Therapeutic HBV minicircle DNA vaccine and preparation method thereof

[0030] Example 1 Preparation of therapeutic HBV microcircle DNA vaccine

[0031] It includes the following steps:

[0032] 1. Primer design and PCR amplification of the target fragment

[0033] Based on recombinant plasmids preS2.S/pcDNA3.1 and hIL2-IFNγ/pcDNA3.1 (see: He Xiaoqian, Chen Guangming, Huang Ying, etc. for the construction method. Construction and identification of therapeutic double-plasmid HBV DNA vaccine. Medical Journal of Chinese People's Liberation Army, 2003 28(6):493-498). Primers are designed upstream of the enhancer of the target gene and downstream of polyA to amplify DNA fragments containing the target gene and its upstream and downstream enhancers, promoters and polyA regulatory sequences. The designed primer sequence is as follows:

[0034] F:5’-GAAGATCTGGCGGGTTGACATTGATTATTGACTAG-3’

[0035] R:5’-ACGCGTCGACCCATAGAGCCCACCGCAT-3’

[0036] Using plasmids preS2.S/pcDNA3.1 and hIL2-IFNγ/pcDNA3.1 as templates, respectively, perform the following PCR reactions: 25μl system contains 10×buffer 2.5μl, dNTP (2.5mmol/L) 2μl, upstream primer (10pM ) 0.5μl, downstream primer (10pM) 0.5μl, template 5μl, DNA polymerase (2.5U/μl) 0.4μl, double distilled water 14.2μl.

[0037] PCR amplification program: 95°C 5min; 95°C 50s, 55°C 50s, 72°C 60s, total 30 cycles; 72°C 5min.

[0038] 2. Construction of the parental plasmid

[0039] Agarose gel electrophoresis to detect the PCR amplification results, cut the target fragment band, use the gel recovery kit (Takara MinBEST Agarose Gel Extraction kit Ver3.0, D823A) to recover the target product, use BglII and SalI (US NEB company) Simultaneously double digestion of the target fragment and the ZY781 vector (see Chen ZY, He CY, Ehrhardt A, Kay MA.minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo.Mol Ther. 2003, 8(3 ): 495-500, gifted by Professor Chen Zhiying, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences). According to the conventional method, the target fragments were cloned into the multiple cloning sites of the vector ZY781, and the parent plasmids preS2.S/ZY781 and hIL2-IFNγ/ZY781 were obtained.

[0040] Transform E. coli ZYCY10P3S2T (see Mar A. Kay, Cheng-Yi He, Zhi-Ying Chen. A robust system for production of minicircle DNA vectors. Nature Biotechnology, (2010). doi:10.1038/nbt.1708, courtesy of the Chinese Academy of Sciences Shenzhen Gifted by Professor Chen Zhiying from Advanced Technology Research Institute), and carried out liquid culture amplification. After restriction enzyme digestion and sequencing to confirm that the inserted fragment sequence is correct, the following parental plasmid reconstitution into minicircle DNA is performed.

[0041] 3. Recombination of parental plasmid into minicircle DNA

[0042] (1) Inoculate the above-mentioned bacterial liquid into TB culture medium containing 50μg/ml kana at an inoculum of 0.25‰, and cultivate overnight at 37°C and 250rpm.

[0043] The preparation method of TB culture broth is as follows: Dissolve 12 g of peptone, 24 g of yeast extract and 4 ml of glycerin in 900 ml of water. After the components are dissolved, autoclave sterilize. Cool to 60℃, then add 100ml of 170mmol/L KH which is autoclaved or filtered with 0.22μm filter membrane 2 PO 4 And 0.72mol/L K 2 HPO 4 The mixture is ready.

[0044] (2) Take the overnight cultured Minicle Induction Mix and mix in equal volume, and react at 32℃ and 250rpm for 5h. If necessary, the reaction can be extended for 1-3h. After the reaction is over, centrifuge, and extract the plasmid with the plasmid extraction kit (Takara miniBEST Plasmid Purification kit ver 4.0, Code No. 9760), which is the minicircle DNA plasmid.

