摘要
目的 构建汉坦病毒浙 37(Z37)株包膜糖蛋白基因G1、G2真核表达质粒 ,并在真核细胞中表达。方法 根据Z37M基因序列设计 6条引物 ,分别以质粒pGEMZ37、pCUMZ37为模板 ,通过聚合酶链反应 (PCR)获得G1及G2片段。将G1、G2片段经BamHⅠ、XhoⅠ双酶切后插入至真核表达载体 pcDNA3 .1(+) ,经酶切鉴定 ,并测序证实。以磷酸钙沉淀法分别将重组质粒转染COS 7细胞 ,用间接免疫荧光法 (IFA)检测瞬时表达的蛋白。结果 获得分别含有编码汉坦病毒 (HV)Z37株包膜糖蛋白G1、G2基因的重组质粒 pcDNA3 .1 G1、pcDNA3 .1 G2 ;在转染的COS 7细胞内 ,用IFA可检测到细胞内有特异性荧光。结论 成功地构建了HVZ37株包膜糖蛋白G1、G2基因真核表达载体 ,并可在COS 7细胞中瞬时表达。
Objective To construct hantavirus Z37 envelope glycoprotein genes G1 and G2 recombinant and express in eukaryocyte. Methods Using three pairs of primers based on hantavirus Z37 M gene sequences. PCR products F1, F2 and G2 were obtained by PCR from the Plasmid pGEMZ37(containing partial cDNA of G1) and Plasmid pCUMZ37(containing partial cDNA of G1 and whole cDNA of G2). G1 PCR products were also obtained by fusing F1 and F2 PCR products. The G1 and G2 PCR products were then digested by BamHⅠ and XhoⅠ and cloned into the corresponding sites of expression vector pcDNA3.1 respectively. The recombinants pcDNA3.1 G1 and pcDNA3.1 G2 were identified by digestion with endonuclease BamHⅠ and XhoⅠ and confirmed by sequencing. After transfecting COS 7 cells by Calcium phosphate/DNA precipitating method, indirect immunofluorescence assay (IFA) was used to verify the transient expression of HV Z37 G1 or G2 protein in COS 7 cells. Results The recombinant expression plasmids pcDNA3.1 G1 and pcDNA3.1 G2 were constructed. After transfection with the above recombinant expression plasmids, specific antigens were detected within cells by IFA. Conclusions The reombinant expression plasmids pcDNA3.1 G1、pcDNA3.1 G2 were constructed successfully and expressed transiently in eukaryocyte.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2003年第1期17-20,共4页
Chinese Journal of Infectious Diseases
