immune-related molecular chaperone protein GroEL2 and its encoding gene and application - Google Patents

Abstract

The invention discloses a Mycobacterium bovis molecular chaperone protein GroEL2 and its encoding gene, belonging to the technical fields of biology and vaccines. The applicant cloned the GroEL2 gene from the genome of Mycobacterium bovis BCG, recombined the gene into Mycobacterium smegmatis for expression, and after confirming its high adhesion and invasion ability, expressed the gene in Escherichia coli, Finally, the recombinant protein GroEL2 was obtained. The protein can improve the body's IgG and IgA antibody levels, as well as the body's Th1 and Th2 cell immunity levels, and has a certain ability to resist Mycobacterium bovis infection. Has application prospects.

Description

Mycobacterium bovis immune-related molecular chaperone protein GroEL2 and its encoding gene are related to application

technical field

The invention belongs to the technical field of biology and vaccines, and in particular relates to a molecular chaperone protein GroEL2 related to Mycobacterium bovis immunity. And Th2-type cellular immunity level, indicating that the protein can trigger high levels of humoral immunity and cellular immunity, has a certain ability to resist Mycobacterium bovis infection.

Background technique

Mycobacterium bovis (M. bovis) is an important pathogenic bacteria in cattle, which can cause progressive weight loss, decreased milk production, and reduced labor capacity, etc., and can be transmitted to humans and other animals from cattle. , causing huge social harm and economic losses. Bovine tuberculosis is endemic in my country. The earliest written report on the epidemic of bovine tuberculosis can be traced back to 1956, when the positive detection rate of tuberculosis in a dairy farm in Xinjiang was reported to be 11%.

"Quarantine + culling" is an effective method used in the control of bovine tuberculosis internationally. However, due to the unclear background of bovine tuberculosis, the economic losses caused by culling are huge. After a lot of economic evaluations, more and more Scholars believe that vaccine immunization can be an important means of prevention and control of bovine tuberculosis. BCG is currently the official vaccine for human tuberculosis, but it is also used in the prevention and control of bovine tuberculosis in some countries. In the prevention of bovine tuberculosis, because cows are dairy animals, and the essence of BCG is a weakened strain of Mycobacterium bovis, it may endanger human health through dairy products, so it needs to be very cautious in application. Subunit vaccines are safe and efficient, and are undoubtedly an important development direction for bovine tuberculosis vaccines. The search for highly immunogenic proteins is the top priority in the development of efficient subunit vaccines.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Mycobacterium bovis immunity-related protein GroEL2, which is a type of molecular chaperone protein and has the ability to improve the immunity level of the body.

In order to achieve the purpose of the present invention, the ruminant pathogenic division of the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, where the applicant is located, amplified the GroEL2 gene from Mycobacterium bovis BCG and cloned it in Mycobacterium smegmatis, It was verified by Western Blot analysis that the protein was successfully expressed, and the cell model confirmed that the recombinant bacteria had very high adhesion and invasion ability. The GroEL2 was further ligated into a prokaryotic expression vector and expressed and purified in E. coli. The Western Blot analysis confirmed that the protein had good immunogenicity. The purified recombinant protein GroEL2 was mixed with adjuvant and then immunized in mice. It was found that the protein could significantly increase the level of IgG, IgA antibody and Th1-type cellular immune response in mice, and had important anti-Mycobacterium bovis infection potential. Important potential targets for new drugs and vaccines.

The immune-related molecular chaperone protein of Mycobacterium bovis provided by the present invention, named GroEL2 protein, is derived from Mycobacterium bovis (Mycobacterium bovis) BCG, and is a protein of the following 1) or 2):

1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 6;

2) A protein derived from 1) with the amino acid sequence of SEQ ID NO: 6 subjected to substitution and/or deletion and/or addition of one or several amino acid residues.

SEQ ID NO:6 consists of 540 amino acid residues.

The above sequence can be synthesized artificially, or can be obtained by first synthesizing its encoding gene and then carrying out biological expression. The encoding gene of GroEL2 in the above 2) can be obtained by deleting the codon of one or several amino acid residues from the DNA sequence shown in SEQ ID NO: 5 from the 1-1623rd base of the 5' end, and and/or by a missense mutation of one or several base pairs.