[0045] The preparation method of Minicle Induction Mix is ​​as follows: Add 4ml of 1M NaOH and 200μl of 20% arabinose to 100ml LB, mix and filter through 0.22μm filter membrane.

[0046] 4. Preparation of HBV minicircle DNA vaccine: strain the positive clones obtained in step 2 by fermentation and culture, and generate minicircle DNA according to the method of step 3, and finally extract the plasmid and purify it.

[0047] by figure 1 It can be seen that the minicircle plasmid pMCS2.S is reconstituted from the parent plasmid preS2.S/ZY781, the size has changed from about 6000bp to about 2500bp, and two specific bands appear after digestion, indicating that the parental plasmid is correctly identified by digestion. And successfully reorganized into a minicircle plasmid.

[0048] The sequencing shows that the target gene and its upstream enhancer, promoter and downstream polyA regulatory sequence have been cloned in the parental plasmid, and the sequence identification is 100% correct. The sequencing results are shown in SEQ ID No.1 and SEQ ID No.2.

[0049] See the flow chart for the construction of minicircle DNA plasmid pMCS2.S and pMCIIF figure 2 , See the electrophoresis results of each plasmid and plasmid digestion image 3.

[0050] Example 2 In vitro cell transfection and expression detection of therapeutic HBV minicircle DNA vaccine

[0051] Cultivate the COS-7 cell line according to the conventional method, digest the adherent cells with trypsin, and resuspend in RPMI1640 medium containing serum, press 3×10 5 Cells/well were seeded on a 6-well plate at 37℃, 5% CO 2 Incubate for 24h in an incubator. When the cell fusion degree reaches about 90-95%, take 4μg pMCS2.S, pMCIIF, preS2.S/pcDNA3.1, hIL2-IFNγ/pcDNA3.1 plasmids respectively, and refer to Invitrogen's liposome transfection reagent Lipofectamine TM2000 operating instructions For cell transfection, make two parallels for each plasmid, and set the cell group without plasmid transfection as the control. Continue to culture, and collect the supernatant after 24h, 48h, 72h, and use the hepatitis B virus surface antigen diagnostic kit (detected at 48h) and human IL2 and IFNγ detection kit to quantitatively detect the expression of the target gene (24h, 48h, Detected at 72h). The results of Table 1 and Table 2 show that the target product was not detected in the plasmid-free cell control group. The plasmids pMCS2.S and pMCIIF of the present invention can both express the target gene in COS-7 cells, and are higher than preS2.S/ The expression levels of pcDNA3.1 and hIL2-IFNγ/pcDNA3.1 plasmids are high ( Figure 4-5 ).

[0052] Table 1 The results of qualitative detection of preS2.S protein after pMCS2.S and preS2.S/pcDNA3.1 plasmids were transfected into COS-7 cells

[0053] Sample

[0054] Judgment criteria: COV: cut-off value reference value; NCx: average OD value of negative control; NCn: negative control OD value n=3; PC: positive control OD value; calculated NCx=(NC1+NC2+NC3)÷3, If NCx<0, then calculate as 0; calculate COV=NCx+0.100; when OD/COV≥1.0, it means HBsAg positive result; when OD/COV<1 means HBsAg negative result.

[0055] Table 2 Quantitative detection of IL2 and IFNγ expression levels after pMCIIF and hIL2+IFNγ/pcDNA3.1 plasmids were transfected into COS-7 cells

[0056]

[0057] Example 3 In vivo animal experiment and immune effect detection of therapeutic HBV microcircle DNA vaccine

