CN116059332B

CN116059332B - A Mycobacterium tuberculosis Ag85B DNA vaccine, its preparation method and application

Info

Publication number: CN116059332B
Application number: CN202211481525.2A
Authority: CN
Inventors: 万巧凤; 杨雨欣; 李慧; 刘琼; 张炜
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2022-11-24
Filing date: 2022-11-24
Publication date: 2025-11-25
Anticipated expiration: 2042-11-24
Also published as: CN116059332A

Abstract

This invention relates to the field of biological vaccine technology, specifically disclosing a Mycobacterium tuberculosis Ag85B DNA vaccine, its preparation method, and its application. The HSV2gD signal peptide gene and the Rv1886c gene are linked between HindⅢ/Xbal on the vector pcDNA3.1+ to obtain the pcD-sRv1886c plasmid, which is the Mycobacterium tuberculosis Ag85B DNA vaccine. The pcD-sRv1886c recombinant plasmid expressing Ag85B protein can effectively induce IFN-γ-secreting CD4 ⁺ T and CD8 ⁺ T cells, accounting for 2.03% and 1.05%, respectively. Furthermore, the expression of Ag85B protein by this plasmid can also stimulate the proliferation of splenic lymphocytes, with a proliferation stimulation index of 5.02. The immune effect generated by the Ag85B protein expressed by this plasmid has a significant killing effect on Mycobacterium tuberculosis H37Ra infection.

Description

Mycobacterium tuberculosis Ag85B DNA vaccine and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological vaccines, in particular to a mycobacterium tuberculosis Ag85B DNA vaccine, a preparation method and application thereof.

Background

Tuberculosis (TB) is a chronic infectious disease that is a serious threat to human health caused by infection with mycobacterium Tuberculosis (Mycobacterium Tuberculosis, m.tb), and about 90% of TB patients are adults. Therefore, prevention and treatment of the infectious disease are urgent. BCG vaccine (Bacillus Calmette-Guerin, BCG) is the only anti-tuberculosis vaccine applied clinically at present, and researches show that BCG immune protection can only be maintained for 10-15 years, has a good immune protection effect on newborns, but has no obvious protection effect on adult tuberculosis. In view of the current situation, the development of vaccines against the disease is significant.

As DNA vaccines can induce a more effective and durable broad-spectrum cellular immunity. In recent years, DNA vaccines constructed based on the gene of the tubercle bacillus immunoprotection antigen are novel vaccines which are studied more. The majority of DNA vaccines selected against tuberculosis are antigens highly expressed in early and logarithmic growth phases of tuberculosis, i.e. in the infectious phase. However, the existing DNA vaccine with high safety for preventing tuberculosis is relatively few.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of a mycobacterium tuberculosis Ag85B DNA vaccine, and the protein expressed by the pcD-sRv1886c recombinant plasmid can effectively induce the CD4 ⁺ T and CD8 ⁺ T cells secreting IFN-gamma, and the percentages are 2.03% and 1.05% respectively. In addition, the plasmid can stimulate the proliferation of spleen lymphocytes, and the proliferation stimulation index is 5.02. Finally, the immune effect produced by the protein stimulation expressed by the plasmid has remarkable killing effect on the infection of tubercle bacillus H37 Ra.

The invention provides a preparation method of a mycobacterium tuberculosis Ag85B DNA vaccine, which comprises the steps of firstly synthesizing a nucleotide chain from HSV2gD signal peptide genes and an Rv1886c partial gene sequence, and then connecting the nucleotide chain between HindIII/Xbal on a vector pcDNA3.1+, so as to obtain a pcD-sRv1886c plasmid, namely the mycobacterium tuberculosis Ag85B DNA vaccine;

The nucleotide chain gene sequence is shown as SEQ ID NO. 3.

Further, the HSV2gD signal peptide gene sequence is shown in SEQ ID NO. 1.

Further, the sequence of the Rv1886c gene is shown as SEQ ID NO. 2.

The invention provides the mycobacterium tuberculosis Ag85B DNA vaccine prepared by the method.

Further, the mycobacterium tuberculosis Ag85B DNA vaccine is formulated with a pharmacologically acceptable excipient.