The encoding gene of the above-mentioned protein also belongs to the protection scope of the present invention.

The encoding gene of the GroEL2 protein can specifically be the gene described in any one of the following 1)-3):

1) its nucleotide sequence is the sequence shown in SEQ ID NO:5;

2) a DNA molecule that can hybridize with the DNA sequence defined by SEQ ID NO: 5 and encode the above-mentioned GroEL2 protein under stringent conditions;

3) A DNA molecule that has more than 90% homology with the gene defined in 1) and encodes the above-mentioned GroEL2 protein.

The gene in 3) preferably has more than 95% homology with the gene defined in 1) or 2).

The sequence shown in SEQ ID NO:5 consists of 1623 base pairs, which is the open reading frame (ORF) of the GroEL2 gene, and the encoded amino acid sequence is the GroEL2 protein shown in SEQ ID NO:6.

Recombinant vectors, transgenic cell lines and recombinant bacteria containing the above-mentioned GroEL2 protein-encoding gene also belong to the protection scope of the present invention.

The specific technical scheme of the present invention is as follows:

Mycobacterium bovis Pasteur strain BCG [American Type Culture Collection (ATCC): 35734] used by the applicant was a gift from Professor Luiz Bermudez of Oregon State University, USA. The preliminary work of the present invention includes the extraction of the preserved genome of Mycobacterium bovis BCG Pasteur strain.

In the present invention, the GroEL2 gene is cloned with the whole genome of the BCG Pasteur strain of Mycobacterium bovis as a template, and is connected to the pMV261 vector to construct a recombinant plasmid pMV261-GroEL2, and the recombinant plasmid is transformed into Mycobacterium smegmatis to obtain The recombinant M. smegmatis was named Ms-pMV-G2. Western Blot results confirmed that the protein was successfully expressed and had reactogenicity. The detection results of adhesion and invasion ability showed that the recombinant M. smegmatis had higher adhesion and invasion ability than the wild strain. The cloned GroEL2 gene was further connected to the pET-30a vector, and a recombinant plasmid pET-30a-GroEL2 was constructed. The recombinant plasmid was transformed into Escherichia coli DH5α to obtain a recombinant Escherichia coli strain, named Escherichacoli pET-30a-GroEL2, in 2019 On May 7, it was sent to the Chinese Collection of Type Culture Collection at Wuhan University, Wuhan City, Hubei Province for preservation, and the preservation number is CCTCC M 2019334. The strain expressed the recombinant protein encoded by the GroEL2 gene under the induction of IPTG, and the results of Western Blot verified the high immunogenicity and reactogenicity of the recombinant protein. In order to verify the immunological effects of the protein in vivo, the purified recombinant protein GroEL2 was mixed with adjuvant and then immunized in mice. It has the potential to resist Mycobacterium bovis infection and prevent bovine tuberculosis.

The GroEL2 protein provided by the present invention can also be used to prepare a diagnostic kit for bovine tuberculosis, such as a Mycobacterium bovis IgG or IgA antibody enzyme-linked immunosorbent assay kit.

The present invention has the following advantages:

1. The GroEL2 recombinant protein of the present invention is encoded by the Mycobacterium bovis GroEL2 gene, which has been confirmed by the inventor to have high adhesion and invasion ability.

2. The GroEL2 recombinant protein of the present invention has been confirmed by the inventor to have immunogenicity.

3. The GroEL2 recombinant protein of the present invention has been confirmed by the inventors in mice that it can cause the body to produce high levels of IgA and IgG antibodies.

4. The GroEL2 recombinant protein of the present invention has been confirmed by the inventors in mice that it can cause the body to produce a high level of Th1-type cellular immunity and Th2-type humoral immunity.

For more detailed technical solutions, please refer to "Specific Implementations".

Sequence Listing Description:

Sequence Listing SEQ ID NO: 1 is the sequence of the primer GroEL2-F1 for amplifying the GroEL2 gene fragment, which was used to construct pMV261-GroEL2.

Sequence Listing SEQ ID NO: 2 is the sequence of the primer GroEL2-R1 for amplifying the GroEL2 gene fragment, which was used to construct pMV261-GroEL2.

