Hepatitis C NS5A Different Immunodominant Region And NS3 Expression And Assay Evaluation

Hepatitis C Virus(HCV)non-structural protein 3(NS3)and non-structural protein 5A(NS5A)were the second and third generation ELISA required fragrnents about HCV diagnosis,NS3 and NS5Awere so important to HCV clinical diagnosis.We obtain two fragments of HCV NS5A and whole region of NS3 using recombinant DNA method,then identified their detection sensitivity by ELISA.The whole fragment of Hepatitis C NS5A and the strong immunoreactive region (aa2212-2313)were amplified by PCR from plasmid pBR^tm/HCV-3011,each were named NS5AL and NS5AS,then,inserted into the plasmid pET-32a to construct recombinant plasmid pET-NS5AL and pET-NS5AS,pET-NS5AL was transformed into E.coli Rosetta(DE3)pLsS,named Rosetta(DE3)pLsS-NS5AL,the pET-NS5AS was transformed into E.coli BL21(DE3),was named BL21(DE3)-NS5AS,the recombinant protein NS5AL and NS5AS were induced by IPTG and analyzed with SDS-PAGE and Western-blot,the result of SDS-PAGE and Western-blot showed that the relative molecular weight of fusion protein NS5AL and NS5AS were some to the expected value,fusion protein NS5AL molecular weight is about 68kDa,NS5AL is about 39 kDa.Fusion protein with 6×His tag was purified using Ni²⁺affinity chromatography,we take Bradford reagent to determine the concentration of purified fusion protein NS5AL and NS5AS,each concentration of purified fusion protein NS5AL and NS5AS were 0.146mg/mL and 0.426mg/mL respectively ,purity of two antigens were more than 96%by SDS-PAGE,ELISA found that fusion protein NS5AL and NS5AS had specific immunoreactivity,and fusion protein NS5AL show better sensitivity than NS5AS.The recombinant plasmid pProEX-HTb-NS3(cloned with NS3 gene)was transfected into E.coli BL21(DE3),named BL21(DE3)-NS3,recombinant protein NS3 was induced with IPTG,the recombinant protein was analyzed by SDS-PAGE and Western-blot,as a result,NS3 gene was expressed in BL21(DE3)with a 72kDa band in SDS-PAGE,we take Ni²⁺affinity chromatography to purify recombinant protein NS3,the concentration of NS3 reach to 0.346mg/mL,purity of NS3 was more than 93%,ELISA found purified fusion protein NS3 had specific immunoreactivity.Our research take prokaryotic expression system,obtain fusion protein NS5A and NS3 successfully,protein NS5A and NS3 were helpful to enhance the sensitivity of diagnosis hepatitis C virus in clinic,what's more,the result establish a foundation about others biological function of protein NS3 and NS5A.