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Research of DC-targed Therapeutic Vaccine for HBV

Author: ZhangPing
Tutor: WuZuZe;WangLiSheng
School: PLA Military Academy of Medical Sciences
Course: Pathology and Pathophysiology
Keywords: therapeutic vaccine HBV immune
CLC: R392
Type: PhD thesis
Year: 2009
Downloads: 282
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Abstract


Viral hepatitis can be seen as an acquired single gene inheritance disease, so it can be treated with gene therapy technique. We constructed DC-targed therapeutic multi-epitope DNA vaccine for HBV, based on the T and B cell epitope for HBV which have been tested active by now, and HTL(T helper lymphocyte) epitope-PADRE, etc. In addition, we added GM-CSF as adjuvant to enhance immune response. We combined fusion protein with CD40L to facilitate viral antigen peptide target DC through CD40-CD40L interaction.Firstly, by using gene synthesize method, we got the aim gene IL-2 signal peptide, HTL peptide(PADRE), CTL peptide HBcAg18-27(HBV special),PreS B peptide and linker which contained isoleucine zipper. Mouse CD40L was got by RT-PCR.Then fusion protein gene HBV-IZ-CD40L was got through connecting these aim genes orderly with PCR method. At last, we cloned HBV-IZ-mCD40L and GM-CSF to pUDK vector, got the recombined plasmid pUDK-HmL-IR-GM.Restriction endonuclease mapping analysis and sequencing proved that the sequence of fusion protein gene, internal ribosome entry site and adapter were correct. Western blot and ELISA analysis proved that the aim genes can express and secrete normally, which suggested that the combinant plasmid constructed successfully.Then normal Balb/c mice and HBV transgenic mice (HBV-tg-m) were immnued successively with the recombinant plasmid using needleless injection machine, immuned one time every 2 weeks,4 times all together. HBV-specific antibody was detected by ELISA, which evaluate the humoral immunity. Lymphocyte proliferation was analyzed with MTT, CTL lethality was detected by LDH release method, cytokines secretion were detected using ELISA, which evaluate the cellular immunity. The HBV DNA copies in HBV transgenic mice serum were deceted by real-time PCR.The recombinant plasmid can induce humoral immunity and cellular immunity by polarize TH1 type immunity and stimulate CD4+T lymphocyte proliferation. Although the HBV DNA copies did not decreased, the level of HBeAg in Tg mice serum decreased after immune.Otherwise, HE staining of live tissues showed that there were obviously inflammatory cells infiltration and hepatocyte cell swelling and particulate degeneration.These results suggested that humoral and cellular immunity can be induced and immunological tolerance can be broken partly after the injection of DNA vaccine. All these provide experimental foundation for the further research of therapeutic HBV DNA vaccine.

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