[0058] Experimental animals, immunization dose, grouping and observation time: 40 healthy Balb/c mice, 18-20g, 4-6 weeks old, SPF grade, randomly divided into 5 groups, 8 mice in each group. The first group is a normal saline control group (100μl/only); the second group is pcDNA3.1/preS2.S plasmid (20μg/only); the third group is pcDNA3.1/preS2.S+pcDNA3.1/hIL2+ IFNγ (10μg+10μg/only) double plasmid; the fourth group is the microcircle vaccine pMCS2.S (20μg/only); the fifth group is the microcircle vaccine pMCS2.S+pMCIIF (10μg+10μg/only) . The mice were injected with bilateral tibialis anterior muscle with in vivo electrical pulse. At the 0th, 2nd, and 4th weeks of inoculation, blood was collected by retro-ocular venous plexus puncture, and the serum was separated by centrifugation at 3000 r/min for 10 min and stored at -20°C. After the last one-time ELISA to detect the level of anti-HBs, calculate the appearance of antibodies Number of positive mice. Four weeks after the inoculation, the mice were sacrificed and lymphocytes were isolated from the spleens for ELISPOT experiments.

[0059] Detection method: (1) Humoral immunity test: Take mouse serum strictly according to the instructions (Shanghai Industrial Kehua Biotechnology Co., Ltd.) for ELISA test; (2) Cellular immunity test: ① Cervical dislocation method kills the mice, puts 75 In a beaker containing 5% ethanol, the mouse body hair is completely moistened for about 3 minutes, then the mouse abdominal cavity is cut open, and the spleen is taken out; ②The separated spleen is placed on a 200-mesh screen with a sterile beaker (with culture droplets). ), use a grinding rod (which can be replaced by a glass syringe core) gently twist the spleen tissue, grind and squeeze out the spleen cells, take 5ml of serum-free 1640 culture medium (IC) and slowly buffer the cells into a beaker. ③Slowly add 5ml of spleen cell suspension to a 14ml centrifuge tube containing 2.5ml of lymphocyte separation solution, centrifuge at 2000-2500r/min for 20min, after collecting and separating lymphocytes, centrifuge to wash the cells twice, each time at 1500r/min for 6min ④Resuspend the separated spleen lymphocytes with an appropriate amount of complete culture medium containing 5% FCS-1640, and count the total number of living cells of separated lymphocytes in each mouse by trypan blue staining, and adjust the cell density to 3×10 5 /ml, 37℃, 5% CO 2 Cultivate in an incubator.

[0060] ELISPOT test: operate strictly according to the method of the BD company's kit instructions, and set the plant lectin (PHA) as the positive control. If the following conditions are met, the ELISPOT detection of HBsAg-specific Th1 cells is considered positive: ①Under certain cell density, the ratio of the same sample HBsAg and non-HBsAg stimulation of the same sample in the ELISPOT detection plate should be not less than 2 ②The density of lymphocytes in the immunization experiment group is 3×10 5 /Well detection, the ratio of HBsAg and non-HBsAg stimulated 3 re-well SFC should not be less than 5, as the positive evaluation standard of mouse specific cellular immunity, the positive rate of cellular immune response of HBV microcircle DNA vaccine immunization group should not be less than 5. Less than 60%.

[0061] The statistical results were processed, and the statistical significance of the mean and percentage difference between the groups were statistically using t-test and statistical summary table method respectively.

[0062] Serum-anti-HBs≥10mIU is considered as a collective protective effective concentration. The results are shown in Table 3. Figure 6-7. The serum anti-HBs concentration of mice in the saline group was less than 10mIU, and the number of anti-HBs mice was 0; preS2.S/pcDNA3.1 group, double plasmid preS2.S/pcDNA3.1+hIL2-IFNγ/pcDNA3.1 group and pMCS2 .S, pMCS2.S+pMCIIF mice can induce anti-HBs in 2 weeks, and produce stronger immune response after 4 weeks. The effect of pMCS2.S combined with adjuvant pMCIIF is more significant, with a positive rate of 100%; microcircle DNA The vaccine pMCS2.S+pMCIIF induced a higher amount of anti-HBs in mice than the double plasmid DNA vaccine pcDNA3.1/preS2.S+pcDNA3.1/hIL2-IFNγ.

[0063] Table 3 Humoral immunity and cellular immunity test results

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