狂犬病毒糖蛋白DNA疫苗的研制及其免疫效果的观察

构建了含有狂犬病毒（RV）CVS株糖蛋白（GP）基因的重组质粒pCMVCVSRG，将其转染至鼠NIH3T3细胞中，用间接免疫荧光法和APAAP法均证实RVGP能在真核细胞中表达。分别将合RV不同毒株的GP基因的质粒（DNA疫苗）及空白载体质粒（对照组）免疫小鼠，仅DNA疫苗免疫的小鼠产生了中和抗体。以RV攻击后，DNA疫苗免疫组小鼠的存活率与对照组相比，差异有极显著性意义（P＜0．01）；不同的启动子（CMV或SV40）与不同GP基因（来源于CVS株或ERA株）对DNA疫苗的免疫效果无明显影响。在注射120d后．用PCR方法仍可检测出RVGP基因。结果表明：狂犬病DNA疫苗能够诱生低水平的中和抗体和记忆性B淋巴细胞，并能保护小鼠抵抗RV的攻击。该疫苗能在体内稳定存在。狂犬病DNA疫苗的研制为狂犬病免疫开辟了一条新途径，并可为防治其他疾病的DNA疫苗的研制奠定基础。

Research on DNA Vaccine Carrying the Rabies Virus Glycoprotein Gene and its Immune Protection Effects

The plasmid p CMVCVSRG containing the full- length cDNA of G protein of rabies virus CVS strain was constructed and transiently transfected into NIH 3T3 cell. The expression of Gprotein in transfected NIH 3T3 was confirmed by indirect immunfluorescent(IIF) and alkaline phosphatase anti-alkaline phosphatase (APAAP ) methods. The mice were inoculated withpCMVCVSRG, pSV2X3CVSRG, pCMV, pSV2X3, respectively. Only mice immuned with the G gene-containing vectors developed neutralizing antibody to rabies virus. After rabies virus challenge, the survival rates of DNA vaccine inoculated group have very significant difference comparing with negative control group (p < 0.01 ). The immune responses to the DNA vaccines containing different promoters(from CMV or SV40) and different G protein genes(from CVS strain or ERA strain) were compared and it was found to be similar in magnitude. The specific glycoprotein gene was detectable by PCR at least 120 days postinjection. Our data shows that rabies DNA vaccine can induce low level rabies virus-neutralizing antibody, memory B lymphocyte and result in protection against a subsequent challenge of rabies virus in mice. Our research works also provide a new approach to develop immunization of rabies virus.