Sequence Listing SEQ ID NO: 3 is the sequence of the primer GroEL2-F2 for amplifying the GroEL2 gene fragment, which was used to construct pET-30a-GroEL2.

Sequence Listing SEQ ID NO: 4 is the sequence of the primer GroEL2-R2 for amplifying the GroEL2 gene fragment, which was used to construct pET-30a-GroEL2.

Sequence Listing SEQ ID NO: 5 is the nucleotide sequence of the GroEL2 gene, which is 1623 bp in length.

SEQUENCE LISTING SEQ ID NO: 6 is the amino acid sequence of the GroEL2 protein, consisting of 540 amino acid residues.

Description of drawings

Figure 1: is the plasmid map of pMV216.

Figure 2: is the map of the recombinant plasmid pMV261-GroEL2 constructed by the present invention. It is composed of pMV261 plasmid and full-length GroEL2 gene after restriction endonuclease digestion and ligation and recombination.

Figure 3: Western blot verification of GroEL2 expression in Ms-pMV-G2. Lane M: high molecular weight protein standard; Lane 1: GroEL2 expression in Ms-pMV-G2; Lane 2: Mycobacterium smegmatis empty control.

Figure 4 is a graph showing the quantitative detection and analysis of the adhesion and invasion ability of A549 cells infected with Ms-pMV-G2. "***" means P<0.001; A: Quantitative test result of adhesion ability; B: Quantitative test result of invasive ability.

Figure 5: is the plasmid map of pET-30a. pET-30a is a commercial plasmid, purchased from Novagen Company.

Figure 6: is the map of the recombinant plasmid pET-30a-GroEL2 constructed by the present invention. It is composed of pET-30a plasmid and full-length GroEL2 gene after restriction endonuclease digestion and ligation and recombination.

Figure 7: is the SDS-PAGE chart of purified Mycobacterium bovis GroEL2 protein. Lane M: high molecular weight protein standard; Lane 1: Uninduced BL21 recombinant bacteria containing pET-30a-GroEL2 plasmid; Lane 2: IPTG-induced BL21 recombinant bacteria containing pET-30a-GroEL2 plasmid; Lane 3: IPTG Expression in the precipitate after induction; Lane 4: Expression in the supernatant after IPTG induction; Lane 5-8: Protein detection after elution; Lane 9: Purified GroEL2 protein.

Figure 8: Western blot analysis of the immunogenicity of GroEL2 protein. Lane M: high molecular weight protein standard; Lane 1: BL21 recombinant bacteria containing pET-30a-GroEL2 plasmid.

Figure 9 shows the changes in body weight of mice in each group after immunization.

Figure 10 shows the changes of total IgG and total IgA antibodies after immunization of mice in each group. A: Test results of total IgG antibodies; B: Test results of total IgA antibodies; **, P<0.01; ***, P<0.001.

Figure 11 shows the changes of specific IgG and specific IgA antibodies after immunization of mice in each group. A: Test results of specific IgG antibodies; B: Test results of specific IgA antibodies; ***, P<0.001.

Figure 12 shows the levels of IFN-γ and IL-6 after immunization of mice in each group. A: Detection of IFN-γ expression level; B: Detection of IL-6 expression level; **, P<0.01; ***, P<0.001.

Detailed ways

Example 1: Construction and verification of recombinant Mycobacterium smegmatis Ms-pMV-G2

1.1 Construction of recombinant Mycobacterium smegmatis Ms-pMV-G2

Using the whole genome of Mycobacterium bovis BCG Pasteur strain BCG (ATCC: 35734) as a template, the GroEL2 gene was cloned using the primers (SEQ ID NO: 1-2 in the sequence listing) designed as follows to obtain the complete sequence of the GroEL2 gene, the sequence length is 1623bp.

The primer sequences for amplifying the GroEL2 gene are shown below:

1. Forward primer GroEL2-F1: GCCAAGACAATTGC GGATCC ATGGCCAAGACAATTGCGTA. Underlined is the restriction site of BamHI. Corresponding to the sequence shown in SEQ ID NO: 1 of the sequence listing.

2. Reverse primer GroEL2-R1: TGGTGGTGGTGGTG GATATC GAAATCCATGCCACCCATGT. The restriction sites of EcoRV are underlined. Corresponding to the sequence shown in SEQ ID NO: 2 of the sequence listing.

