流行性乙型脑炎病毒非结构蛋白1基因的克隆及其在昆虫细胞中的表达

Cloning and Expression of the Nonstructural Protein 1 Gene of Japanese Encephalitis Virus in Insect Cell

目的获得流行性乙型脑炎(乙脑)病毒非结构蛋白1(Non-structural Protein 1,NS1)基因并在昆虫细胞中表达,为研制乙脑病毒基因工程疫苗奠定基础。方法逆转录-聚合酶链反应扩增乙脑病毒NS1基因并测序列,定向克隆入杆状病毒(Baculovirus,BV)转移载体pFastBac HTa,获得重组转移载体pFastBac-NS1。并将pFastBac-NS1转化到DH10Bac感受态细胞中,筛选到重组转座子rBacmid-NS1。在脂质体介导下将rBacmid-NS1转染粉蚊夜蛾(Trichoplusia ni,TN)细胞获得重组BV(rBV-NS1)。rBV-NS1感染TN细胞后,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulfate-Polyacryamide Gel Eleetrophoresis,SDS-PAG)、间接免疫荧光试验(Indirect Immunofluorescence Assay,IFA)和蛋白质印迹法(Western blotting,WB)检测NS1重组蛋白。结果成功克隆乙脑病毒NS1基因,SDS-PAGE、IFA和WB检测表明,NS1基因在昆虫细胞得到表达,能与乙脑患者血清发生特异性免疫反应,具有免疫原性。结论乙脑病毒NS1在昆虫细胞中表达,为研究NS1的生物学特性和研制基因工程疫苗奠定了基础。

Objective To express the non-structral protein 1（NS1）genes of Japanese encephalitis virus,and study the antigenicity and establish the basis for developing the diagnosis kit.Methods The NS1 cDNA was amplified with RT-PCR and cloned into the transfer plasmid pFastBac HTa,then the recombianat transfer plasmid pFastBac-NS1 was transformed into DH10Bac competent cells.The recombinant transposition rBacmid-NS1 was obstained by screening of white plaque and was identified by PCR.After the rBacmid-NS1 transfected into TN cells,the recombinant baculovirus rBV-NS1 was harvested.The expressed protein was detected by SDS-PAGE and Western Blot.Results The SDS-PAGE and Western blot showed that the NS1 gene was well expressed,and the expressed product had antigenicity.Conclusions The stable expression of this protein and the analysis of its antigenic specificity provide the foundation to develop the vaacine for Japanese encephalitis virus.