A recombinant DNA vaccine of Mycobacterium tuberculosis and preparation method thereof

Abstract

The present invention discloses a recombinant DNA vaccine of Mycobacterium tuberculosis and a preparation method thereof. The recombinant DNA vaccine of the present invention is obtained by recombining genes Ag85B, Rv2029c and Rv1738 into a eukaryotic expression vector. The present invention has found that after immunizing 6-8 week old C57BL / 6 female mice, the DNA vaccine Ag85B-Rv2029c-Rv1738-pVAX1 can significantly increase the number of CD4 + , CD8 + The proportion of T lymphocytes was increased, the secretion of antigen-specific Th1 cytokines IL-2, IFN-γ, and TNF-α of mouse spleen lymphocytes was increased, and the BCG bacterial counts in the lungs and spleens of mice were reduced, indicating that the recombinant DNA vaccine can effectively control the growth and reproduction of mycobacteria in mice. Therefore, the recombinant DNA vaccine of the present invention can be used for the prevention and treatment of Mycobacterium tuberculosis infection.

Description

[0001] This application is a divisional application with an application date of September 29, 2020, application number CN202011047690.8, and invention name “A recombinant DNA vaccine for Mycobacterium tuberculosis and its preparation method”. Technical Field

[0002] The present invention relates to the technical field of gene engineering and recombinant DNA vaccine development, and in particular to a recombinant DNA vaccine of Mycobacterium tuberculosis and a preparation method thereof. Background Art

[0003] Tuberculosis (TB) is a serious respiratory infectious disease caused by Mycobacterium tuberculosis (MTB). Although various types of new vaccines have been widely studied, including recombinant BCG vaccine, nutritional deficiency Mycobacterium tuberculosis vaccine, protein peptide vaccine, DNA vaccine, virus-based tuberculosis subunit vaccine, etc., the existing vaccines have more or less defects. For example, although BCG can effectively prevent miliary tuberculosis and tuberculous meningitis. However, its protective effect on adults is limited, and revaccination is ineffective. Moreover, BCG is a preventive vaccine and cannot provide effective protection for individuals with latent infection. Therefore, the development of a new tuberculosis vaccine is imminent. Summary of the invention

[0004] The purpose of the present invention is to provide a recombinant DNA vaccine of Mycobacterium tuberculosis and a preparation method thereof. The vaccine can supplement the performance of commercially available BCG vaccine and control latent tuberculosis infection. It is expected to be used as a vaccine for booster immunization after initial BCG immunization; a therapeutic vaccine for controlling latent infection; and a therapeutic vaccine for controlling tuberculosis in combination with drugs.

[0005] In the present invention, complete Ag85B, Rv2029c and Rv1738 in the standard strain H37Rv are selected and connected to the expression vector by enzyme digestion; wherein, the recombinant Rv1738 (285bp, 1965657..1965941) and 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 period, so these genes can be used as candidate genes for vaccines, and the present invention recombines the above genes into the vector pVAX1 as a DNA vaccine. The technical scheme of the present invention is specifically described as follows.

[0006] A recombinant DNA vaccine for Mycobacterium tuberculosis is obtained by recombining genes Ag85B, Rv2029c and Rv1738 into an expression plasmid. Preferably, the expression plasmid is pVAX1, and the recombinant vaccine is Ag85B-Rv2029c-Rv1738-pVAX1 (abbreviated as B21).

[0007] The present invention further provides a method for preparing the above-mentioned recombinant DNA vaccine of Mycobacterium tuberculosis, comprising the following steps:

[0008] (1) Amplification of Ag85B, Rv2029c and Rv1738 genes;

[0009] (2) digesting the Ag85B gene and expression plasmid with double enzymes, respectively, and recovering and connecting them by gel extraction to obtain the recombinant plasmid;

[0010] (3) transforming, amplifying and expressing the recombinant plasmid obtained in step (2);

[0011] (4) using the recombinant plasmid obtained in step (3) and the Rv2029c gene to form a recombinant plasmid by gel recovery and ligation;

[0012] (5) transforming, amplifying and expressing the recombinant plasmid obtained in step (4);

[0013] (6) using the recombinant plasmid obtained in step (5) and the Rv1738 gene to form a recombinant plasmid by gel recovery and ligation;

[0014] (7) Transforming, amplifying and expressing the recombinant plasmid obtained in step (6) to obtain a recombinant DNA vaccine.