The PCR reaction system is as follows:

Template DNA 2 μL, 2×planta ^TM Master Mix 25 μL, primers 2 μL each, and ultrapure water 19 μL.

PCR amplification conditions are as follows:

Pre-denaturation at 98 °C for 5 min; denaturation at 98 °C for 30 sec, annealing at 58 °C for 30 sec, extension at 72 °C for 2 min, 30 cycles of reaction; complete extension at 72 °C for 10 min.

The PCR amplification product was recovered and digested with BamHI and EcoRV, while the pMV261 plasmid (Fig. 1) was double digested with the same enzymes. The digested GroEL2 gene and pMV261 plasmid were ligated with TreliefTM SoSoo Cloning Kit (TSINGKE Company) to obtain recombinant plasmid pMV261-GroEL2 (Fig. 2). After the recombinant plasmid pMV261-GroEL2 was transformed into Mycobacterium smegmatis mc ² 155, it was placed in a shaker at 37°C and cultured at 110 r/min to log phase (OD ₆₀₀ =0.8-1.0). After the correct identification by bacterial liquid PCR, Western blot was used to determine the expression.

1.2 Western blot identification of the expression of GroEL2 gene in recombinant Mycobacterium smegmatis Ms-pMV-G2

The recombinant Mycobacterium smegmatis Ms-pMV-G2 prepared in 1.1 was heat-shocked at 42°C for 3-4 hours, and then the bacterial liquid was collected. After centrifugation at 4°C for 5 min at 10000 rpm/min, the cells were washed 3 times with PBS, and the cells were resuspended. After ultrasonication, the cells were disrupted for SDS-PAGE electrophoresis, and the gel was transferred to the PVDF membrane. After the membrane was washed, the membrane was placed in 5% BSA diluted with TBST, and sealed at room temperature for 3 hours; after thorough washing, the PVDF membrane was placed in a 2000-fold dilution Incubate the HIS antibody at 4°C overnight; after thorough washing, put the PVDF membrane into 10,000-fold diluted horseradish peroxidase-labeled goat anti-mouse IgG for 1 h at room temperature; after washing three times with TBST, the mixed ECL luminescence solution It was evenly dropped on the membrane, and the signal was detected using a Kodak Image Station chemiluminescence detector. The results showed that Ms-pMV-G2 could react with His antibody, and a specific reaction band could be detected with a molecular weight of about 62kDa (Figure 3, lane 1). At the same time, the M. smegmatis control could not react with the His antibody. , no signal was detected (Figure 3, lane 2), so the GroEL2 gene was successfully expressed in Ms-pMV-G2.

Example 2: Detection of Adhesion and Invasion Ability of Recombinant Mycobacterium smegmatis Ms-pMV-G2

2.1 Detection of the adhesion ability of recombinant M. smegmatis Ms-pMV-G2

A549 cells (gift from Professor Luiz Bermudez of Oregon State University, USA) were cultured in a 12-well cell culture plate, and after growing into a monolayer and reaching 2×10 ⁵ cells/well, the recombinant smegmatis branches were added according to the infection ratio of 10:1 Bacillus Ms-pMV-G2, set up blank Mycobacterium smegmatis as a control, washed thoroughly with PBS after 30 min at 4°C, then added Triton X-100 (Bio-Rad Company) to lyse the cells, collected intracellular bacteria and coated with 7H11 The solid (purchased from BD company) plate was cultured in a 37°C, 5% CO ₂ incubator for 5-7 days. It was found by bacterial count that the adhesion ability of the recombinant strain Ms-pMV-G2 was significantly higher than that of the wild strain Ms, P<0.001 (Fig. 4A), which was 2.51 times that of the wild strain. It indicated that GroEL2 has the function of improving bacterial adhesion ability.

