High-yield expression of nucleoprotein gene of Xinjiang hemorrhagic fever virus in bacteria and establishment of serological methodology for diagnosis and preliminary applications

Objective To clone and sequence of nucleoprotein (NP) gene of Crimean Congo hemorrhagic fever virus (CCHFV) Chinese isolates (Xinjiang hemorrhagic fever virus, XHFV) BA88166 and to express it in bacteria for application to clinical diagnosis. Methods Viral RNA was RT PCR amplified using the proof reading DNA polymerase to produce the complete NP gene. The PCR product was sequenced,analyzed for phylogenesis,and cloned into expression vector pET32a and the recombinant plasmid was expressed in E.coli BL 21 with high yield. The primarily purified fused protein was used to coat ELISA plates to detect antibody. Results The similarities between NP gene of BA88166 and other XHFVs in nucleotide level and in amino acid level were very high. They formed an independent genetic branch. The NP gene of BA88166 encoded a 482 amino acid nucleoprotein, with the deduced molecular weight of 54kD. Western blot showed that the fusion protein expressed in bacteria possessed good antigenicity. The ELISA results for detecting the human and animal sera collected in endemic areas were identical with those by IFA and in good accordance to clinical diagnosis. Conclusion The relations of NP genes of XHFV BA88166 and other XHFVs were evolutionally close. Combined with the analysis of M gene, the human origin BA88166 might be a variant of tick borne BA8402. The nucleoprotein expressed in bacteria could be used as a safe diagnostic antigen and the established methodology is accurate, specific, simple and fast and so is applicable to clinical examination and epidemiological survey. [