甲型肝炎病毒P1B、P2A、P3AB和P3D蛋白在原核系统中的表达和抗原活性分析

目的研究甲型肝炎病毒P1B、P2A、P3AB和P3D在原核系统中的表达,并探讨这些蛋白的抗原活性和作为诊断抗原的应用价值。方法采用PCR方法,从克隆有HAV HM175株全长cDNA基因的克隆载体pHAV16H1上扩增出目的基因,以M47作为表达载体,构建N端带硫氧还蛋白的重组表达质粒。重组质粒在大肠埃希菌BL21(DE3)中经IPTG诱导表达。采用DEAE阴离子交换和镍离子柱螯和亲和层析纯化的方法对重组蛋白进行纯化。以Western Blot和间接ELISA的方法检测重组蛋白的抗原活性。结果四个重组质粒经测序证明构建正确无误,在大肠埃希菌中表达四个蛋白的相对分子质量也正确无误。Western Blot分析和间接ELISA方法证实四个蛋白中只有p2a有抗原活性。结论原核表达的P2a蛋白,具有较好的抗原活性,在间接ELISA方法中检测出了所有的24份阳性血清和24份阴性血清,非常有希望做成诊断抗原用于诊断急性甲肝患者和区分甲型肝炎自然感染与注射疫苗所产生的不同免疫。

Expression of the P1B,P2A,P3AB and P3D protein of hepatitis A virus in prokaryotic cell and antigenicity analysis

Objective To express P1B,P2A,P3AB and P3D cDNA gene fragments in prokaryotic system using thioredoxin fusion expression system; to investigate the antigenicity and application of recombinant protein.Methods By using PCR technique, P1B,P2A,P3AB and P3D gene fragments were cloned. Choosing M47 as the expressive vector, the recombinant plasmid P1B,P2A,P3AB and P3D was constructed and expressed in Escherichia coli after inducing by IPTG. By anion exchange and affinity chromatography, purified recombinant protein was obtained. By using Western Blot analysis and indirect ELISA to detect its antigenic activity.Results Four recombinant plasmids was proved to be constructed successfully by sequencing and the correct molelar weight of their expression products. Recombinant proteins were obtained in BL21(DE3) and purified after Ni~ 2 affinity chromatography. Western Blot analysis and indirect ELISA showed that P2a had specific antigenicity.Conclusion The P2a protein expressed in prokaryotic system was proved to have specific antigencity.The indirect ELISA distinguished 24 positive sera from 24 negtive sera. It is very likely that P2a can be a antigen to diagnose acute patients of hepatitis A and differentiate inactivated vaccine-induced immunity from an infection.