2.2 Detection of invasive ability of recombinant Mycobacterium smegmatis Ms-pMV-G2

A549 cells were plated into a 12-well cell culture plate at 2×10 ⁵ cells/well, and the recombinant Mycobacterium smegmatis Ms-pMV-G2 was inoculated into the A549 cells with an infection ratio of 10:1, and the wild strain smegmatis was set. Mycobacterium Ms served as a control. Bacteria and A549 cells were treated in a 37° C., 5% CO ₂ incubator for 1 h, then Triton X-100 (Bio-Rad Company) was added to lyse the cells, and the number of colonies was quantitatively determined by colony counting method. The results showed that the invasion ability of recombinant M. smegmatis Ms-pMV-G2 was significantly higher than that of the wild strain, P<0.001 (Fig. 4B), which was 4.21 times that of the wild strain. It indicated that GroEL2 had the function of enhancing the ability of bacterial invasion.

Example 3: Cloning, expression and purification of GroEL2 gene in E. coli

3.1 Cloning and expression of recombinant pET-30a-GroEL2 plasmid

Using the whole genome of Mycobacterium bovis BCG Pasteur strain (ATCC: 35734) as a template, the GroEL2 gene was cloned using the primers (SEQ ID NO: 3-4 of the sequence listing) designed as follows to obtain the full sequence of the GroEL2 gene, and the sequence length was 1623bp.

The primer sequences for amplifying the GroEL2 gene are shown below:

1. Forward primer GroEL2-F2: CG GAATTC ATGGCCAAGACAATTGCGTA. Underlined is the restriction site of EcoRI. Corresponding to the sequence shown in SEQ ID NO: 3 of the sequence listing.

2. Reverse primer GroEL2-R2: CC AAGCTT GAAATCCATGCCACCCATGT. Underlined is the restriction site of HindIII. Corresponding to the sequence shown in SEQ ID NO: 4 of the sequence listing.

The PCR reaction system is as follows:

Template DNA 2 μL, 2×planta ^TM Master Mix 25 μL, primers 2 μL each, and ultrapure water 19 μL.

PCR amplification conditions are as follows:

Pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 sec, annealing at 62 °C for 30 sec, extension at 72 °C for 45 s, 30 cycles of reaction; complete extension at 72 °C for 10 min.

The PCR amplification product was recovered and digested with EcoRI and HindIII, while the pET30a(+) plasmid (Fig. 5) was double digested with the same enzymes. The digested GroEL2 gene and pET-30a(+) plasmid were ligated with T4 DNA ligase to obtain a recombinant plasmid pET-30a-GroEL2 (Fig. 6). After the recombinant plasmid pET-30a-GroEL2 was transformed into Escherichia coli DH 5α, it was placed on a shaker at 37°C and cultivated at 180 r/min for 12 hours. The sequence shown) was transformed into Escherichia coli BL21, and the recombinant Escherichia coli was cultured in LB liquid medium to OD=0.6, and 1 mL of bacterial liquid was taken as the pre-induction control, and isopropyl thiogalactoside (IPTG) was added to The final concentration was 0.8 mM, and the expression was induced for 4 h at 37 °C on a shaker. The sample was then centrifuged at 12,000 r/min for 1 min, the supernatant was discarded, resuspended in PBS, and centrifuged at 12,000 r/min for 1 min. The supernatant was discarded, and 600 μL of PBS was added to resuspend the cells. The cells were clarified; then centrifuged at 12,000 r/min for 10 min to separate the supernatant and the precipitate. The precipitate was resuspended with an equal volume of 600 μL PBS, sampled and added with an appropriate amount of loading buffer, boiled in boiling water for 10 min, and centrifuged at 4,000 r/min for 2 min. The supernatant and precipitate were taken and subjected to SDS-PAGE to observe the existence and expression of the target protein. The results showed that GroEL2 was successfully expressed in the supernatant (Fig. 7, lane 4). The amino acid sequence corresponds to the sequence shown in SEQ ID NO: 6 of the sequence table.

The applicant named the Escherichia coli recombinant strain as Escherichia coli pET-30a-GroEL2; on May 7, 2019, it was sent to the Chinese Collection of Type Cultures (CCTCC) located in Wuhan University, Wuhan City, Hubei Province for preservation, and the deposit number is CCTCC M 2019334.

