新疆出血热病毒核蛋白基因的高效表达及其在诊断中的初步应用

目的　克隆并测定了克里米亚 刚果出血热病毒 (CCHFV)中国分离株 (新疆出血热病毒 ,XHFV)BA8816 6株核蛋白 (NP)基因的序列并实现其在细菌中的高效表达与临床诊断的应用。方法　病毒RNA经RT PCR扩增出完整的NP基因。将扩增产物进行序列分析并克隆至融合表达载体pET32a ,使重组质粒在大肠杆菌BL 2 1中高效表达。将融合蛋白经初步纯化后包被ELISA板用于抗体检测。结果　XHFVBA8816 6株NP基因序列以及推导的氨基酸序列与其它XHFV的NP基因和蛋白序列同源性较高 ,在进化树上形成独立的分支。BA8816 6株NP基因编码 4 82个氨基酸的核蛋白 ,推测的相对分子质量 (Mr)约为 5 4× 10 3。在细菌中表达的融合蛋白经印迹试验证明具有良好的抗原性。以所建立的ELISA方法检测疫区人和动物血清的结果与IFA一致 ,并与临床诊断有很好的符合率。结论　BA8816 6株与其它XHFVBA6 6 0 19、BA84 0 2的NP基因在进化上关系密切 ,综合M基因的序列分析结果 ,人源分离株BA8816 6可能是来自蜱的BA84 0 2变异株。表达于细菌中的核蛋白可作为安全的诊断性抗原用于临床检测及流行病学调查 ,所建立的方法准确、特异、简便、快速

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.