CN112190701A - Recombinant DNA vaccine of mycobacterium tuberculosis and preparation method thereof - Google Patents
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Abstract
The invention discloses a recombinant DNA vaccine of mycobacterium tuberculosis and a preparation method thereof. The recombinant DNA vaccine is obtained by recombining genes Ag85B, Rv2029c and Rv1738 into a eukaryotic expression vector. The research of the invention finds that after C57BL/6 female mice of 6-8 weeks old are immunized, the DNA vaccine Ag85B-Rv2029C-Rv1738-pVAX1 can obviously improve CD4 in peripheral blood of the mice+，CD8+The proportion of T lymphocytes increases the secretion of Th1 type cell factors IL-2, IFN-gamma and TNF-alpha of mouse spleen lymphocyte antigen specificity, reduces BCG bacterial count of mouse lung and spleen, and prompts the recombinationThe DNA vaccine can effectively control the growth and the propagation of the mycobacteria in the mouse body. Therefore, the recombinant DNA vaccine of the present invention can be used for the prevention and treatment of Mycobacterium tuberculosis infection.
Description
Technical Field
The invention relates to the technical field of genetic engineering and recombinant DNA vaccine development, in particular to a recombinant DNA vaccine of mycobacterium tuberculosis and a preparation method thereof.
Background
Tuberculosis (TB) is a serious respiratory infectious disease caused by Mycobacterium Tuberculosis (MTB). According to the worldwide tuberculosis report of 2019 of the world health organization, about 1000 new registered tuberculosis cases exist worldwide, the number of tuberculosis deaths of Human Immunodeficiency Virus (HIV) negative people reaches 120 ten thousand, and the number of tuberculosis deaths of other HIV positive people reaches 25.1 ten thousand. In addition, it is estimated that about one third of the world's population suffers from latent tuberculosis infection (LTBI).
BCG (BCG) is the only tuberculosis vaccine available at present, and can effectively prevent the miliar tuberculosis and the tuberculous meningitis. But the protective effect on adults is limited and the multiple cropping is ineffective. And the BCG vaccine is a preventive vaccine and cannot provide effective protection for individuals with latent infection, so that the development of a novel tuberculosis vaccine is urgent.
Various types of novel vaccines have been widely studied, including recombinant BCG vaccines, auxotrophic mycobacterium tuberculosis vaccines, protein polypeptide vaccines, DNA vaccines, tuberculosis subunit vaccines using viruses as vectors, and the like.
Disclosure of Invention
The invention aims to provide a recombinant DNA vaccine of mycobacterium tuberculosis and a preparation method thereof, the vaccine can supplement the performance of commercial bacille calmette-guerin, control tuberculosis latent infection, and is expected to be used as a vaccine for strengthening immunity after the initial immunity of the bacille calmette-guerin; therapeutic vaccines to control latent infection; a therapeutic vaccine for controlling tuberculosis in combination with a drug.
In the invention, complete Ag85B, Rv2029c and Rv1738 in a standard strain H37Rv are selected and connected to an expression vector through enzyme digestion; the recombinant Rv1738(285bp,1965657..1965941), Rv2029c (1020bp,2275405..2276424) can be used for serological detection of latent tuberculosis, and Ag85B (978bp, 2134897..2135874) can be used for serological detection of acute infection, so that the genes can be used as candidate genes of a vaccine, and the genes are recombined in a vector pVAX1 to be used as a DNA vaccine. The technical scheme of the invention is specifically introduced as follows.
A recombinant DNA vaccine of Mycobacterium tuberculosis is prepared by recombining genes Ag85B, Rv2029c and Rv1738 into expression plasmid. Preferably, the expression plasmid is pVAX1, and the recombinant vaccine is Ag85B-Rv2029c-Rv1738-pVAX1 (abbreviated as B21).
The invention further provides a preparation method of the recombinant DNA vaccine of the mycobacterium tuberculosis, which comprises the following steps:
(1) amplifying Ag85B, Rv2029c and Rv1738 genes;
(2) performing double digestion on the Ag85B gene and the expression plasmid respectively, and performing gel recovery and connection to obtain a recombinant plasmid;
(3) transforming, amplifying and expressing the recombinant plasmid obtained in the step (2);
(4) carrying out gel recovery and connection on the recombinant plasmid obtained in the step (3) and the Rv2029c gene respectively to form a recombinant plasmid;
(5) transforming, amplifying and expressing the recombinant plasmid obtained in the step (4);
(6) carrying out gel recovery and connection on the recombinant plasmid obtained in the step (5) and the Rv1738 gene respectively to form a recombinant plasmid; (7) and (4) transforming, amplifying and expressing the recombinant plasmid obtained in the step (6) to obtain the recombinant DNA vaccine.