The invention provides application of the mycobacterium tuberculosis Ag85B DNA vaccine in preparing medicaments for treating or preventing tuberculosis.

Further, the Ag85B DNA vaccine can be used for preparing inducers of IFN-gamma secreting CD4 ⁺ T and CD8 ⁺ T cells.

Further, the Ag85B DNA vaccine can be used for preparing medicines for stimulating the proliferation of spleen lymphocytes.

Furthermore, the Ag85B DNA vaccine can specifically express Ag85B protein, and the Ag85B protein can be used for preparing medicines for inhibiting tuberculosis infection.

Further, the tubercle bacillus is H37Ra.

The invention provides application of the mycobacterium tuberculosis Ag85B DNA vaccine in preparing medicaments used as carriers or adjuvants.

Compared with the prior art, the invention has the beneficial effects that:

1. The invention uses molecular biology technology to connect HSV2-gD signal peptide gene and Rv1886c gene 963bp nucleotide sequence to pcDNA3.1+ with strong promoter, and constructs eukaryotic expression plasmid pcD-sRv1886c, namely mycobacterium tuberculosis Ag85B DNA vaccine.

2. The invention successfully constructs eukaryotic expression plasmid pcD-sRv1886c and detects the specific expression of Ag85B protein, and preliminarily evaluates the humoral immunity and the cellular immunity effect generated by the immunization of the recombinant plasmid pcD-sRv1886c with the immunization dose of 50 mug/mouse. Upon immunization, the recombinant plasmid pcD-sRv1886c was able to induce the production of IgG and IgG 1-specific antibodies, with increasing antibody titers of IgG and IgG1 over time. The increased levels of IgG and IgG1 potency at 42d compared to 28d indicate that Ag85B stimulates the body to produce a durable humoral immune response. Since m.tb belongs to intracellular parasitic bacteria, cellular immunity is important against m.tb infection. IFN-gamma is produced primarily by activated T cells, which can up-regulate NK cell and CD8+ T cell activity. The result of the invention shows that the recombinant plasmid of pcD-sRv1886c can effectively induce the CD4 ⁺ T and CD8 ⁺ T cells secreting IFN-gamma after immunization, and the ratio is 2.03% and 1.05% respectively. In addition, the plasmid can also stimulate the proliferation of spleen lymphocytes, and the proliferation stimulation index is 5.02, which shows that the plasmid can effectively induce and promote the cell immune effect. Finally, the immune effect produced by the protein stimulation expressed by the plasmid has remarkable killing effect on the infection of tubercle bacillus H37 Ra.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows an electrophoresis diagram of the digestion and identification of a recombinant eukaryotic expression plasmid pcD-sRv1886c of the present invention;

The meanings of each lane are M:15kb DNA marker;1:pcDNA3.1+ empty plasmid, 2: pcD-sRv1886c recombinant plasmid, 3: hindIII single cleavage product of plasmid, 4: xbal single cleavage product of plasmid, 5: hindIII/Xbal double cleavage product of plasmid.

FIG. 2 shows the Western blot detection results after plasmid transfection in the invention;

the meanings of each lane are M protein marker, CHO cells transfected with 1:pcD-sRv1886c recombinant plasmid, and CHO cells transfected with 2:pcDNA3.1+ empty plasmid.

FIG. 3 shows the results of detection of antibody titers in the present invention.

FIG. 4 shows the effect of recombinant plasmid pcD-sRv1886c on IFN-gamma secreting CD4+ T and CD8+ T cells according to the present inventionn=5);

Wherein, panel a represents a PBS group cd3+cd ⁺IFN-γ⁺ cell proportional flow chart, circling a population of secreting IFN- γ lymphocytes;

Panel B shows a pcD-sRv1886c immunoset CD3+CD4 ⁺IFN-γ⁺ cell proportional flow chart, panel B outlines the CD3 ⁺ T cell population;

Panel C shows a flow-through statistical analysis of the CD3 ⁺CD4⁺IFN-γ⁺ cell fraction and CD3 ⁺CD8⁺IFN-γ⁺ cell fraction of the pcD-sRv1886C immunized group.

