HCVCE2融合蛋白在家蚕培养细胞、蚕幼虫中的表达及其初步应用

Expression of HCV CE2 fused protein in cultured cells and larvae of silkworm and initial application

目的:高效表达HCVCE2融合蛋白并进行初步应用.方法:将2a型HCV全长CE2基因的cDNA重组于质粒pBacPAK8,构建重组转移载体pBacPAK CE2,与经线性化修饰的家蚕核型多角体病毒(BmNPV)DNA共转染家蚕培养细胞株,构建重组病毒.双酶切及PCR鉴定重组病毒基因组中目的片段的表达.重组病毒感染BmN细胞株及五龄家蚕幼虫后,SDS PAGE分析细胞培养上清、细胞抽提物及幼虫体液样品中,HCVCE2融合蛋白的特异性条带.并用间接ELISA法初步检测表达产物的生物活性.结果:双酶切及PCR鉴定重组病毒基因组含有约1.6kb的目的片段,重组病毒感染后的BmN细胞培养上清、细胞抽提物及幼虫体液样品中,均可见一Mr约90×103的特异性条带;用间接ELISA检测证明表达产物具有较好的免疫原性.结论:HCVCE2融合基因在家蚕培养细胞及蚕幼虫中获得了高效表达,并具有生物活性,为进一步进行疫苗的研究及临床诊断试剂的开发奠定基础.

AIM: To highly express HCV CE2 fused protein and to study its initial application. METHODS: A recombinant transfer vector pBacPAK-CE2, containing total cDNA of CE2 gene with 2a HCV strain, was constructed and co-transfected into BmN cells with linearized BmBacPAK (modified BmNPV) DNA for the construction of a recombinant baculovirus carrying CE2 fused gene. The recombinants were identified by restriction endonucleases digestion and PCR amplification. After being infected by the recombinant baculovirus, all the samples of cell extract, culture supernatant, and hemolymph of larvae were detected for specific HCV CE2 bands by SDS-PAGE. The biologic activity of the expressed product was detected with indirect ELISA. RESULTS: The genome of recombinant baculovirus contained a 1.6 kb inserted fragment identified by restriction endonucleases digestion and PCR amplification. From SDS-PAGE analysis, a 90 ku band was determined in cell extract, culture supernatant and hemolymph of larvae. Indirect ELISA showed that the expression products had strong immunogenicity. CONCLUSION: HCV CE2 fused protein with biologic activity is highly expressed in cell culture and larvae of silkworm, which provides basis for further study on the vaccine against HCV infection and the development of diagnosis reagent.