Example 1 Preparation of therapeutic HBV microcircle DNA vaccine
 It includes the following steps:
 1. Primer design and PCR amplification of the target fragment
 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:
 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.
 PCR amplification program: 95°C 5min; 95°C 50s, 55°C 50s, 72°C 60s, total 30 cycles; 72°C 5min.
 2. Construction of the parental plasmid
 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.
 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.
 3. Recombination of parental plasmid into minicircle DNA
 (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.
 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.
 (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.
 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.
 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.
 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.
 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.
 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.