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目的观察戊型肝炎病毒(HEV)基因Ⅲ型(GⅢ)ORF2全长片段在昆虫细胞中的表达和病毒样颗粒(VLP)的装配情况,对纯化的VLP进行抗原性分析。方法应用含III型HEV全长ORF2的pVL1393-GⅢORF2质粒,体外与杆状病毒同源重组后,转染昆虫细胞Sf9细胞与在Tn5细胞中表达,用聚丙烯酰胺凝胶电泳和westernblot检测表达产物,超速离心法纯化,检测是否形成病毒样颗粒,用免疫电镜和ELISA分析病毒样颗粒的抗原性。结果Ⅲ型ORF2全长基因在Sf9和Tn5昆虫细胞中表达可获得72、64、58、56、53kD的蛋白质,在Tn5细胞中有56、53kD的蛋白质分泌到培养液。纯化后可获得6.7mg/mL的蛋白质,分子量为53kD,电镜观察发现了直径约为25nm的VLP,浮密度为1.291g/cm3。检测基因IV型戊肝感染病人血清,GⅢVLP与GIVLP具有同样的灵敏度,均能检测到滴度分别为1∶204800和1∶102400的IgM和IgG抗体。同时GⅢVLP在戊肝病人血清和IgM阳性猪血清孵育发生凝集反应。结论戊型肝炎病毒基因Ⅲ型ORF2全长基因片段可以在昆虫细胞表达系统高效表达。形成的VLP具有HEV病毒颗粒类似的抗原活性,可用于制备HEV抗体诊断试剂和开发预防用疫苗。
OBJECTIVE: To observe the expression of full-length ORF2 fragment of hepatitis E virus (HEV) gene type Ⅲ (G Ⅲ) in insect cells and the assembly of virus-like particles (VLPs). Antigen analysis of purified VLPs was performed. Methods Plasmid pVL1393-GⅢORF2 containing full-length ORF2 of type III HEV was co-inoculated with baculovirus in vitro. Sf9 cells were transfected with Sf9 cells and expressed in Tn5 cells. The expression of the expressed product was detected by polyacrylamide gel electrophoresis and Western blotting , Purified by ultracentrifugation, and tested for the formation of virus-like particles. The antigenicity of the virus-like particles was analyzed by immunoelectron microscopy and ELISA. Results The full - length ORF2 gene of type Ⅲ expressed 72, 64, 58, 56 and 53 kD protein in Sf9 and Tn5 insect cells, and 56 and 53 kD of protein in Tn5 cells were secreted into the culture medium. 6.7mg / mL of protein was obtained after purification, molecular weight of 53kD, electron microscopy found a VLP diameter of about 25nm, the floating density of 1.291g / cm3. The serum of patients with genotype V hepatitis E virus infection, GⅢVLP and GIVLP had the same sensitivity, and both of IgM and IgG antibodies with titers of 1:204800 and 1:102400 were detected. At the same time GⅢVLP in hepatitis E patient serum and IgM positive pig serum incubated agglutination. Conclusion The full-length ORF2 gene of hepatitis E virus gene type Ⅲ can be highly expressed in insect cell expression system. The resulting VLPs have similar antigenic activity as HEV virus particles and can be used in the preparation of HEV antibody diagnostic reagents and in the development of vaccines for prophylaxis.