[0015] Preferably, in step (2), the expression plasmid is pVAX1.

[0016] Preferably, in step (2), the enzymes used for double digestion are Nhe I and Hind III; in step (4), the enzymes used for double digestion are Hind III and Eco RI; in step (6), the enzymes used for double digestion are Eco RI and Not I.

[0017] Preferably, in step (3), step (5) and step (7), the recombinant plasmid is transformed, amplified and expressed in Escherichia coli.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The vaccine of the present invention uses the nucleic acid vaccine vector pVAX1 approved by the US FDA, which can express proteins in mammalian cells.

[0020] The DNA vaccine Ag85B-Rv2029c-Rv1738-pVAX1 uses the nucleic acid vaccine vector pVAX1 to fuse and express the Mycobacterium tuberculosis antigens Ag85B, Rv2029c and Rv1738. After the vaccine is transfected into macrophages, the fusion protein Ag85B-Rv2029c-Rv1738 can be expressed in macrophages, effectively activate macrophages, and increase the secretion levels of IL-6 and TNF-α.

[0021] The present invention found that after immunization of 6-8 week old C57BL / 6 female mice for 2 weeks, the DNA vaccine Ag85B-Rv2029c-Rv1738-pVAX1 can significantly increase the number of CD4 + , CD8 + The proportion of T lymphocytes, CD4 + , CD8 + T lymphocytes play an important role in the anti-tuberculosis immune process.

[0022] The present invention found that 18 weeks after immunization of 6-8 week old C57BL / 6 female mice, 5×10 6 Four weeks after CFU BCG / mouse infection, the secretion of TH1 cytokines IL-2, IFN-γ, and TNF-α by splenic lymphocytes increased after stimulation with Ag85B, Rv2029c, and Rv1738 specific antigens, and the BCG counts in the lungs and spleens of mice in the vaccine immunization group were significantly reduced, indicating that DNA vaccines can effectively control the growth and reproduction of Mycobacterium tuberculosis and other bacteria in mice, and can be used for the prevention and treatment of Mycobacterium tuberculosis infection.

[0023] The present invention used DNA vaccine Ag85B-Rv3425-Rv1813c-pVAX1 (B31) as a control and found that the DNA vaccine (B21) of the present invention can more effectively activate macrophages and enhance TH1 type cellular immune response compared with B31. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 To verify the construction of the recombinant DNA vaccine by enzyme digestion, Lane 1: DNAMarker, Lane 2: control vector pVAX1, Lane 3: recombinant Ag85B-Rv2029c-Rv1738-pVAX1, Lane 4: recombinant Ag85B-Rv3425-Rv1813-pVAX1;

[0025] Figure 2 It is the expression of recombinant B21 DNA and B31 DNA vaccines in eukaryotic cells, among which Lane 1: protein Ag85B control, Lane 2: pVAX1 vector transfection, Lane 3: B21 transfection, Lane: B31 transfection;

[0026] Figure 3 The results of IL-6 (a) and TNF-α (b) secretion after recombinant B21 DNA vaccine transfected Raw264.7 cells;

[0027] Figure 4 This is the flow cytometry staining result of the recombinant B21 DNA vaccine;

[0028] Figure 5 (a) shows the results of IL-2, IFN-γ, and TNF-α content detection in the cell supernatant after mice were challenged with BCG; (b) shows the results of CFU counts in the lungs and spleen after mice were challenged with BCG. DETAILED DESCRIPTION

[0029] The reagents and conditions not specifically marked in the present invention all adopt conventional products and methods well known to those of ordinary skill in the art.

[0030] Example 1

[0031] Preparation of recombinant DNA vaccines

[0032] 1.1 Preparation of genomic DNA of Mycobacterium tuberculosis H37Rv strain

[0033] Mycobacterium tuberculosis (H37Rv) was cultured in 7H9 Broth medium for 4 weeks and inactivated at 80℃ for 2h. Genomic DNA was extracted using a bacterial DNA (small amount) extraction kit. Due to the thick wall of tuberculosis bacteria, the bacterial digestion time was extended to 3 to 5h.

[0034] 1.2 Amplification of the genes of Mycobacterium tuberculosis H37Rv strain Ag85B, Rv2029c, and Rv1738.

