丙型肝炎病毒HCV E1区基因在真核细胞中的表达及表达产物的初步应用  

Expression of HCV E1 glycoprotein in mammalian cell line SP2/0 and the application of the expression product

作　　者：高建恩[1] 陶其敏[1] 马大龙[2] 冯百芳[1] 蒋栋[1] 

机构地区：[1]北京医科大学人民医院肝病研究所,北京100044 [2]北京医科大学免疫学系

出　　处：《北京医科大学学报》2000年第1期22-25,共4页Journal of Peking University(Health Sciences)

基　　金：国家自然科学基金! ( 3 940 0 116 );美国中华医学基金会! ( 93 5 82 )

摘　　要：目的:建立丙型肝炎病毒包膜区基因的真核表达载体,并对表达产物进行鉴定,同时测定表达产物与抗HCV阳性血清的反应情况。方法:用限制性内切酶XbaⅠ和EcoRⅠ从原核表达载体pMSCVE1中切下HCVE1区的基因片段,并将其克隆到真核表达载体PcDNA3.1中,阳性克隆经SmaⅠ和XbaⅠ双酶切鉴定后,用磷酸钙共沉淀法将其转染入小鼠骨髓瘤细胞SP2/0中,同时利用WesternBlot方法对表达产物进行鉴定;测定表达产物与20份抗HCV阳性血清标本和10份抗HCV阴性的血清标本的反应性。结果:经酶切过夜后从原核表达载体上切下的基因片段与经过同样双酶切的真核表达载体PcDNA3.1连接,阳性克隆经SmaⅠ和XbaⅠ酶切后产生了预期大小的354bp的片段。经磷酸钙转染,产生了具有对G418抗性的细胞克隆,经WesternBlot方法用抗HCVE1区合成肽抗原的单抗检测表达产物,产生了特异的反应;并证实该表达产物与HCV患者的血清有特异反应。经WesternBlot证实在抗HCV阳性血清中有35%(7/20)可与表达产物产生特异的反应;阳性细胞经3个月传代后,仍可检测到HCVE1的表达产物。结论:此项研究所建立的能够表达HCVE1基因细胞系表达产物可与阳性血清发生特异性反应,该细胞系的建立为进一步研究抗HCVE1抗体的临床意义及进行有关HCV核酸免疫的研究创造了条件。Objective: To express HCV E1 gene in the mammalian cell line and apply the expression product in the detection of anti HCV E1 antibody in anti HCV positive serum. Methods: The DNA fragment of HCV E1 gene was obtained by digesting the prokaryotic expression vector of pMS CVE1, containing the HCV E1 gene, with Eco RⅠ and Xba Ⅰ and was ligased into the eukaryotic expression vector of PcDNA3.1 by T4 DNA ligase. The recombinant eukaryotic expression vector was then transfected into the murine myoloma cell line by phosphate calcium method. The expression protein was visualized by Western Blot analysis using the monoclonal antibody generated by immunizing the BALB/c mouse with peptides derived from HCV E1 sequence and the prevalence of anti HCV E1 antibody in 20 anti HCV positive sera and 10 anti HCV negative sera determined by Western Blot assay. Results: There was the product of 489 bp after the digestion with Eco RⅠ and Xba Ⅰ. There was also a product of 354 bp after the digestion of the recombinant plasmid extracted from the ampicillin resistant bacteria colony with the restricted endonuclease Sma Ⅰ and Xba Ⅰ. The Western Blot result indicated that after transfection of the expression vector into SP 2/0 cells there are 4 strains of cell lines that express HCV E1 protein, the molecular weight of the expression protein was about 28 000 , but the expression efficiency of the recombinant protein was relatively low. The expression protein could react with the HCV infected serum and the prevalence of anti HCV E1 expression protein in the anti HCV positive serum was 35%(7/20). Conclusion: the HCV E1 glycoprotein expressed in the mammalian expression system was specific, and the cell line which expressed the HCV E1 protein might be used as a tool for the investigation of the clinical significance of anti HCV E1 antibody.

关 键 词：HCVE1 真核细胞 丙型肝炎病毒 基因表达 

分 类 号：R373.21[医药卫生—病原生物学]