HIV-1 B'亚型gag-env融合基因的DNA疫苗构建和免疫原性分析

目的 构建HIV-1 B′亚型中国流行株gag和env融合基因的DNA疫苗，对其免疫原性进行研究。方法 根据已报道的HIV-1 B′亚型RIA2分离株gag和env基因的氨基酸序列按哺乳动物密码子使用频率进行优化并人工合成基因，插入真核表达载体pDRVISV1．0中，构建表达RL42 gag-env，融合蛋白的DNA疫苗，pSVRL／GE。用Western blot和抗gag p55抗体细胞内染色的方法体外检测pSVRUGE的表达效率。DNA疫苗pSVRL/GE免疫BALB／c小鼠后，用ELISPOT检测小鼠的细胞免疫反应。结果 限制性内切酶鉴定表明融合基因已成功插入pDRVISV1．0载体中，Western blot证实融合基因可有效表达融合蛋白；细胞内染色结果表明，pSVRL/GE转染的293T细胞中49．8％表达gag p55，荧光强度均值为924；而空载体pDRVISV1．0转染的293T细胞中非特异背景染色只有0．5％。经免疫的小鼠脾细胞体外用H-2^d限制性表位肽AMQMIKET刺激后，ELISPOT检测显示，pSVRUGE免疫小鼠每10^6脾细胞形成226个斑点（SD=140），而空载对照组每1驴脾细胞形成29个斑点（SD=16）（P〈0．05）。结论 所构建的DNA疫苗pSVRL/GE可高效表达相应抗原蛋白，并可有效激活机体的细胞免疫反应。

Immunogenicity of a new DNA vaccine against HIV-1 B' clade

Objective To construct a DNA vaccine against gag and em gene of Thailand B clade (B') and to study its immunogenicity. Methods A DNA vaccine, pSVRL/GE, was constructed by inserting a synthetic fusion gene, which were humanized and synthesized according to the amino acid sequence of the gag and env genes of HTV-1 B' isolate RL42, into plasmid vector pDRVISV1.0. Western blot and intracellular anti-gag p55 staining were used to determine the expression efficacy in vitro . BALB/c mice were immunized with the DNA vaccine and thereafter T cell immune responses were determined with EIISPOT. Results Enzyme-restricted digestion showed that the fusion gene was successfully inserted into vector pDRVISV1.0. Fluorescence-activated cell sorter ( FACS) analysis after intracellular staining displayed 49.8 % pSVRL/GE-transfected 293T cells was gag p55 positive with a mean fluorescence intensity(MFI) of 924, whereas the controls only showed 0.5% positive cells with a mean MFI of 78.67. Immunization with DNA vaccine pSVRL/GE(n=5) stimulated 226(SD=140) spot forming cells(SFCs) per 106 splenocytes targeting at the H-2d restricted epitope AMQMLKET, whereas the control group only showed 29(SD = 16) SFCs per 106 splenocytes(P<0.05) . Conclusion The DNA vaccine pSVRL/GE is able to efficiently expresse its inserted fusion gene and induce vigrous cellular immune responses.