Preferably, in step (2), the expression plasmid is pVAX 1.
Preferably, in the step (2), the enzymes used in the double enzyme digestion are NheI and HindIII; in the step (4), the enzymes used in the double enzyme digestion are HindIII and EcoR 1; in step (6), the enzymes used for the double digestion were EcoR1 and Not 1.
Preferably, in the step (3), the step (5) and the step (7), the recombinant plasmid is transformed, amplified and expressed in E.coli.
Compared with the prior art, the invention has the beneficial effects that:
in the vaccine of the present invention, the protein can be expressed in mammalian cells using the nucleic acid vaccine vector pVAX1 approved by the FDA in the united states.
DNA vaccine Ag85B-Rv2029c-Rv1738-pVAX1 uses nucleic acid vaccine vector pVAX1 to fuse and express tubercle bacillus antigens Ag85B, Rv2029c and Rv1738, after the vaccine transfects macrophages, fusion protein Ag85B-Rv2029c-Rv1738 can be expressed in the macrophages, and effectively activate the macrophages, and improve the secretion level of IL-6 and TNF-alpha.
The research of the invention finds that after 2 weeks of C57BL/6 female mice with the age of 6-8 weeks are immunized, the DNA vaccine Ag85B-Rv2029C-Rv1738-pVAX1 can obviously improve CD4 in peripheral blood of the mice+，CD8+Proportion of T lymphocytes, CD4+，CD8+T lymphocytes play an important role in the process of anti-tuberculosis immunity.
The research of the invention finds that after C57BL/6 female mice with the age of 6-8 weeks are immunized for 18 weeks, tail vein injection is adopted for 5 multiplied by 1064 weeks after CFU BCG/mouse infection, the secretion of TH1 type cell factors IL-2, IFN-gamma and TNF-alpha is increased after the spleen lymphocytes are stimulated by specific antigens of Ag85B, Rv2029c and Rv1738, and the BCG count of lung and spleen of mice in a vaccine immunization group is obviously reduced, which indicates that the DNA vaccine can effectively control the growth and reproduction of mycobacterium tuberculosis and the like in the mice and is used for preventing and treating mycobacterium tuberculosis infection.
The DNA vaccine Ag85B-Rv3425-Rv1813c-pVAX1(B31) is used as a control in the research of the invention, and the DNA vaccine (B21) is found to be more effective in activating macrophages and improving TH1 type cellular immune response compared with B31.
Drawings
FIG. 1 shows the construction of recombinant DNA vaccine by enzyme digestion. Lane 1: DNA Marker, Lane 2: control vector pVAX1, Lane 3: recombinant Ag85B-Rv2029c-Rv1738-pVAX1, Lane 4: recombinant Ag85B-Rv3425-Rv1813-pVAX 1.
FIG. 2 recombinant B21 DNA, B31 DNA vaccine can be expressed in eukaryotic cells. Lane 1: protein Ag85B control, Lane 2: transfection of pVAX1 vector, Lane 3: b21 transfection, Lane: b31 transfection.
FIG. 3 shows the secretion results of IL-6(a), TNF-alpha (B) after recombinant B21 DNA vaccine transfection of Raw264.7 cells.
FIG. 4 shows the flow-type staining results of recombinant B21 DNA vaccine.
FIG. 5 shows the cell supernatant IL-2, IFN-. gamma.and TNF-. alpha.contents of mice challenged with BCG (a), and CFU counts in lung and spleen (b).
Detailed Description
Reagents and conditions not specifically identified in the present invention are conventional products and methods well known to those of ordinary skill in the art.
EXAMPLE 1 preparation of recombinant DNA vaccine
1.1 preparation of genomic DNA of Mycobacterium tuberculosis H37Rv Strain
Mycobacterium tuberculosis (H37Rv) was inactivated by culturing in 7H9 Broth medium for 4 weeks at 80 ℃ for 2 hours. Genomic DNA was extracted using a bacterial DNA (small) extraction kit. The digestion time of the bacteria is extended to 3 to 5 hours due to the wall thickness of tubercle bacillus.
1.2 amplification of genes of Mycobacterium tuberculosis H37Rv strain Ag85B, Rv2029c and Rv 1738.