FIG. 5 shows the effect of recombinant plasmid pcD-sRv1886c on proliferation of spleen lymphocytes in the present inventionn=5)。

FIG. 6 shows the killing effect of recombinant plasmid pcD-sRv1886c on tubercle bacillus H37Ra in the present invention.

Detailed Description

The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified, and materials, reagents, etc. used in the examples described below are commercially available.

Example 1

1. Materials and methods

1. Cell, strain and plasmid construction

Chinese hamster ovary cells (CHO-K1) were purchased from the national academy of sciences of Chinese medicine. The clone strain E.coli DH 5. Alpha. Was given away by Ma Guorong teacher. A963 bp nucleotide sequence of HSV2gD signal peptide gene and Rv1886c gene was synthesized by Shanghai Biotechnology Co., ltd and ligated between HindIII/Xbal on pcDNA3.1+.

The HSV2gD signal peptide gene sequence is shown in SEQ ID NO. 1;

SEQ ID NO.1:

AAGCTTACCATGGGGCGTTTGACCTCCGGCGTCGGGACGGCGGCCCTGCTAGTTGTCGCGGTGGGACTCCGCGTCGTCTGCGCC

The sequence of the Rv1886c gene is shown as SEQ ID NO. 2;

SEQ ID NO.2:

TTCTCCCGGCCGGGGCTGCCGGTCGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGCGACATCAAGGTTCAGTTCCAGAGCGGTGGGAACAACTCACCTGCGGTTTATCTGCTCGACGGCCTGCGCGCCCAAGACGACTACAACGGCTGGGATATCAACACCCCGGCGTTCGAGTGGTACTACCAGTCGGGACTGTCGATAGTCATGCCGGTCGGCGGGCAGTCCAGCTTCTACAGCGACTGGTACAGCCCGGCCTGCGGTAAGGCTGGCTGCCAGACTTACAAGTGGGAAACCTTCCTGACCAGCGAGCTGCCGCAATGGTTGTCCGCCAACAGGGCCGTGAAGCCCACCGGCAGCGCTGCAATCGGCTTGTCGATGGCCGGCTCGTCGGCAATGATCTTGGCCGCCTACCACCCCCAGCAGTTCATCTACGCCGGCTCGCTGTCGGCCCTGCTGGACCCCTCTCAGGGGATGGGGCCTAGCCTGATCGGCCTCGCGATGGGTGACGCCGGCGGTTACAAGGCCGCAGACATGTGGGGTCCCTCGAGTGACCCGGCATGGGAGCGCAACGACCCTACGCAGCAGATCCCCAAGCTGGTCGCAAACAaCACCCGGCTATGGGTTTATTGCGGGAACGGCACCCCGAACGAGTTGGGCGGTGCCAACATACCCGCCGAGTTCTTGGAGAACTTCGTTCGTAGCAGCAACCtGAAGTTCCAGGATGCGTACAACGCCGCGGGCGGGCACAACGCCGTGTTCAACTTCCCGCCCAACGGCACGCACAGCTGGGAGTACTGGGGCGCTCAGCTCAACGCCATGAAGGGTGACCTGCAGAGTTCGTTAGGCGCCCATCATCACCATCACCATTAATCTAGA

the nucleotide chain gene sequence obtained by synthesis is shown as SEQ ID NO. 3;

SEQ ID NO.3:

