军事医学2016，Vol.40Issue(9)：713-716,737,5.DOI:10.7644/j.issn.1674-9960.2016.09.005
含5种烈性出血热病毒的假病毒阳性参考品的制备
Construction of pseudovirus reference standard containing five kinds of hemorrhagic fever virus
摘要
Abstract
Objective To construct the pseudovirus containing nucleic acid(NA)fragments of Marburg virus,Zaire Ebola virus,Sudan Ebola virus,Lassa fever virus and Yellow fever virus by using a lentiviral vector system in order to provide a reference standard for the detection of the five viruses.Methods The gene fragments of the above five viruses were synthesized in vitro,connected into a single gene by fusion PCR technique,and cloned into lentiviral vectors with its auxiliary vector.After co-transfecting into 293T cells,the supernatants were collected on 48 h and 72 h post transfection. The naked NA was cleaned from the supernatants using DNase and RNase digestion before pseudotype virus was purified and concentrated.After the NA of the pseudotype virus,were extracted normal PCR and real-time PCR were conducted. Results Sequence analysis showed that the five target genes in vitro synthesis were properly connected and inserted into lentivirus vectors.Using the NA of the pseudotype virus as the template,both normal PCR and real-time PCR could sensitively amplify the target gene with the primers and probes of the above five,viruses respectively.The result indicated that the pseudovirus particles containing the five kinds of hemorrhagic fever virus target genes were successfully packaged. Conclusion The pseudovirus particles containing gene fragments of five viruses are constructed,which can be used as a common reference standard for NA detection.关键词
马尔堡病毒/扎伊尔型埃博拉病毒/苏丹型埃博拉病毒/拉沙热病毒/黄热病毒/假病毒/阳性参考品Key words
Marburg virus/Zaire Ebola virus/Sudan Ebola virus/Lassa fever virus/yellow fever virus/pseudovirus/reference standard分类
医药卫生引用本文复制引用
曹雪锋,康晓平,冉鑫,霍耐凡,吴晓燕,李裕昌,杨银辉..含5种烈性出血热病毒的假病毒阳性参考品的制备[J].军事医学,2016,40(9):713-716,737,5.基金项目
国家自然科学基金资助项目（81501789）；军队重大专项课题 （）
