人巨细胞病毒糖蛋白gB基因重组腺病毒载体的构建及包装

目的构建携带有人巨细胞病毒(HCMV)糖蛋白gB基因的重组腺病毒载体,并在HEK293细胞中进行包装。方法将gB基因克隆至穿梭质粒Track-CMV,构建重组质粒Track-CMV/gB,亚克隆至转移载体pAD-Easy-1,构建重组质粒pAD-Easy-1/gB,将该重组骨架质粒转染HEK293细胞,利用HEK293细胞产生重组腺病毒。空斑法及PCR法挑选重组病毒,荧光显微镜观察标志蛋白(绿色荧光蛋白)的表达,利用噬斑法检测病毒滴度。结果重组腺病毒载体经酶切鉴定证明构建正确,转染重组腺病毒载体的HEK293细胞经PCR扩增,可见约700bp的目的基因片段,荧光显微镜观察可见绿色荧光蛋白表达,空斑试验检测病毒滴度为2×106PFU/ml。结论已成功构建了含有gB基因的重组腺病毒载体,为进一步研制重组腺病毒载体HCMV疫苗提供了条件。

Construction and Packaging of Recombinant Adenovirus Vector Carrying gB Gene of Human Cytomegalovirus

Objective To construct the recombinant adenovirus vector carrying gB gene of human cytomegalovirus (HCMV) for packaging in HEK293 cells. Methods Clone gB gene to shuttle plasmid Track-CMV, then co-transform adenovirus skeleton plasmid pAD-Easy-1 and the constructed recombinant plasmid Track-CMV / gB to E. coli BJ5183 cells. Screen positive clones, extract recombinant plasmid pAD-Easy-1 / gB and transfect to HEK293 cells. Recombinant adenovirus was screened by plaque formation test and PCR, observed for the expression of green fluorescence protein (GFP) as marker by fluorescent microscopy, and determined for titer by plaque assay. Results Restriction analysis proved that recombinant adenovirus vector was constructed correctly. The target gene fragment at a length of about 700 bp was amplified by PCR from the HEK293 cells transfected with pAD-Easy-1 / gB. Fluorescent microscopy proved the expression of GFP. The titer of recombinant adenovirus was 2 × 106 PFU / ml. Conclusion The recombinant adenovirus carrying gB gene of HCMV was successfully constructed, which provided a basis for further preparation of recombinant adenovirus-based HCMV vaccine.