AAGCTTACCATGGGGCGTTTGACCTCCGGCGTCGGGACGGCGGCCCTGCTAGTTGTCGCGGTGGGACTCCGCGTCGTCTGCGCCTTCTCCCGGCCGGGGCTGCCGGTCGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGCGACATCAAGGTTCAGTTCCAGAGCGGTGGGAACAACTCACCTGCGGTTTATCTGCTCGACGGCCTGCGCGCCCAAGACGACTACAACGGCTGGGATATCAACACCCCGGCGTTCGAGTGGTACTACCAGTCGGGACTGTCGATAGTCATGCCGGTCGGCGGGCAGTCCAGCTTCTACAGCGACTGGTACAGCCCGGCCTGCGGTAAGGCTGGCTGCCAGACTTACAAGTGGGAAACCTTCCTGACCAGCGAGCTGCCGCAATGGTTGTCCGCCAACAGGGCCGTGAAGCCCACCGGCAGCGCTGCAATCGGCTTGTCGATGGCCGGCTCGTCGGCAATGATCTTGGCCGCCTACCACCCCCAGCAGTTCATCTACGCCGGCTCGCTGTCGGCCCTGCTGGACCCCTCTCAGGGGATGGGGCCTAGCCTGATCGGCCTCGCGATGGGTGACGCCGGCGGTTACAAGGCCGCAGACATGTGGGGTCCCTCGAGTGACCCGGCATGGGAGCGCAACGACCCTACGCAGCAGATCCCCAAGCTGGTCGCAAACAaCACCCGGCTATGGGTTTATTGCGGGAACGGCACCCCGAACGAGTTGGGCGGTGCCAACATACCCGCCGAGTTCTTGGAGAACTTCGTTCGTAGCAGCAACCTGAAGTTCCAGGATGCGTACAACGCCGCGGGCGGGCACAACGCCGTGTTCAACTTCCCGCCCAACGGCACGCACAGCTGGGAGTACTGGGGCGCTCAGCTCAACGCCATGAAGGGTGACCTGCAGAGTTCGTTAGGCGCCCATCATCACCATCACCATTAATCTAGA.

2. The first group of experimental animals is that 10 SPF-class C57BL/6J mice, female, 6-7 weeks old, and weight (18+ -1) g. In the second group, 12 SPF-grade C57BL/6J female mice, 6-7 weeks old, body weight (19+ -2) g, all purchased from Ningxia university laboratory animal center (qualified certificate number: SCXK (Ning) 2020-0001), were bred and used for scientific purposes, and according to Ningxia university animal ethics related regulations (2021-N019).

3. Main reagent

DL15000 DNA MARKER (Takara Bio), restriction enzymes HindIII and XbaI (NEB), kanamycin sulfate (Kan), IPTG, agarose, tryptone and yeast extract (Ningxia Bo Biotechnology Co., ltd.), plasmid miniextraction and gel recovery kits (Tiangen Biotechnology Co., ltd.), endotoxin-free plasmid miniextraction kit (Omega Bio-Tek), murine anti-Ag 85B mab (Beijing Boossen Biotechnology Co., ltd.), lipofectamine ^TM transfection reagent (Thermo Fisher), protein marker (Beijing full gold Biotechnology Co., ltd.), 1.25% aver Ding Mazui (Nanj Aibei Biotechnology Co., ltd.), HRP-labeled goat anti-mouse IgG secondary antibody (Merck), HRP-labeled goat anti-mouse IgG1 secondary antibody (Abcam), and internal fixation rupture membrane solution 、Protein Transport Inhibitor Cocktail(500X)、PE Anti-Mouse CD8a、FITC Anti-Mouse CD4、eFluor 450Anti-Mouse CD3e、APC Anti-Mouse IFN-γ(Thermo Fisher);CCK8 kit (cloud).

4. Identification of eukaryotic expression plasmids

DH5 alpha competent cells were transformed with the constructed plasmid, plated on kanamycin-resistant LB plates, cultured overnight at 37 ℃, single white colonies were selected from the transformed kanamycin LB plates, and inoculated into kanamycin-containing LB liquid medium for culture overnight. The plasmids were extracted using a small amount of plasmid extraction kit, and subjected to double enzyme digestion and identification, and the plasmids were sent to the Boxing family biotechnology company, inc. for sequencing. The expression plasmid was designated pcD-sRv1886c.

5. Eukaryotic expression plasmid transfection CHO cells

(1) Mass extraction of pcD-sRv1886c plasmid endotoxin-free plasmids were extracted using the endotoxin-free plasmid extraction kit. The eukaryotic expression plasmid pcD-sRv1886C and empty plasmid pcDNA3.1+ identified as correct were extracted, the concentration and purity were determined, adjusted to 1. Mu.g/. Mu.L with PBS and stored at-20℃for further use.

