P[1]型A群牛轮状病毒VP8重组蛋白的表达与免疫原性研究

doi:10.16431/j.cnki.1671-7236.2022.05.038

摘要/Abstract

摘要： 【目的】利用原核表达系统表达P[1]型A群牛轮状病毒(BRVA) VP8重组蛋白，并评价其反应原性与免疫原性。【方法】根据国内流行毒株RVA/Cow-tc/CHN/SDA2/2018/G6P[1] VP8基因序列(GenBank登录号:MN937517)进行密码子偏好性优化，构建P[1]型BRVA VP8大肠杆菌原核表达系统，将重组质粒转化大肠杆菌BL21(DE3)感受态细胞，IPTG诱导表达，通过SDS-PAGE分析重组蛋白表达形式。镍琼脂糖亲和层析纯化重组蛋白，Western blotting鉴定重组蛋白与兔抗G6P[1]型BRVA血清特异性结合能力，并将P[1]型BRVA VP8重组蛋白免疫兔，首免后2周加强免疫1次，共免疫2次。分别用间接ELISA方法与中和试验评价其免疫原性。【结果】本研究成功构建P[1]型BRVA VP8重组蛋白的原核表达系统;SDS-PAGE结果显示，高效表达大小为20 ku的P[1]型BRVA VP8重组蛋白，以可溶形式存在;Western blotting结果显示，P[1]型BRVA VP8重组蛋白能与兔抗G6P[1]型BRVA血清特异性结合;间接ELISA结果显示，P[1]型BRVA VP8重组蛋白亚单位疫苗能诱导兔产生抗体，兔首免1周后抗体开始出现并迅速上升，于第3周达到高峰，至第5周仍维持在较高水平;中和试验结果显示，第2次免疫后抗体最高的兔血清对G6P[1]型和G8P[1]型BRVA的中和效价分别为1∶17 783和1∶64。【结论】本研究成功构建原核表达系统表达P[1]型BRVA VP8重组蛋白，并证实其具有良好的反应原性及免疫原性，为研制BRVA亚单位疫苗奠定基础。

关键词: A群牛轮状病毒(BRVA); P[1]型; VP8蛋白; 原核表达; 免疫原性

Abstract: 【Objective】 The purpose of this study was to express P[1] Bovine rotavirus A (BRVA) VP8 recombinant protein by prokaryotic expression system,and evaluate its reactivity and immunogenicity.【Method】 The codon preference was optimized according to the VP8 gene sequence of domestic epidemic strain RVA/Cow-tc/CHN/SDA2/2018/G6P[1] (GenBank accession No.:MN937517),the prokaryotic expression system of P[1] BRVA VP8 E.coli was constructed,the recombinant plasmid was transformed into E.coli BL21(DE3) competent cells,the expression was induced by IPTG,and the recombinant protein was purified by nickel agarose affinity chromatography.Western blotting was used to identify the specific recognition ability of recombinant protein and rabbit anti-G6P[1] BRVA serum,and P[1] BRVA VP8 recombinant protein was used to immunize rabbits.Two weeks after the first immunization,the immunization was strengthened once and twice in total.The immunogenicity was evaluated by indirect ELISA and neutralization test.【Result】 The prokaryotic expression system of P[1] type BRVA VP8 recombinant protein was successfully constructed,SDS-PAGE showed that the recombinant protein was highly expressed with a size of 20 ku,and the recombinant protein existed in soluble form.Western blotting results showed that the recombinant protein could specifically bind to rabbit anti-G6P[1] BRVA serum.Indirect ELISA results showed that P[1] type BRVA VP8 recombinant protein could induce antibody production in rabbits.The antibody began to appear and increased rapidly in the 1st week after the first immunization,peaked in the 3rd week and remained at a high level until to the 5th week.The neutralization test results showed that the neutralization titers of immunized rabbit serum to G6P[1] and G8P[1] BRVA were 1∶17 783 and 1∶64,respectively.【Conclusion】 This study successfully constructed a prokaryotic expression system to express P[1] BRVA VP8 recombinant protein,and confirmed that it had good reactivity and immunogenicity,which laid a foundation for the development of BRVA subunit vaccine.

Key words: Bovine rotavirus A (BRVA); P[1]; VP8 protein; prokaryotic expression; immunogenicity

中图分类号:

S82.65^＋3

姜晓明, 汤承, 岳华. P[1]型A群牛轮状病毒VP8重组蛋白的表达与免疫原性研究[J]. 中国畜牧兽医, 2022, 49(5): 1970-1976.

JIANG Xiaoming, TANG Cheng, YUE Hua. Expression and Immunogenicity of P[1] Bovine Rotavirus A VP8 Recombinant Protein[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1970-1976.

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