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获得稳定表达RSV病毒NS1基因的HEp-2-NS1细胞株并对其生物学特性进行初步研究。采用RT-PCR方法从RSV病毒中获得NS1全长基因,克隆到逆转录病毒载体pBABE-puro中,重组载体与包装质粒PIK通过磷酸钙共沉淀法转染包装细胞293FT细胞,产生的逆转录病毒颗粒感染HEp-2细胞,经嘌呤霉素筛选后得到稳定表达细胞株,用QPCR、细胞病变染色法、RT-PCR和间接免疫荧光法检测细胞中NS1 mRNA和蛋白表达情况。结果表明重组逆转录病毒载体pBABE-NS1经双酶切及测序鉴定正确;筛选获得5株阳性单克隆细胞株,QPCR结果显示HEp-2-NS1细胞有NS1基因扩增,其中d株的相对表达量是正常细胞对照组的8 483倍;在外源干扰素作用下,HEp-2-NS1细胞仍对VSV病毒保持敏感,细胞感染病毒48h后染色结果显示全部死亡;对第3代及第30代细胞的RT-PCR和间接免疫荧光法检测结果显示,导入的NS1基因在HEp-2细胞中不仅能转录成相应的mRNA而且能成功稳定地表达。成功构建了稳定表达RSV病毒非结构蛋白NS1的HEp-2-NS1改造细胞株,为建立适用于临床分离的转基因细胞提供良好的基础。
The HEp-2-NS1 cell line stably expressing the NS1 gene of RSV virus was obtained and its biological characteristics were studied. The NS1 full-length gene was obtained from RSV virus by RT-PCR and cloned into the retroviral vector pBABE-puro. The recombinant vector and the packaging plasmid PIK were transfected into 293FT cells of packaging cells by calcium phosphate co-precipitation method. The resulting retrovirus The cells were infected with HEp-2 cells. After puromycin selection, stable expression cell lines were obtained. The expression of NS1 mRNA and protein was detected by QPCR, cytopathic stain, RT-PCR and indirect immunofluorescence. The results showed that the recombinant retroviral vector pBABE-NS1 was identified by double enzyme digestion and sequencing. Five positive monoclonal cell lines were screened. QPCR results showed that NS1 gene was amplified in HEp-2-NS1 cells and the relative expression of d The amount of HEp-2-NS1 cells was still sensitive to VSV virus under the action of exogenous interferon, and all cells died after being infected with virus for 48 hours. On the 3rd and 30th generation The results of RT-PCR and indirect immunofluorescence assay showed that the introduced NS1 gene could not only transcribe into corresponding mRNA but also stably expressed in HEp-2 cells. The recombinant HEp-2-NS1 cell line stably expressing NS1, a non-structural protein of RSV virus, was successfully constructed and provided a good foundation for the establishment of transgenic cells suitable for clinical isolation.