(2) Plasmid transfection CHO cells were inoculated in 12 well cell culture plates and when the cells were grown to 80% -90% monolayer in DMEM nutrient solution containing 10% fetal bovine serum, the supernatant was discarded, washed 2-3 times with PBS buffer, and recombinant plasmid pcD-sRv1886c and empty plasmid pcdna3.1+ were transfected separately according to liposome transfection reagent instructions. After culturing at 37 ℃ in a 5% CO2 incubator for 5 hours, the DMEM culture solution containing 5% fetal bovine serum is replaced for further culturing until 48 hours.

6. Western blot detection of recombinant protein expression

Collecting 40 μl of CHO cell culture liquid 48h after transfection, adding 5×sample buffer solution at a volume ratio of 1:4, loading after boiling water bath for 5min, separating by 12% SDS-PAGE, transferring onto PVDF membrane with semi-dry electrotransfer membrane instrument, sealing with 5% skimmed milk powder for 1h, washing the membrane with TBST for 10min for 3 times, adding mouse anti-Ag 85B monoclonal antibody (1:1 000 dilution), adding HRP-labeled rabbit anti-mouse IgG (1:2000), oscillating for 60min at 37deg.C, adding ECL luminescent liquid, developing, fixing, and analyzing the result.

7. Immunization of animals

(1) The first time, after 1 week of adaptive feeding, the mice in the first group were randomly divided into 2 groups of 5 mice each. The PBS group and pcD-sRv1886c recombinant plasmid immunization group (50. Mu.g/dose) were divided. Mice were immunized by thigh intramuscular injection, 2 weeks after initial immunization, 2 total immunizations. After 14, 28 and 42 days of primary immunization, blood is collected through a retroorbital venous plexus, and serum is separated.

(2) The second time, after 1 week of adaptive feeding, the mice in the second batch were randomly divided into 2 groups of 6 mice each. The group was divided into PBS+H237 Ra-infected group and pcD-sRv1886 c-immunized+H237 Ra-infected group.

8. Antibody titer detection

An indirect ELISA method is adopted. The Ag85B protein prepared in the early stage of the experiment is prepared into a solution with the concentration of 2 mug/mL, a plurality of 96-well ELISA plates are coated, the concentration is 0.1 mL/well, the temperature is 4 ℃ overnight, 2% BSA blocking solution is added, the concentration is 0.1 mL/well, the temperature is 37 ℃ and is kept stand for 2 hours, TBST washing plates are 4 times, immune serum (with the final concentration of 1:100, 1:200, 1:400, 1:800, 1:1:600, 1:3 200, 1:6:400, 1:12, 1:25:600 and 1:51) is added, PBS negative control is arranged in column 1, the temperature is 37 ℃ and is incubated L h, HRP-labeled goat anti-mouse IgG (1:1 000 dilution), the temperature is 0.1 mL/well, the temperature is 40 minutes at 37 ℃ and the temperature is PBST washing plates, the substrate is added, the temperature is 0.1 mL/well and the temperature is kept away from light and the temperature is 37 ℃ and is 15 minutes, the reaction is stopped by adding 50 mug 2mol/L H SO4 to each well, and the OD 450A value is measured by the ELISA plate. The highest sample dilution at >2.1 times the OD of the test well/the OD of the negative control was defined as antibody titer.

9. Flow Cytometry (FCM) detection of IFN-gamma secreting CD4 ⁺ T and CD8 ⁺ T lymphocytes from the spleen of mice

After blood collection, the mice are killed by cervical dislocation, single cell suspension is prepared after spleen is taken, the operation is strictly performed according to lymphocyte separation liquid instruction, and finally, the cell concentration is adjusted to be 2X 10 ⁶/mL. Taking 100 mu L of cell suspension in 1mL of culture solution, processing the culture solution to be used for stimulating and culturing for 24 hours at the concentration of 2 mu g/mLAg B protein, centrifuging for 5 minutes at room temperature, discarding supernatant, re-suspending cells by using 100 mu L of flow cell staining solution, adding 4 mu LeFluor-CD 3, 0.5 mu L of FITC-CD4 and 1.25 mu L of PE-CD8,4 DEG photophobic incubation for 30 minutes, adding 400 mu L of flow cell staining solution, centrifuging for 5 minutes at room temperature, adding 500 mu L of membrane breaker to re-suspend cells, 4 DEG photophobic incubation for 1 hour, centrifuging for 5 minutes at room temperature, re-suspending cells by using 100 mu L of flow cell staining solution, adding 2.5ulAPC-IFN-r antibody, re-suspending cells by using 300 mu L of flow cell staining solution after room temperature photophobic incubation for 30 minutes, adding 400 mu L of membrane breaking washing solution, centrifuging for 5 minutes at room temperature, and performing detection by using a machine after the supernatant is discarded.

