目的 对原核表达发热伴血小板减少综合征病毒（SFTSV）蛋白Gc免疫原性进行初步研究。方法 参照SFTSV HB29病毒株包膜蛋白Gc编码基因，通过大肠埃希菌密码子偏好性优化后化学合成方法得到Gc目的基因。将目的基因导入到载体pET-His中，得到表达载体pET-His-Gc。经测序确认后转化到BL21（DE3）感受态细胞中表达，纯化后进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western blot验证。蛋白免疫新西兰大白兔后收集兔血清进行抗体效价测定。结果 成功构建了pET-His-Gc融合蛋白表达载体，并在大肠埃希菌中得到可溶性表达，且得到的Gc蛋白具有良好的免疫原性。动物实验结果表明，3次免疫后兔血清的抗体效价可达到1 ∶ 512 000。结论 实现了SFTSV包膜蛋白Gc的原核表达，为后续SFTSV包膜蛋白的研究奠定了基础。
Objective To express severe fever with thrombocytopenia syndrome virus (SFTSV) glycoprotein Gc in prokaryotic cells, and to preliminarily study the immunogenicity of glycoprotein Gc. Methods The DNA sequence encoding envelope glycoprotein Gc in SFTSV HB29 strain was chemically synthesized after codon optimization based on Escherichia coli codon preference. The expression vector pET-His-Gc was constructed by cloning the target gene into the vector pET-His. The sequence of the vector was confirmed by sequencing. Protein expression was carried out by transforming the vector into BL21 (DE3) competent cells. Target protein was purified and verified by SDS-PAGE and Western blot. Purified protein was used to immunize New Zealand white rabbits. The rabbit sera were collected for determination of antibody titer. Results The expression vector pET-His-Gc was successful constructed. Glycoprotein Gc was expressed in E. coli as a soluble protein. The obtained Gc protein showed good immunogenicity. Animal experiments showed that the rabbit serum antibody titer reached 1:512 000 after three immunizations. Conclusion In this study, SFTSV envelope glycoprotein Gc is successfully expressed in prokaryotic cells, which paves the way for subsequent studies of SFTSV envelope proteins.
[1] Yu XJ,Liang MF,Zhang SY,et al. Fever with thrombocytopenia associated with a novel bunyavirus in China[J]. N Engl J Med,2011,364(16):1523-1532. DOI:10.1056/NEJMoa1010095.
[2] Liu Q,He B,Huang SY,et al. Severe fever with thrombocytopenia syndrome,an emerging tick-borne zoonosis[J]. Lancet Infect Dis,2014,14(8):763-772. DOI:10.1016/s1473-3099(14)70718-2.
[3] 施泽纶,乾虎,马世良,等. 新型布尼亚病毒病原学和诊断学进展[J]. 新发传染病电子杂志,2018,3(3):140-142.
[4] 吕沐天,孙颖,刘沛,等. 发热伴血小板减少综合征布尼亚病毒研究进展[J]. 微生物学杂志,2013,33(2):86-88.
[5] Takahashi T,Maeda K,Suzuki T,et al. The first identification and retrospective study of severe fever with thrombocytopenia syndrome in Japan[J]. J Infect Dis,2014,209(6):816-827. DOI:10.1093/infdis/jit603.
[6] Shin J,Kwon D,Youn SK,et al. Characteristics and factors associated with death among patients hospitalized for severe fever with thrombocytopenia syndrome,South Korea,2013[J]. Emerg Infect Dis,2015,21(10):1704-1710. DOI:10.3201/eid2110.141928.
[7] McMullan LK,Folk SM,Kelly AJ,et al. A new phlebovirus associated with severe febrile illness in Missouri[J]. N Engl J Med,2012,367(9):834-841. DOI:10.1056/NEJMoa1203378.
[8] Arikawa J,Yao JS,Yoshimatsu K,et al. Protective role of antigenic sites on the envelope protein of Hantaan virus defined by monoclonal antibodies[J]. Arch Virol,1992,126(1/4):271-281. DOI:10.1007/BF01309700.
[9] Överby AK,Pettersson RF,Grünewald K,et al. Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus[J]. Proc Natl Acad Sci USA,2008,105(7):2375-2379. DOI:10.1073/pnas.0708738105.
[10] 张文帅,迟莹,张黎,等. 发热伴血小板减少综合征布尼亚病毒Gn和Gc蛋白的分段表达[J]. 江苏预防医学,2013,24(1):4-6.DOI:10.3969/j.issn.1006-9070.2013.01.002.
[11] 徐菱遥,韩亚萍,周宜庆,等. SFTSV糖蛋白Gn重组质粒的构建及其体液免疫原性研究[J]. 南京医科大学学报:自然科学版,2016,36(5):554-558. DOI:10.7655/NYDXBNS20160509.
[12] 陈蕾,戴新宪,郝春生,等. 发热伴血小板减少综合征布尼亚病毒糖蛋白抗原(Gn)定量检测方法的建立及验证[J]. 中国生物制品学杂志,2017,30(10):1066-1072. DOI:10.13200/j.cnki.cjb.001747.