1) The specific primer design is as follows:
ag 85B-S: the 5 'aaagctagcgccaccatggcattctcccggccggggct-3' enzyme cutting site is as follows: nhe1
Ag 85B-A: 5'-tttaagcttgccggcgcctaacgaactct-3' the cleavage sites are: HindIII
Rv2029 c-S: 5'-tttaagcttatgacggagccagcggcgt-3' the cleavage sites are: HindIII
Rv2029 c-A: 5'-aaagaattctggcgaggcttccgggttaacga-3' the cleavage sites are: EcoR1
Rv 1738-S: 5'-aaagaattcatgtgcggcgaccagt-3' the cleavage sites are: EcoR1
Rv 1738-A: 5'-aaagcggccgcctactatcaatacaacaatcgcgccgg-3' the cleavage sites are: not1
2) PCR reaction (takaraPCR reagents):
3) PCR reaction conditions
The product was identified by electrophoresis on a 1% agarose gel.
1.3 digestion, ligation and transformation of vectors pVAX1 and Ag85B
The PCR products of the vectors pVAX1, Ag85B were double digested with NheI and HindIII at 37 ℃ for 3 hours. The enzyme digestion product was purified with a gel recovery kit. T4 DNA ligase was ligated overnight at 4 ℃. Coli DH5 alpha strain, selecting clone plasmid for sequencing verification.
The PCR product of the vector constructed successfully and Rv2029c was digested simultaneously with HindIII and EcoR1 at 37 ℃ for 3 hours. The enzyme digestion product was purified with a gel recovery kit. T4 DNA ligase was ligated overnight at 4 ℃. Coli DH5 alpha strain, selecting clone plasmid for sequencing verification.
The PCR product of the vector constructed successfully and Rv1738 was digested simultaneously with EcoR1 and Not1 at 37 ℃ for 3 hours. The enzyme digestion product was purified with a gel recovery kit. T4 DNA ligase was ligated overnight at 4 ℃. Coli DH5 alpha strain, selecting clone plasmid for sequencing verification. Thus, the recombinant DNA vaccine Ag85B-Rv2029c-Rv1738-pVAX1(B21) was constructed (FIG. 1).
Simultaneously, Ag85B-Rv3425-Rv1813c-pVAX1(B31) was constructed using the above method.
The construction of the recombinant DNA vaccine verified by enzyme digestion is shown in FIG. 1, Lane 1: DNA Marker, Lane 2: control vector pVAX1, Lane 3: recombinant Ag85B-Rv2029c-Rv1738-pVAX1, Lane 4: recombinant Ag85B-Rv3425-Rv1813c-pVAX 1. Restriction enzymes Nhe1 and Not1 were used for cleavage. As can be seen from the figure, the recombinant plasmid B21 carries 2163bp of exogenous fragment, and the recombinant plasmid B31 carries 1823bp of fragment. The result shows that the recombinant plasmid recombinant Ag85B-Rv2029c-Rv1738-pVAX1, Ag85B-Rv3425-Rv1813c-pVAX1 are successfully constructed.
1.4 verification of cell expression of recombinant DNA vaccines
The DNA vaccine plasmid successfully constructed is used for transfecting HEK293t cells, cell samples are collected, and whether eukaryotic expression exists is detected. Specifically, each of the 12-well plates was filled with 1 × 106HEK293t cells were transfected overnight with 3ul Lipofectamine 2000(Thermo Scientific) per well in serum free medium and cell samples were harvested 4h later by culturing in DMEM + 10% FBS for 48h and using anti-Ag85B antibody for western blot detection.
As shown in fig. 2, Lane 1: protein Ag85B control, Lane 2: transfection of pVAX1 vector, Lane 3: b21 transfection, Lane: b31 transfection. The results show that: 48h after transfection, the recombinant B31 DNA vaccine expresses fusion protein Ag85B-Rv3425-Rv1813c in eukaryotic cells, the recombinant B21 DNA vaccine expresses fusion protein Ag85B-Rv2029c-Rv1738 in eukaryotic cells, and the unloaded group has no band at the corresponding position.
Example 2 cellular level immunization of vaccines and determination of immunological indices
Transfecting Raw264.7 cells by DNA vaccine plasmids, collecting culture supernatant and cell samples, and detecting related immunity indexes. Specifically, each well of a 24-well plate is filled with 2 × 105Raw264.7 cells were transfected overnight with 1. mu.l jet OPTIMUS (polypuls) per well in serum-free medium and cell supernatants were harvested 4h later by DMEM + 10% FBS for 24h, 48h, 72 h.
The treated cell supernatants were collected and assayed using the Mouse IL-6ELISA Kit, Mouse TNF-. alpha.ELISA Kit (Dake) as indicated in the Kit. As shown in FIG. 3, it was found by comparison that, after transfection, the DNA vaccine B21 of the present invention can induce macrophage to secrete IL-6 and TNF-alpha more effectively than the DNA vaccine B31, and the secretion of TNF-alpha is increased significantly compared with the IL-6 of PBS control group.