10. Spleen lymphocyte proliferation assay

Lymphocytes prepared in 9 above and having a cell density of 2X 10 ⁶/mL were seeded in 96-well plates. Each experimental well was treated with 2 μg/mL Ag85B protein for stimulation culture, no protein was added to the negative control wells, and no lymphocytes were seeded in the zeroing wells. 6 duplicate wells were set, incubated at 37℃for 24h, and CCK8 was added 4h before termination of the incubation, with 10uL per well. After the culture, the OD value at 450nm is measured by an enzyme-labeled instrument, and the lymphocyte proliferation stimulation index (Simulation Index SI) is calculated, wherein the formula=the OD value of the experimental group/the OD value of the control group.

11. Culture and infection experiment of tubercle bacillus H37Ra

H37Ra stored in Roche medium was inoculated into 7H9 liquid medium (containing 100 g/LOADC), shake-cultured at 37℃for 3w, centrifuged at 5 000r/min for 10min, and the bacteria were collected and stored at-20℃for use. After normal feeding for 42d, H37 Ra-infected mice were infected with 10 ⁶ CFU/H37 Ra via the tail vein, and pcD-sRv1886c immunized +H237 Ra-infected mice were immunized by thigh intramuscular injection (100 μg/mouse), 1 week, 3 consecutive immunizations, and 42d after the 1 st immunization, H37 Ra-infected mice were infected with the same dose of H37Ra via the tail vein as PBS +H237 Ra-infected mice. After 28d infection, the mice of each group were sacrificed by cervical scission, and after the lung tissue was fixed with formaldehyde for 24h, paraffin-embedded sections were acid-fast stained to observe the number of tubercle bacillus in the lung tissue sections.

12. Statistical analysis

Experimental data were analyzed and collated using GraphPad-Prism 5 statistical mapping software and SPSS 20.0 statistical analysis software. The metrology data is expressed as mean ± standard deviation. For normal distribution compliant and variance-aligned metrology data, two-sample t-test is used for comparison between two sets. For detection of log-normal distribution data-antibody titers, t-test was performed after log-transformation. Flow cytometry results were analyzed using Flowjo software. P <0.05 is statistically significant for the differences.

2. Results

1. Cleavage of eukaryotic expression plasmid the double cleavage (HindIII and Xbal) products of eukaryotic recombinant expression plasmid pcD-sRv1886c were analysed by 1% agarose gel electrophoresis and bands of approximately 1000bp in size were seen, the size being in accordance with the expected (963 bp) size, see FIG. 1. The gene sequencing result is completely consistent with the HSV2gD signal peptide gene and the M.tb Rv1886c gene sequence, which shows that the plasmid construction is correct.

2. Expression of recombinant proteins

48H after the recombinant plasmid pcD-sRv1886c is transfected into CHO cells, cell supernatants are collected for Western blot, and the result shows that a band appears at about 35kDa, which is consistent with the size of the predicted result, and no band appears in the empty vector, as shown in FIG. 2.

3. Effect of recombinant plasmid on serum IgG and IgG1 antibody titers

The indirect ELISA results showed that specific IgG and IgG1 antibodies were detected in the serum of immunized mice at 14d after one immunization, antibody titers were 1:1720 and 1:460, respectively, igG and IgG1 titers at 28d were 1:5760 and 1:2560, respectively, and IgG and IgG1 titers were 1:6080 and 1:2720, respectively, as shown in FIG. 3. The results show that the pcD-sRv1886c plasmid can continuously induce humoral immunity.

