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目的:建立呼吸道合胞病毒快速、敏感、特异、廉价的早期血清学检测方法.方法:从北京儿童医院患者鼻咽分泌物分离的呼吸道合胞病毒中,用RT-PCR的方法扩增出RSVF蛋白的F1区部分基因片段F1-1(AA137～363),此区域包含F蛋白的主要中和抗原位点.将该基因片段克隆到pMD18-T载体,将连接质粒经PCR鉴定,证实插入目的片段.酶切后将该片段与pET-32a表达质粒连接,用限制性内切酶酶切及序列测定,证实插入片段的正确性.利用pET-32a表达质粒在大肠杆菌BL21表达正确的RSVF1-1蛋白.重组蛋白N端包含有6个连续的组氨酸标签,通过镍离子金属鏊合亲和层析纯化,获得了高纯度的目的重组蛋白RSVF1-1,然后将重组RSVF1-1包被建立间接ELISA法检测人血清中的RSVF蛋白IgG抗体.结果:RT-PCR产物的片段大小为678bp.经酶切和测序鉴定后,插入序列无误.Western Blot分析显示:重组蛋白具有较好的抗原性.用该蛋白建立的间接ELISA方法检测结果与进口Euroimmun和维润试剂盒检测结果比较,差异无显著性.结论:F蛋白基因片段的成功表达为进一步开发诊断试剂盒提供了实验依据.
OBJECTIVE: To establish a rapid, sensitive, specific and inexpensive early serological test for respiratory syncytial virus.Methods: RSVF was amplified by RT-PCR from respiratory syncytial virus isolated from nasopharyngeal secretions from Beijing Children’s Hospital The F1 region of the protein was partly F1-1 (AA137 ~ 363), which contained the major neutralizing antigenic site of F protein.The fragment was cloned into pMD18-T vector and the ligated plasmid was identified by PCR and confirmed the purpose of insertion The fragment was ligated with pET-32a expression plasmid, and the correctness of the inserted fragment was confirmed by restriction enzyme digestion and sequence analysis.Using pET-32a expression plasmid to express RSVF1- 1 protein.The N-terminus of the recombinant protein contains 6 consecutive histidine tags, which were purified by nickel ion affinity chromatography to obtain a highly purified recombinant protein of interest, RSVF1-1, which was then coated with recombinant RSVF1-1 The indirect ELISA was used to detect the IgG antibody of RSV F protein in human serum.RESULTS: The fragment size of RT-PCR product was 678 bp.The inserted sequence was confirmed by enzyme digestion and sequencing.Western Blot analysis showed that the recombinant protein had good antigen Use this egg The results of indirect ELISA assay established by Bai et al were not significantly different from those of imported Euroimmun and Wei Run kit.Conclusion: The successful expression of F protein gene fragment provided experimental basis for further development of diagnostic kit.