[0035] 1) The specific primer designs are as follows:

[0036] Ag85B-S (SEQ ID NO. 1): 5'-AAAGCTAGCGCCACCATGGCATTCT CCCGGCCGGGGCT-3', restriction site: NheⅠ

[0037] Ag85B-A (SEQ ID NO. 2): 5'-TTTAAGCTTGCCGGCGCCTAACGAA CTCT-3', restriction site: HindⅢ

[0038] Rv2029c-S (SEQ ID NO. 3): 5'-TTTAAGCTTATGACGGAGCCAGCG GCGT-3', restriction site: HindⅢ

[0039] Rv2029c-A (SEQ ID NO. 4): 5'-AAAGAATTCTGGCGAGGCTTCCGG GTTAACGA-3', restriction site: EcoRI

[0040] Rv1738-S (SEQ ID NO. 5): 5'-AAAGAATTCATGTGCGGCGACCAGT-3', restriction site: EcoRI

[0041] Rv1738-A (SEQ ID NO.6): 5'-AAAGCGGCCGCCTACTATCAATACA ACAATCGCGCCGG-3', restriction site: NotⅠ

[0042] 2) PCR reaction system (takaraPCR reagent):

[0043]

[0044] 3) PCR reaction conditions

[0045]

[0046] The product was identified by 1% agarose gel electrophoresis.

[0047] 1.3 Enzyme digestion, ligation and transformation of vectors pVAX1 and Ag85B

[0048] The PCR products of vector pVAX1 and Ag85B were double-digested with NheⅠ and HindⅢ at 37℃ for 3h. The digested products were purified with a gel recovery kit. T4 DNA ligase was used to ligate overnight at 4℃. The E. coli DH5α strain was transformed, and the clones were selected to extract the plasmids for sequencing verification.

[0049] The above-mentioned successfully constructed vector and PCR product of Rv2029c were double-digested with HindⅢ and EcoRI at 37℃ for 3h. The digested product was purified with a gel recovery kit. T4 DNA ligase was used to connect overnight at 4℃. The E. coli DH5α strain was transformed, and the clones were selected to extract the plasmid for sequencing verification.

[0050] The above-mentioned successfully constructed vector and the PCR product of Rv1738 were double-digested with EcoRⅠ and NotⅠ at 37℃ for 3h. The digested product was purified with a gel recovery kit. T4 DNA ligase was used to connect overnight at 4℃. The E. coli DH5α strain was transformed, and the clones were picked and the plasmids were sequenced and verified. At this point, the recombinant DNA vaccine Ag85B-Rv2029c-Rv1738-pVAX1(B21) was constructed ( Figure 1 ).

[0051] The above method was used to construct Ag85B-Rv3425-Rv1813c-pVAX1 (B31).

[0052] The construction of the recombinant DNA vaccine was verified by enzyme digestion. Figure 1 As shown, Lane 1: DNA Marker, Lane 2: Control vector pVAX1, Lane 3: Recombinant Ag85B-Rv2029c-Rv1738-pVAX1, Lane 4: Recombinant Ag85B-Rv3425-Rv1813c-pVAX1. Use restriction endonucleases NheⅠ and NotⅠ for digestion. As can be seen from the figure, the recombinant plasmid B21 carries a 2163bp exogenous fragment, and the recombinant plasmid B31 carries a 1823bp fragment. This shows that the recombinant plasmids Ag85B-Rv2029c-Rv1738-pVAX1 and Ag85B-Rv3425-Rv1813c-pVAX1 were successfully constructed.

[0053] 1.4 Verification of recombinant DNA vaccine cell expression

[0054] HEK293t cells were transfected with the DNA vaccine plasmid constructed above, and cell samples were collected to detect whether eukaryotic expression was present. Specifically, 1×10 6 HEK293t cells were cultured overnight, and 2 μL of plasmid was transfected in each well using 3 μL Lipofectamine 2000 (Thermo Scientific) in serum-free medium. After 4 hours, DMEM + 10% FBS was used for 48 hours to collect cell samples, and anti-Ag85B antibodies were used for western blot detection.

[0055] like Figure 2 As shown, Lane 1: protein Ag85B control, Lane 2: pVAX1 vector transfection, Lane 3: B21 transfection, Lane: B31 transfection. The results showed that 48 hours after transfection, the recombinant B31 DNA vaccine expressed the fusion protein Ag85B-Rv3425-Rv1813c in eukaryotic cells, and the recombinant B21 DNA vaccine expressed the fusion protein Ag85B-Rv2029c-Rv1738 in eukaryotic cells, while the empty group had no bands at the corresponding positions.