3.2 Purification of GroEL2 protein

After the recombinant Escherichia coli BL21 was induced to express and split according to the above method, and after SDS-PAGE gel electrophoresis, it was determined that the GroEL2 protein was expressed in the supernatant. Purify it using a nickel column:

(1) the ethanol used to seal the column flows out through the column;

(2) Add 12 mL of sterile deionized water to the affinity chromatography column for washing;

(3) Add 50mL of Binding buffer to balance the column;

(4) Add the protein supernatant filtered through a 0.45 μm pore size filter, and collect the first few drops of the filtered liquid, numbered 1.

(5) Add 30 mL of Binding buffer to equilibrate the column, collect the first few drops of filtrate, numbered 2.

(6) Add 50 mL of Washing buffer to wash off impurities, and collect the first few drops, numbered 3.

(7) Add 6 mL of Elution buffer to elute the target protein, collect the first few drops, numbered 4.

(8) Add 50 μL of loading buffer to each of the tubes numbered 1-4 and boil for 10 min.

(9) Configure SDS-PAGE polyacrylamide gel, add the treated samples into the wells (20 μL/well), and electrophoresis (the conditions for electrophoresis on concentrated gels are DC voltage of 80 volts, and the conditions for separation gel electrophoresis are DC voltages of 120 volts). Volt), after the electrophoresis was completed, the gel was removed and stained with Coomassie brilliant blue overnight. After destaining, it was confirmed that the purified target protein was obtained (Fig. 7, lane 9).

3.3 Detection of immunogenicity of GroEL2 protein

The immunogenicity of GroEL2 protein was identified by Western blot method. The main steps were as follows: the purified recombinant protein GroEL2 (2.0 μg/lane) was subjected to SDS-PAGE electrophoresis, and the protein on the gel was transferred to PVDF membrane. Put it into 5% BSA diluted with TBST, and block it for 3 hours at room temperature; after thorough washing, put the PVDF membrane into 5000-fold diluted bovine positive serum antibody, and incubate at 4°C overnight; after thorough washing, put the PVDF membrane into 5000-fold diluted horseradish peroxidase-labeled goat anti-mouse IgG at room temperature for 1 h; after washing three times with TBST, the mixed ECL luminescent solution was evenly dropped on the membrane, and the signal was detected by a Kodak Image Station chemiluminescence detector. The results showed that GroEL2 protein could react with bovine positive serum, and a specific reaction band could be detected with a molecular weight of about 62 kDa (Fig. 8), so GroEL2 protein had good immunogenicity.

Example 4: Detection of GroEL2 protein-induced immunological response in mice

4.1 Immunization and body weight monitoring of mice

GroEL2 protein and adjuvant (MONTANIDETM IMS 1313 VG N) were mixed 1:1, and after complete emulsification, 3 female Balb/C mice aged 6-8 weeks with 16-18 g were intranasally immunized, each with 25 μg. At the same time, a single immune adjuvant group and an immune PBS group were set up as controls. Weighed once on the 0th day, and then weighed once a week and recorded it to make a weight change curve table. The results showed that with the prolongation of time, the body weight of the mice in the GroEL2 protein immunization group, the adjuvant immunization group and the PBS group increased slowly from 18g to 22g, and there was no significant difference in body weight between the groups (P>0.05) (Figure 9). , the mice were in good mental state.

4.2 Detection of total IgG and IgA antibodies in mice after immunization

The mouse IgG ELISA kit and IgA ELISA kit of Xinbosheng Company were used to detect the total mouse IgG and IgA antibodies on the 0, 7, 14, 21, and 28 days after immunization. According to the instructions for testing, the steps are briefly described as follows:

The diluted serum was added to the warmed reaction plate and incubated at 37°C for 90 min. After extensive washing, biotinylated antibody working solution was added. Incubate at 37°C for 60min. After thorough washing, the enzyme conjugate working solution was added and incubated at 37°C for 30 min in the dark. After thorough washing, chromogenic substrate (TMB) was added and incubated at 37°C for 15 min in the dark. OD450 values were measured immediately after the addition of stop solution.

The results showed that from the 14th day after immunization, the total IgG antibody level of the mice in the GroEL2+ adjuvant immunization group was significantly higher than that in the simple immune adjuvant and PBS groups, P<0.01. The difference was significant on days 21 and 28, P<0.001 (Figure 10A). The total IgA antibody continued to increase significantly from the 7th day, P<0.001. Total IgG and total IgA antibody levels showed a rapid upward trend (Figure 10B). It indicated that GroEL2 protein could induce high levels of total IgG and total IgA antibodies in mice at the early stage of immunization.