Example 3 vaccine animals level immune index and protection level
After C57BL/6 female mice with 6-8 weeks of age are immunized by the recombinant vaccine, the immunization is carried out once every two weeks for three times. Two weeks after completion of immunization, blood was collected from the orbit and red blood cells were usedDetecting CD4 in peripheral blood after cracking red of lysate+，CD8+Proportion of T lymphocytes, as shown in fig. 4, flow staining results indicate: the recombinant DNA vaccine B21 can promote CD4 in peripheral blood of mice+And CD8+The T cell ratio increased and was significantly different from the PBS control group.
After 18 weeks of immunization, tail vein injection of BCG 5X 10 was used6CFU/mouse, dissect after 4 weeks. Splenic lymphocytes were isolated and spiked with 5. mu.g/ml Ag85B, Rv2029c, Rv1738 proteins, respectively, and the cell supernatants were assayed for secretion levels of IL-2, IFN-. gamma.and TNF-. alpha.after 36h (FIG. 5 a). The results showed that the cytokine secretion level of the DNA vaccine B21 group was significantly increased compared to the PBS group. The lung and spleen CFU counts were performed simultaneously (fig. 5B), and the DNA vaccine B21 group significantly reduced the lung and spleen bacterial load compared to the PBS group. And FIG. 5 shows that the spleen lymphocytes in the group B21 of the DNA vaccine secrete cytokines higher than that in the group B31, and the bacterial load of the lung and the spleen is lower than that in the group B31.
Claims (6)
1. A recombinant DNA vaccine of mycobacterium tuberculosis is characterized in that the recombinant DNA vaccine is obtained by recombining genes Ag85B, Rv2029c and Rv1738 into a eukaryotic expression vector.
2. The recombinant DNA vaccine of claim 1, wherein the expression plasmid is pVAX1 and the recombinant vaccine is Ag85B-Rv2029c-Rv1738-pVAX 1.
3. A method for preparing a recombinant DNA vaccine of Mycobacterium tuberculosis as described in claim 1, comprising the steps of:
(1) amplifying Ag85B, Rv2029c and Rv1738 genes;
(2) performing double digestion on the Ag85B gene and the expression plasmid respectively, and performing gel recovery and connection to obtain a recombinant plasmid;
(3) transforming, amplifying and expressing the recombinant plasmid obtained in the step (2);
(4) carrying out gel recovery and connection on the recombinant plasmid obtained in the step (3) and the Rv2029c gene respectively to form a recombinant plasmid;
(5) transforming, amplifying and expressing the recombinant plasmid obtained in the step (4);
(6) carrying out gel recovery and connection on the recombinant plasmid obtained in the step (5) and the Rv1738 gene respectively to form a recombinant plasmid;
(7) and (4) transforming, amplifying and expressing the recombinant plasmid obtained in the step (6) to obtain the recombinant DNA vaccine.
4. The method according to claim 3, wherein in the step (2), the expression plasmid is pVAX 1.
5. The process according to claim 3, wherein in the step (2), the enzymes used for the double digestion are NheI and HindIII; in the step (4), the enzymes used in the double enzyme digestion are HindIII and EcoR 1; in step (6), the enzymes used for the double digestion were EcoR1 and Not 1.
6. The method according to claim 3, wherein in the steps (3), (5) and (7), the recombinant plasmid is transformed, amplified and expressed in E.coli.
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| CN113930450A (en) * | 2021-09-27 | 2022-01-14 | 复旦大学 | Optimized recombinant DNA vaccine of tubercle bacillus and preparation method thereof |
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| US20110189208A1 (en) * | 2008-09-24 | 2011-08-04 | Andrew William Heath | Tb vaccine |
| CN111655281A (en) * | 2018-01-26 | 2020-09-11 | 加利福尼亚大学董事会 | Intranasal delivery of vaccines containing cyclodipeptide adjuvants for tuberculosis |
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| US20110189208A1 (en) * | 2008-09-24 | 2011-08-04 | Andrew William Heath | Tb vaccine |
| CN111655281A (en) * | 2018-01-26 | 2020-09-11 | 加利福尼亚大学董事会 | Intranasal delivery of vaccines containing cyclodipeptide adjuvants for tuberculosis |
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| CN113930450A (en) * | 2021-09-27 | 2022-01-14 | 复旦大学 | Optimized recombinant DNA vaccine of tubercle bacillus and preparation method thereof |
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