戊型肝炎病毒Ⅳ型ORF2蛋白在汉逊酵母中的表达

Expression of ORF2 Protein of HEV Genotype Ⅳ in Hansenula polymorpha

摘要:

为了寻求新型表达系统来研制戊型肝炎基因工程疫苗，利用甲基营养型汉逊酵母（Hansenula polymorpha）系统表达戊型肝炎病毒（Hepatitis E virus,HEV）Ⅳ型结构区ORF2编码蛋白第112-607氨基酸片段。为实现目的基因在汉逊酵母中的高效表达，根据汉逊酵母偏爱密码子优化设计目的基因，用搭桥PCR法合成优化后的基因序列，并克隆到多拷贝表达载体上，转化汉逊酵母营养缺陷宿主菌ATCC26012(Ura₃^-)，在选择培养基上培养，运用PCR法筛选得到携带外源基因的重组菌株，然后用含甲醇的培养基诱导表达，对表达产物进行SDS-PAGE、ELISA和Western blot检测和鉴定。SDS-PAGE实验结果表明目的蛋白分子量约为56kD，表达量占菌体总蛋白的12%；ELISA检测结果表明表达产物为具有免疫反应性的HEV ORF2蛋白，ELISA效价最高可达1∶2048，目的蛋白表达量随着基因拷贝数的增加呈升高的趋势；Western blot鉴别实验结果证实表达产物与HEV 多抗有特异性抗原抗体结合反应。HEV结构区ORF2蛋白在汉逊酵母中的成功表达，为研制基因工程戊型肝炎疫苗奠定了基础。

Abstract:

Hepatitis E, an acute infectious disease transmitted via the fecal-oral route, is caused by hepatitis E virus. However, no effective treatment currently exists for hepatitis E, and the only epidemic control approach is vaccination. But so for there are no commercial vaccine for hepatitis E available in the world. To find a new expression system to develop recombinant hepatitis E vaccine, in this study the expression system of methylotrophic yeast Hansenula polymorpha was used to express the gene encoding amino acid 112-607 of the open reading frame 2 (ORF2) of hepatitis E virus (HEV) genotype Ⅳ. In order to achieve high expression level，the coding sequence was optimized according to codon usage bias of Hansenula polymorpha and synthesized through overlapping PCR. Subsequently the gene was subcloned into the multi-copy expression vectors of Hansenula polymorpha，which include pDGXHP1.0 (MOX promotor),pDGXHP2.0 (MOX promotor) and pDGXHP2.1（FMD promotor）.The series of one-copy and multi-copy recombinant plasmids were transformed into ATCC26012(Ura3-) by electroporation. The transformants were cultured in selection media MDL and screened for the existence of foreign gene by PCR. Then the strains were induced in MM media and the expression products were detected by SDS-PAGE, ELISA and Western blot assays to select the high-level expression strains. The result of SDS-PAGE showed that the HEV ORF2 expression product was accumulated up to 12% of total cellular protein and its molecular weight is 56kD. The expression product showed high immunoreactivity detected by ELISA and the highest titer is 1:2048. The result of Western blot demonstrated that the expression product could be specifically recognized by the polyclonal antibody against HEV. The successful expression of HEV ORF2 protein in Hansenula polymorpha provides foundation for the further development of recombinant subunit vaccine against hepatitis E.