论文部分内容阅读
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 prokaryoticly express recombinant fever virus with nucleated capsid protein of Bunyavirus and to establish a preliminary IgM immunoassay for detecting fever with thrombocytopenia syndrome (Bunyavirus), to provide an efficient reagent for scientific research and clinical diagnosis, or to provide a preparation method . Methods The nucleocapsid protein gene of fever virus with thrombocytopenia syndrome was amplified from NCBI by bioinformatics method. The nucleocapsid protein gene of fever virus with thrombocytopenia syndrome was amplified by PCR and cloned into corresponding prokaryotic expression vector by double enzyme digestion, and the purification process was optimized. Efficient recombinant expression of fever with thrombocytopenia syndrome Bunyavirus nucleocapsid protein coating antigen, and then study the enzyme-linked immunosorbent assay fever with thrombocytopenia syndrome Bunyavirus IgM reagent. Results fever with thrombocytopenia syndrome Bunyavirus nucleocapsid protein expression in PET30a, PGEX4T-3 prokaryotic vector can be verified by sequencing the open reading frame expression of the target protein gene sequence correct purified by column chromatography 16 degrees IPTG 0.2 ml IPTG were used to induce the supernatant of PET30a-rSFTSN (P1, P2, P3) / BL21DE3 cells after 12 h, respectively. The SF-N1 transcripts of SFTSN P1 were further optimized for the detection of fever with thrombocytopenia IgM detection of the labeled antigen, and research scientific research fever with thrombocytopenia syndrome Bunia virus Ig M reagent. Conclusion Prokaryotic expression of fever with severe thrombocytopenia syndrome Bunyavirus nucleocapsid protein, and the establishment of immune detection of fever with thrombocytopenia syndrome Bunyavirus IgM, for the scientific research, clinical diagnosis and other laid the foundation.