含轮状病毒NSP3基因病毒样颗粒的构建和表达

Construction and expression of virus-like particles containing rotavirus NSP3gene

目的:通过改变MS2噬菌体衣壳蛋白RNA包装位点的亲和力,构建并表达含轮状病毒NSP3基因耐RNA酶的病毒样颗粒,并探讨其稳定性。方法:设计含PvuⅠ和KpnⅠ酶切位点的上、下游引物,扩增1 049 bp轮状病毒NSP3基因片段,并将包装位点-5位的U替换为C,提高与MS2衣壳蛋白作用的亲和力。用PvuⅠ和KpnⅠ双酶切扩增的目的基因和pACYC-MS2表达载体,连接获得重组载体pACYC-MS2-NSP3,转化TOP10感受态细胞后用PCR验证阳性克隆并测序。阳性克隆转化BL21(DE3)细胞后表达含轮状病毒NSP3基因病毒样颗粒,用超声破碎、纯化获得表达产物,鉴定并讨论其稳定性。结果:成功构建pACYC-MS2-NSP3表达载体并获得了含轮状病毒NSP3基因病毒样颗粒,其可耐受DNA酶和RNA酶的降解,并在-20℃、4℃、室温25℃下10 d保持稳定。结论:通过改变MS2噬菌体衣壳蛋白RNA包装位点的亲和力,可以成功构建耐RNA酶的病毒样颗粒,本研究所构建的病毒样颗粒具有耐RNA酶的特性和良好的稳定性,为轮状病毒实时荧光定量逆转录-PCR的标准品和质控品的研究提供了一个可行的方法。

Objective： To construct and express ribonuclease-resistant virus-like particles containing rotavirus NSP3 gene by changing the affinity of MS2 bacteriophage coat protein pac site and to discuss the stability.Methods： In the study,1 049 bp rotavirus NSP3 gene fragments were amplified by PCR using the primers containing PvuⅠ and KpnⅠ restriction enzyme sites and the uridine at position-5 in the pac site was replaced with cytosine to increase the affinity.The gel-purified PCR-amplified DNA fragments and pACYC-MS2 vector were digested with PvuⅠ and KpnⅠ and then ligated to generate recombinant plasmid pACYC-MS2-NSP3.The expression vector was transformed into competent Escherichia coli strain TOP 10,and was verified by PCR and sequencing.The positive bacteria were transformed into competent E.coli strain BL21（DE3）.Then the cells were lysed by ultrasonic disruption,virus like particles（VLPs） were harvested after purification and their stability was discussed.Results： The expression plasmid containing mutant pac site（uridine at position-5 in the pac site replaced with cytosine） was constructed successfully and the ribonuclease-resistant virus-like particles containing rotavirus NSP3 gene were expressed successfully.The VLPs were resistant to ribonuclease and deoxyribonuclease,and were stable at-20 ℃,4 ℃ and room temperature（25 ℃）,respectively.Conclusion： The methods used to increase the affinity of pac site could successfully construct and express the VLPs.The VLPs containing rotavirus NSP3 gene are stable and could be used as surrogates for positive controls and standards in rotavirus real time fluorescent quantitative reverse transcription-PCR kits.