XJ-160病毒复制子型表达载体的构建

Construction of Replicon Expression Vector Derived from XJ-160 Virus

XJ-160病毒是我国首次分离的辛德毕斯病毒,全基因组测序已经完成。本文利用该病毒全基因序列首先构建了全基因组cDNA克隆质粒,在此基础上,利用基因重组技术将病毒结构基因序列替换为含有多个单酶切位点的序列,得到复制子表达载体质粒pRepxj160。为验证载体的功能,将报告基因绿色荧光蛋白(EGFP)和β-半乳糖苷酶基因(lacZ)分别插入到载体的多克隆位点,得到两个表达质粒;经体外转录获得的转录体RNA转染BHK-21细胞后14h,可检测到报告基因的表达。结果表明我们构建的XJ-160病毒复制子型表达载体具有自主复制功能,可以表达异源基因。本研究为进一步开发具有我国自主知识产权的甲病毒载体奠定了基础。

XJ-160 is the first Chinese isolate of a Sindbis-like virus, and its genome was completely sequenced in our previous work. In this investigation, a full-length genomic cDNA clone was firstly constructed from RT-PCR products of the viral RNA. Then, the plasmid of replicon expression vector (pRepxj160) was derived from this cDNA clone replacing viral structural sequence with multiclonal sequence by DNA recombination technique.To verify function of this vector, reporter genes, EGFP and lacZ, were cloned into this plasmid, respectively. And RNA transcripted from expression plasmid was inducted into BHK-21 cell line, resulting in expression of green fluorescent protein and β -galactosidase 14 hours later. The results indicated that the replicon vector derived from XJ-160 virus was self-replicating and that the following gene expression was efficient. Our study settled the basis for developing alphavirus vector system with Chinese intellectual property.