[0056] Example 2

[0057] Vaccine cell-level immunity and determination of immune indicators

[0058] The DNA vaccine plasmid was transfected into Raw264.7 cells, and the culture supernatant and cell samples were collected to detect relevant immune indicators. Specifically, each well of a 24-well plate was filled with 2×10 5Raw264.7 cells were cultured overnight in serum-free medium and transfected with 1 μg of plasmid per well using 1 μL jet OPTIMUS (polypuls). After 4 hours, the cells were cultured in DMEM+10% FBS for 24 hours, 48 ​​hours, and 72 hours, and the cell supernatant was collected.

[0059] The treated cell supernatant was taken and tested using Mouse IL-6 ELISA Kit and Mouse TNF-α ELISA Kit (Dakoway) according to the instructions in the kit. Figure 3 As shown, after comparison, it was found that after transfection, the DNA vaccine B21 of the present invention can more effectively induce macrophages to secrete IL-6 and TNF-α than the DNA vaccine B31, and the secretion of IL-6 and TNF-α increased significantly compared with the PBS control group.

[0060] Example 3

[0061] Vaccine animal level immune indicators and protection level

[0062] The recombinant vaccine was used to immunize 6-8 week old C57BL / 6 female mice, and then immunized once every two weeks for a total of three times. Two weeks after the immunization, blood was collected from the eye sockets, and the CD4 + , CD8 + The proportion of T lymphocytes, such as Figure 4 As shown in the flow cytometry results, the recombinant DNA vaccine B21 can promote the expression of CD4 + and CD8 + The proportion of T cells increased, which was significantly different from the PBS control group.

[0063] Eighteen weeks after the immunization, BCG 5×10 6 CFU / mouse, dissected after 4 weeks. Splenic lymphocytes were isolated and stimulated with 5μg / ml Ag85B, Rv2029c, and Rv1738 proteins, respectively. After 36h, the secretion levels of IL-2, IFN-γ, and TNF-α in the cell supernatant were detected ( Figure 5 a). The results showed that the cytokine secretion level of the DNA vaccine B21 group was significantly higher than that of the PBS group. At the same time, lung and spleen CFU counts were performed ( Figure 5 b) Compared with the PBS group, the DNA vaccine B21 group significantly reduced the bacterial load in the lungs and spleen. Figure 5 The results showed that the cytokine secretion of spleen lymphocytes in DNA vaccine B21 group was higher than that in B31 group, and the bacterial load in lung and spleen was lower than that in B31 group.

[0064] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of Ag85B gene, Rv2029c gene and Rv1738 gene in the preparation of recombinant DNA vaccine for the treatment of tuberculosis; The recombinant DNA vaccine is obtained by recombining the Ag85B gene, the Rv2029c gene and the Rv1738 gene into a eukaryotic expression vector; the eukaryotic expression vector is pVAX1, and the recombinant DNA vaccine is Ag85B-Rv2029c-Rv1738-pVAX1; The tuberculosis is latent tuberculosis.

2. The use according to claim 1, It is characterized in that The method for preparing the recombinant DNA vaccine comprises the following steps: (1) Amplification of Ag85B, Rv2029c and Rv1738 genes; (2) The Ag85B gene and expression plasmid were digested with double enzymes, respectively, and the recombinant plasmid was obtained by gel recovery and ligation; (3) transforming, amplifying and expressing the recombinant plasmid obtained in step (2); (4) using the recombinant plasmid obtained in step (3) and the Rv2029c gene to separate and gel-recover and connect to form a recombinant plasmid; (5) transforming, amplifying and expressing the recombinant plasmid obtained in step (4); (6) Respectively use the recombinant plasmid and Rv1738 gene obtained in the double enzyme digestion step (5) to gel-recover and connect to form a recombinant plasmid; (7) Transforming, amplifying and expressing the recombinant plasmid obtained in step (6) to obtain a recombinant DNA vaccine.

3. The use according to claim 2, It is characterized in that In step (2), the enzymes used for double digestion are NheⅠ and HindⅢ; in step (4), the enzymes used for double digestion are HindⅢ and EcoRⅠ; in step (6), the enzymes used for double digestion are EcoRⅠ and NotⅠ.

4. The use according to claim 2, It is characterized in that In step (3), step (5) and step (7), the recombinant plasmid is transformed, amplified and expressed in Escherichia coli.

Images