【摘要】 利用RT-PCR法扩增猪繁殖与呼吸综合征病毒(PRRSV)ORF5和ORF6基因,分别构建pcDNA-ORF5、pcD-NA-ORF5/6和pcDNA-ORF5-ORF6真核表达载体,磷酸钙共沉淀法瞬时转染293T细胞,48h后收集细胞,流式细胞仪检测,结果表明:PRRSVORF5基因编码囊膜蛋白(E蛋白)能在在293T细胞表面表达,而由共表达M蛋白(ORF6基因编码的基质蛋白)介导的E蛋白的表达量比单独E蛋白表达量高,串联表达与单独E蛋白相比较低。将pcDNA-ORF5、pcDNA-ORF5/6和pcDNA-ORF5-ORF6分别与MuLV假病毒构建体系的两种骨架载体pHIT60(包括MuLV的结构蛋白基因,即gag和pol)和pHIT111(为MuLV的基因组,还包括一个报告基因LacZ)瞬时共转染293T细胞,48h后收集假病毒上清,超速离心后通过Westernblot证实E蛋白能够在此假病毒颗粒表面表达,证明E蛋白已整合到此假病毒粒子表面。将整合PRRSVE蛋白的假病毒粒子分别感染Marc-145和PAM宿主靶细胞,均能检测到LacZ基因的表达,结果表明:所构建的假病毒粒子具有感染性,且由M蛋白介导的MuLV-E/M感染性比MuLV-E假病毒感染性高。更多还原

【Abstract】 For constructing pcDNA-ORF5,pcDNA-ORF5-ORF6,pcDNA-ORF5/6,the ORF5 and ORF6 of porcine reproductive and respiratory syndrome virus(PRRSV)were amplified by RT-PCR,and transiently transfected into 293T cells by calciumphosphate co-precipitation.After 48 h,293T cells were collected and surveyed by flow cytometry examination.The result indicated that the expression level of the E protein that mediated by the M protein was higher than that of the E protein expressed independently.Then pcDNA-ORF5,pcDNA-ORF5-ORF6,pcDNA-ORF5/6 were respectivly co-transfected into 293T cells with pHIT60(include MuLV structural genes,namely gag and pol)and pHIT111(retroviral genome,containing LacZ as a reporter).The supernatants were harvested 48 h post-transfection,and the analysis of the characteristic of the pseudotyping virions was performed by Western blot and infection test.The result indicated that the E proteins were expressed on the virions,and incorporatedlinto the retroviral virions.Infection test were performed on Marc-145 and PAM,all the cells infected were Lac Z positive.These results indicated the pseudotype virions of MuLV-E and MuLV-E/M were infectious,and higher infectivity was achieved by MuLV-E/M.更多还原