4. Effect of plasmid on IFN-gamma secreting CD4 ⁺ T and CD8 ⁺ T cells

FCM results showed that the proportion of cd3+cd ⁺IFN-γ⁺ cells in pcD-sRv1886c and PBS groups was 2.03% ± 0.23% and 0.14% ± 0.02%, respectively, the differences were very significant (p < 0.001) and the proportion of cd3+cd ⁺IFN-γ⁺ cells was 1.05% ± 0.11% and 0.13% ± 0.01%, respectively, the differences were significant (p < 0.01), see fig. 4.

5. Effect of recombinant plasmid on proliferation of spleen lymphocytes

The detection result shows that pcD-sRv1886c immune group spleen lymphocytes can generate remarkable proliferation capacity after being stimulated by Ag85B specific antigen. The splenic lymphocyte proliferation stimulation index of pcD-sRv1886c immunized group was 5.02, and the difference was statistically significant (P < 0.01) compared to PBS group, see FIG. 5.

6. Experiment of killing effect of recombinant plasmid on tubercle bacillus H37Ra

4 Weeks after H37Ra infection, the bacterial count (gram staining in red) was significantly reduced in the pcD-sRv1886c immunized +H237 Ra-infected group compared to the PBS +H237 Ra-infected group, as shown in FIG. 6.

In conclusion, the results of the present invention show that eukaryotic expression plasmid pcD-sRv1886c was successfully constructed and specific expression of Ag85B protein was detected. The invention preliminarily evaluates that the pcD-sRv1886c recombinant plasmid with the immune dose of 50 mug/dose generates humoral immunity and cellular immunity effect after muscle immunity. Upon immunization, the recombinant plasmid pcD-sRv1886c was able to induce the production of IgG and IgG 1-specific antibodies, with increasing antibody titers of IgG and IgG1 over time. The increased levels of IgG and IgG1 potency at 42d compared to 28d indicate that Ag85B stimulates the body to produce a durable humoral immune response. Since m.tb belongs to intracellular parasitic bacteria, cellular immunity is important against m.tb infection. IFN-gamma is produced primarily by activated T cells, which can up-regulate NK cell and CD8+ T cell activity. The results of this study showed that a post-immunization 42d, pcD-sRv1886c recombinant plasmid was able to effectively induce IFN-gamma secreting CD4 ⁺ T and CD8 ⁺ T cells at a ratio of 2.03% and 1.05%, respectively. In addition, the plasmid can also stimulate the proliferation of spleen lymphocytes, and the proliferation stimulation index is 5.02, which shows that the plasmid can effectively induce and promote the cell immune effect. The immune effect produced by the stimulation of the protein expressed by the plasmid pcD-sRv1886c constructed by the invention has remarkable killing effect on the infection of tubercle bacillus H37 Ra.

It should be noted that the Ag85B DNA vaccine prepared by the invention can also be used for model organisms with relatively close relation with human species sources, such as rats, rabbits, pigs, dogs and the like, so that the Ag85B DNA vaccine prepared by the invention has potential for human application.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. The application of a Mycobacterium tuberculosis Ag85B DNA vaccine in the preparation of drugs for the prevention of pulmonary tuberculosis, characterized in that the Mycobacterium tuberculosis Ag85B DNA vaccine is prepared by first synthesizing the HSV2gD signal peptide gene sequence and the Rv1886c partial gene sequence into nucleotide chains, and then linking them between Hind Ⅲ / Xbal on the vector pcDNA3.1+ to obtain the pcD-sRv1886c plasmid, which is the Mycobacterium tuberculosis Ag85B DNA vaccine;

The HSV2gD signal peptide gene sequence is shown in SEQ ID NO.1;

The partial gene sequence of Rv1886c is shown in SEQ ID NO.2;

The nucleotide chain gene sequence is shown in SEQ ID NO.3.

2. The use of the Mycobacterium tuberculosis Ag85B DNA vaccine according to claim 1 in the preparation of a drug for the prevention of pulmonary tuberculosis, characterized in that the Mycobacterium tuberculosis Ag85B DNA vaccine is formulated with a pharmacologically acceptable excipient.