DNA fragments of Core(300bp)and NS4(400bp),encoding the nucleocapsid region(Core)protein CS and nonstructural region(NS4)protein NS4a of HCV,have been obtained from HCV genome by PCR,both of the two fragments were liked with C33c(700bp)and formed a chineric gene C33c-Core-NS4(HCV-CCN).The chimeric gene was recombined with expression vector pET24a(+)and was expressed in Escherichia coli.The expressed protein was purified by Ni(+)NTB affinity chromatography.Its molecular weight was about 58kD.Western blotting analysis showed that the chimerical antigen had good antigenicity,which could play an important role in anti-HCV assay.
In order to improve the diagnostic efficiency of the hepatitis C in China, the DNA fragments of core, C33c,NS4 in Hepatitis C virus(HCV) were connected and subcloned to pET24a(+). The pET24a-CCN was highly expressed with the inducement IPTG in the E.coli BL21(DE3). In 1L bacterial culture medium,30mg purified recombinant proteins were available using His Binding Resin under denatured condition. Used as diagnostic antigen to detect the human hepatitis C by ELISA, The results showed that the 100% positive coincidence rate (66/66) and 94.5% negative coincidence rate (86/91) were obtained. To detect 46 positive HAV sera and 46 positive HBs sera, all the results were negative. At the same time, the freeze-dried recombinant antigen detects 66 HCV positive sera, all the results were positive. Therefore, the recombinant antigen was sensitivity, specialty and stability to detect hepatitis C. It was an ideal reagent to detect hepatitis C.