4.3 Detection of mouse-specific IgG and IgA antibodies after immunization

The mice were tailed and blood collected on days 0, 7, 14, 21, and 28 after immunization, respectively, and serum was collected for IgA, IgG antibody detection and cytokine detection. Specific steps are as follows:

The GroEL2 protein (final concentration of 2 μg/mL) was used as the antigen to coat the microtiter plate, washed overnight at 4°C, and then blocked at 37°C for 1 h after adding blocking solution; after thorough washing, 100 μL of 100-fold diluted serum to be tested was added, and incubated at 37°C 2h; after thorough washing, add horseradish peroxidase-labeled goat anti-mouse IgG or IgA, incubate at 37°C for 2h, add substrate TMB after thorough washing for 10min; add stop solution 2M sulfuric acid, read with enzyme-linked immunosorbent assay , and measure the OD value at a wavelength of 450 nm.

The results showed that the levels of specific IgG (FIG. 11A) and specific IgA (FIG. 11B) antibodies in the GroEL2+ adjuvant immunized group showed a sustained and significant increase. Since the 7th day after immunization, the difference was extremely significant with the simple immune adjuvant group and the PBS group (P<0.001). It indicated that GroEL2 protein could induce high levels of specific IgG and specific IgA antibodies in mice at the early stage of immunization.

4.4 Detection of cytokines in mice after immunization

The mouse IFN-γ ELISA kit and IL-6 ELISA kit of Xinbosheng Company were used to detect the levels of IFN-γ and IL-6 in mice on days 0, 7, 14, 21, and 28 after immunization. According to the instructions for testing, the steps are briefly described as follows:

The diluted serum was added to the warmed reaction plate and incubated at 37°C for 90 min. After extensive washing, biotinylated antibody working solution was added. Incubate at 37°C for 60min. After thorough washing, the enzyme conjugate working solution was added and incubated at 37°C for 30 min in the dark. After thorough washing, chromogenic substrate (TMB) was added and incubated at 37°C for 15 min in the dark. OD450 values were measured immediately after the addition of stop solution.

The results showed that from the 14th day and the 7th day after immunization, the total IFN-γ and IL-6 levels of the mice in the GroEL2+ adjuvant immunization group were significantly higher than those in the control group (P<0.001) (Figure 12). It shows that GroEL2 protein can cause mice to produce high levels of IFN-γ and IL-6 at the early stage of immunization, namely Th1 and Th2 cellular immunity levels.

In conclusion, the GroEL2 protein can improve the adhesion and invasion ability of mycobacteria, indicating that the protein can improve the virulence of bacteria; its prokaryotic expression protein can induce the production of high levels of IgA and IgG antibodies in mice. Although there is still some debate on the role of antibodies against tuberculosis, the significant increase in the expression levels of IFN-γ and IL-6 verifies that the GroEL2 protein can trigger both high levels of Th1-type cellular immunity and high levels of Th2 The level of humoral immunity confirmed that the protein has a certain anti-mycobacterial infection potential, and can be used as an important target for vaccine development and diagnostic preparations.

sequence listing

<110> Huazhong Agricultural University

<120> Mycobacterium bovis immune-related molecular chaperone protein GroEL2 and its encoding gene and application

