本文選題：乙型肝炎 + DNA疫苗��； 參考：《中國人民解放軍軍醫(yī)進修學(xué)院》2005年博士論文

【摘要】：慢性乙型肝炎(CHB)嚴重危害人類健康,目前尚無特效治療手段。乙型肝炎慢性化的主要原因之一是被感染者缺乏有效的特異性細胞免疫應(yīng)答,不能清除其體內(nèi)的乙型肝炎病毒(HBV)。DNA疫苗將編碼外源蛋白基因的質(zhì)粒DNA直接導(dǎo)入機體組織,外源基因在體細胞中表達后,表達產(chǎn)物被遞呈,與主要組織相容性復(fù)合物結(jié)合,可刺激機體產(chǎn)生相應(yīng)的細胞毒性T淋巴細胞(CTL)和抗體,介導(dǎo)細胞和體液免疫應(yīng)答。 乙肝DNA疫苗可誘導(dǎo)機體產(chǎn)生抗原特異性細胞及體液免疫,接種后可誘導(dǎo)多種實驗動物產(chǎn)生特異性CTL及保護性抗體,清除HBV轉(zhuǎn)基因鼠血清HBsAg及HBV DNA,接種于對重組亞單位疫苗無應(yīng)答的健康志愿者可產(chǎn)生保護性抗體。由于可誘導(dǎo)特異性細胞免疫,乙肝DNA疫苗有可能抑制或清除CHB患者體內(nèi)的HBV,但要作為臨床治療CHB的工具尚需解決許多難題,主要是確保安全性并增強免疫效果。既往的研究大多通過聯(lián)合使用免疫佐劑、細胞因子及采用不同的接種方法增強疫苗的免疫原性。本研究在乙肝DNA疫苗中插入HBV自身復(fù)制調(diào)控元件—增強子(ENH)及前S2(PreS2)、前C(PreC)抗原基因片段,通過基因調(diào)控的方法增強乙肝DNA疫苗目的基因的表達以增強DNA疫苗的免疫原性,并采用FDA已批準用于臨床試驗的載體VR1012構(gòu)建疫苗,所構(gòu)建的疫苗確保安全性,可進入臨床試驗進行深入研究。 本研究采用常規(guī)PCR法從adr亞型HBV全基因DNA序列中分別擴增HBsAg,PreS2-HBsAg,HBsAg—ENH Ⅰ,PreS2-HBsAg-ENH Ⅰ,HBcAg和ENH Ⅱ-PreC-HBcAg基因片段,重組到載體VR1012中,構(gòu)建6種HBV重組質(zhì)粒(重組質(zhì)粒是乙肝DNA疫苗的主體),轉(zhuǎn)染HepG2細胞及COS-7細胞,免疫BALB/C小鼠及HBV轉(zhuǎn)基因鼠。采用細胞內(nèi)酶免疫染色、ELISA、ELISPOT等方法檢測其在HepG2細胞、COS-7細胞內(nèi)的表達,BALB/C小鼠的體液、細胞免疫及HBV轉(zhuǎn)基因鼠血清HBsAg、HBV DNA及肝組織病理改變。發(fā)現(xiàn)轉(zhuǎn)染的HepG2細胞、COS-7細胞均表達相應(yīng)的目的蛋白—
[Abstract]:Chronic hepatitis B (CHB) is a serious hazard to human health. One of the main causes of chronic hepatitis B infection is the lack of specific cellular immune response in infected patients, and the hepatitis B virus (HBV) virus DNA vaccine can not be removed directly into the tissues of the body by introducing the plasmid DNA encoding foreign protein gene into the body tissue. After the expression of exogenous gene in somatic cells, the expressed product was presented and combined with the major histocompatibility complex, which could stimulate the production of cytotoxic T lymphocyte (CTL) and antibodies, and mediate cellular and humoral immune responses. Hepatitis B DNA vaccine can induce antigen-specific cells and humoral immunity, and induce many experimental animals to produce specific CTL and protective antibodies after inoculation. The serum HBsAg and HBV DNA of HBV transgenic mice were cleared, and protective antibodies were produced in healthy volunteers who did not respond to the recombinant subunit vaccine. Hepatitis B DNA vaccine may inhibit or eliminate HBV in patients with CHB due to its ability to induce specific cellular immunity. However, as a tool for clinical treatment of CHB, many problems need to be solved, mainly to ensure safety and enhance the immune response. Previous studies have mostly enhanced the immunogenicity of vaccines by combined use of immune adjuvants, cytokines and different inoculation methods. In order to enhance the immunogenicity of hepatitis B DNA vaccine, we inserted HBV self-replicating regulatory element (Enh) and pre-S _ 2 PreS _ (2) and pre-C _ (2) prec _ (2) gene fragments into hepatitis B DNA vaccine, and enhanced the expression of target gene of DNA vaccine by means of gene regulation, so as to enhance the immunogenicity of DNA vaccine. The vaccine was constructed with VR1012, which has been approved by FDA for clinical trials. The constructed vaccine ensures safety and can be further studied in clinical trials. In this study, HBcAg and ENH 鈪，

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