发热伴血小板减少综合征布尼亚病毒IgM抗体检测试剂研究

Prokaryotic expression of SFTSV nucleocapsid protein and establishment of ELISA kit for detection of total IgM

目的原核重组表达高效发热伴血小板减少综合征布尼亚病毒核衣壳蛋白,并初步建立发热伴血小板减少综合征布尼亚病毒IgM免疫检测方法,为科研、临床诊断提供高效试剂或提供制备方法。方法利用生物信息学方法从NCBI中筛选并合成发热伴血小板减少综合征布尼亚病毒核衣壳蛋白基因,通过PCR扩增、双酶切构建到相应原核表达载体中,优化纯化工艺、制备出高效重组表达发热伴血小板减少综合征布尼亚病毒核衣壳蛋白包被抗原,进而研究酶联免疫检测发热伴血小板减少综合征布尼亚病毒IgM试剂。结果发热伴血小板减少综合征布尼亚病毒核衣壳蛋白在PET30a、PGEX4T-3原核载体中均能表达,测序验证开放阅读框表达的目的蛋白基因序列正确,通过柱层析纯化16度IPTG 0.2 mm IPTG分别诱导12 h PET30a-rSFTSN(P1、P2、P3)/BL21DE3菌体超声后上清,通过进一步优化酶免检测试剂,最终选r SFTSN P1做为发热伴血小板减少综合征布尼亚病毒IgM检测的标记抗原,并研究科研用免疫检测发热伴血小板减少综合征布尼亚病毒Ig M试剂。结论原核高效重组表达发热伴血小板减少综合征布尼亚病毒核衣壳蛋白,并建立免疫检测发热伴血小板减少综合征布尼亚病毒IgM,为科研、临床辅助诊断等奠定了基础。

Objective To highly prokaryotic express the nucleocapsid protein of the severe fever with thrombocytopenia syndrome bunyavirus（ SFTSV） and to establish the ELISA reagent kit for the detection of Ig M,expecting to provide efficient ELISA reagent kit or preparation method for the research and clinical diagnosis. Methods The SFTSV nucleocapsid protein gene was screened from NCBI by bioinformatics methods and synthesised. The target fragment were amplified by PCR,and cloned to the prokaryotic expression vector by restriction digest. After the exploring and optimization of the purification process,the SFTSV nucleocapsid protein was obtained and the ELISA regent kit were developed.Results The expression vector PET30 a-rSFTSN（ P1、P2） and PGEX4 T-3-r SFTSN（ P3） was constucted successfully.The ET30 a-rSFTSN（ P1-P3） were expressed very effectively in BL21 DE3 respectively,induced by 0. 2 m MIPTG at 16℃. Finally the recombinant rSFTSNP1 was screened as HRP labeled antigen and the ELISA reagent kit for detecting the SFTSV Ig M was developed. Conclusion The SFTSV nucleocapsid protein was highly expressed by Prokaryotic expression system and the ELISA reagent kit for detecting the SFTSV IgM were successly developed. This study laid the foundation for the research and clinical assistant diagnosis.