A组轮状病毒NSP1基因克隆表达及免疫学性质研究

Expression of NSP1 of a Group A Human Rotavirus and Immunological Properties

轮状病毒(rotavirus,RV)非结构蛋白1(non structural protein1,NSP1)在病毒与宿主的相互作用中发挥着重要的功能。运用基因克隆和表达技术在大肠杆菌中表达了TB-Chen株RVNSP1蛋白,进行了NSP1的免疫学性质和RV感染细胞中NSP1蛋白的合成与分布以及NSP1的系统进化和基因分型研究。结果表明,大肠杆菌BL21(DE3)能高效表达重组NSP1蛋白(rNSP1),rNSP1表达量约占菌体总蛋白的34.4%。rNSP1能诱导免疫豚鼠产生特异性血清抗体。Western blot及免疫荧光检测结果表明,抗rNSP1血清抗体能特异性识别自身蛋白,对SA11、Wa株的NSP1蛋白有交叉反应性;免疫荧光结果还表明,SA11感染的MA104细胞中合成的NSP1蛋白在细胞质中区域化聚集形成辐射状排列的颗粒状结构,而Wa株的NSP1不能形成此样结构。至今发现的A组RV至少可以分为16个不同的NSP1基因型,TB-Chen株NSP1为A2型。不同基因型有独特的敏感宿主范围,同一基因型可能感染不同种动物,同一种动物也可能感染不同基因型。基因型A4型和A16型仅在鸟类病毒株中出现;而且鸟类中只有A4型和A16型。研究结果为进一步研究NSP1蛋白质的结构功能及其应用开发奠定了很好的基础。

The non structural protein 1 （NSP1） of rotavirus （RV） plays a very important role in interaction between virus and host.The full-length gene of the NSP1 of strain TB-Chen was expressed in E.coli BL21（DE3） cells by using gene cloning and expression technologies,and immunogenicity,synthesis and subcellular distribution of the NSP1 protein in RV infected cells,and phylogenesis and genotypes of the NSP1 were studied.The results showed that the recombinant NSP1 protein （rNSP1） could be highly expressed in E.coli BL21（DE3） cells,which possesses 34.4% of total cell proteins.The rNSP1 could elicit specific antibodies in guinea pigs,the antibodies could react with the rNSP1 itself and NSP1 protein expressed in SA11 infected or Wa infected MA104 cells by Western blot and immunofluoresence assay.The results of immunofluoresence also showed that NSP1 protein was aggregated in the cytoplasm and formed radiate granular structure in SA11 infected MA104 cells but not in Wa infected MA104 cells.At least 16 recognized genotypes of NSP1 were classified in group A RVs,based on diversity of sequences of the NSP1 open reading frame,and TB-Chen strain NSP1 was defined as genotype A2.RVs with different NSP1 genotype have specific host ranges,RVs possessing the same NSP1 genotype can infect different species,the same species can be infected with RVs possessing different NSP1 genotypes.Genotypes A4 and A16 were only found in birds and only A4 and A16 were found in birds.The results obtained in the present study are important for further studying on structure,function and immunological properties of the NSP1.