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 40

<212> DNA

<213> Artificial sequence

<400> 1

gccaagacaa ttgcggatcc atggccaaga caattgcgta 40

<210> 2

<211> 40

<212> DNA

<213> Artificial sequence

<400> 2

tggtggtggt ggtggatatc gaaatccatg ccacccatgt 40

<210> 3

<211> 28

<212> DNA

<213> Artificial sequence

<400> 3

cggaattcat ggccaagaca attgcgta 28

<210> 4

<211> 28

<212> DNA

<213> Artificial sequence

<400> 4

ccaagcttga aatccatgcc acccatgt 28

<210> 5

<211> 1623

<212> DNA

<213> Mycobacterium bovis

<400> 5

atggccaaga caattgcgta cgacgaagag gcccgtcgcg gcctcgagcg gggcttgaac 60

gccctcgccg atgcggtaaa ggtgacattg ggccccaagg gccgcaacgt cgtcctggaa 120

aagaagtggg gtgcccccac gatcaccaac gatggtgtgt ccatcgccaa ggagatcgag 180

ctggaggatc cgtacgagaa gatcggcgcc gagctggtca aagaggtagc caagaagacc 240

gatgacgtcg ccggtgacgg caccacgacg gccaccgtgc tggcccaggc gttggttcgc 300

gagggcctgc gcaacgtcgc ggccggcgcc aacccgctcg gtctcaaacg cggcatcgaa 360

aaggccgtgg agaaggtcac cgagaccctg ctcaagggcg ccaaggaggt cgagaccaag 420

gagcagattg cggccaccgc agcgatttcg gcgggtgacc agtccatcgg tgacctgatc 480

gccgaggcga tggacaaggt gggcaacgag ggcgtcatca ccgtcgagga gtccaacacc 540

tttgggctgc agctcgagct caccgagggt atgcggttcg acaagggcta catctcgggg 600

tacttcgtga ccgacccgga gcgtcaggag gcggtcctgg aggaccccta catcctgctg 660

gtcagctcca aggtgtccac tgtcaaggat ctgctgccgc tgctcgagaa ggtcatcgga 720

gccggtaagc cgctgctgat catcgccgag gacgtcgagg gcgaggcgct gtccaccctg 780

gtcgtcaaca agatccgcgg caccttcaag tcggtggcgg tcaaggctcc cggcttcggc 840

gaccgccgca aggcgatgct gcaggatatg gccattctca ccggtggtca ggtgatcagc 900

gaagaggtcg gcctgacgct ggagaacgcc gacctgtcgc tgctaggcaa ggcccgcaag 960

gtcgtggtca ccaaggacga gaccaccatc gtcgagggcg ccggtgacac cgacgccatc 1020

gccggacgag tggcccagat ccgccaggag atcgagaaca gcgactccga ctacgaccgt 1080

gagaagctgc aggagcggct ggccaagctg gccggtggtg tcgcggtgat caaggccggt 1140

gccgccaccg aggtcgaact caaggagcgc aagcaccgca tcgaggatgc ggttcgcaat 1200

gccaaggccg ccgtcgagga gggcatcgtc gccggtgggg gtgtgacgct gttgcaagcg 1260

gccccgaccc tggacgagct gaagctcgaa ggcgacgagg cgaccggcgc caacatcgtg 1320

aaggtggcgc tggaggcccc gctgaagcag atcgccttca actccgggct ggagccgggc 1380

gtggtggccg agaaggtgcg caacctgccg gctggccacg gactgaacgc tcagaccggt 1440

gtctacgagg atctgctcgc tgccggcgtt gctgacccgg tcaaggtgac ccgttcggcg 1500

ctgcagaatg cggcgtccat cgcggggctg ttcctgacca ccgaggccgt cgttgccgac 1560

aagccggaaa aggagaaggc ttccgttccc ggtggcggcg acatgggtgg catggatttc 1620

tga 1623

<210> 6

<211> 540

<212> RNA

<213> Mycobacterium bovis

<400> 6

Claims (9)

1. Mycobacterium bovis immune-related molecular chaperone protein GroEL2, having the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing. 2 . The gene encoding the immune-related molecular chaperone protein GroEL2 of Mycobacterium bovis according to claim 1 . 3. The gene according to claim 2, wherein the gene has the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing. 4. A recombinant expression vector containing the gene of claim 2 or 3. 5. The recombinant bacteria containing the gene of claim 2 or 3. 6 . The recombinant expression vector according to claim 4 , wherein the recombinant expression vector is obtained by inserting the gene of claim 2 or 3 between the multiple cloning sites of pET30a. 7 . 7 . The recombinant bacteria according to claim 5 , wherein the recombinant bacteria is Escherichia coli carrying the gene of claim 2 or 3 . 8 . 8. Use of the molecular chaperone protein GroEL2 of claim 1 in the preparation of bovine tuberculosis vaccine. 9. Use of the molecular chaperone protein GroEL2 of claim 1 in the preparation of a diagnostic kit for bovine tuberculosis.