霍乱毒素B亚基与草鱼呼肠孤病毒VP7融合基因的合成和原核表达  

Synthesis and Prokaryotic Expression of the Fusion Gene of the Cholera Toxin B Subunit and Grass Carp Reovirus VP7

作　　者：王桐桐 金姗姗 韩玉行 索美涛 郭新琦 张秋胜 WANG Tongtong;JIN Shanshan;HAN Yuxing;SUO Meitao;GUO Xinqi;ZHANG Qiusheng(School of Agriculture,Ludong University,Yantai 264039,China)

机构地区：[1]鲁东大学农学院,山东烟台264039

出　　处：《鲁东大学学报（自然科学版）》2023年第3期203-208,共6页Journal of Ludong University:Natural Science Edition

基　　金：山东省科技攻关计划(2018GSF121035)。

摘　　要：草鱼呼肠孤病毒(grass carp reovirus, GCRV)是一种致病力强,严重危害水产养殖业健康发展的病毒。免疫接种疫苗是预防GCRV的有效途径,而口服免疫接种对生活在水中的鱼类是一种理想的接种方式。GCRV衣壳蛋白VP7具有较好的免疫原性。霍乱毒素B亚基(cholera toxin B subunit, CTB)是一种较好的黏膜佐剂。本研究采用PCR技术,将CTB基因和VP7基因进行柔性融合,获得1260 bp的CTB-VP7融合基因,构建重组表达质粒pET28a-CTB-VP7,转化大肠杆菌BL21 (DE3),获得了表达CTB-VP7融合蛋白的菌株。当采用异丙基硫代半乳糖苷(IPTG)诱导时,获得CTB-VP7融合蛋白的分子量约49 kDa,与预期的分子量大小一致。当IPTG浓度为0.08 mmol·L^(-1),诱导温度为37℃,诱导转速为100 r·min~(-1),诱导表达5.0 h时,CTB-VP7融合蛋白的表达量最大,达到2.45 mg·L^(-1)。CTB-VP7融合蛋白在菌体中主要以包涵体的形式存在。在融合蛋白纯化时,洗脱缓冲液中咪唑浓度为200 mmol·L^(-1),目标蛋白纯度达96%。本研究通过基因合成和原核表达得到CTB-VP7融合蛋白,为进一步验证CTB-VP7融合蛋白能否作为抵抗GCRV的粘膜疫苗奠定了基础。Grass carp reovirus(GCRV)is a type of virus with strong pathogenicity that seriously endangers the healthy development of aquaculture.Immunization is an effective way to prevent GCRV.GCRV capsid protein VP7 has good immunogenicity.Cholera toxin B subunit(CTB)is a good mucosal adjuvant.In this study,the CTB gene and VP7 gene were fused by PCR technology to obtain a 1260 bp of CTB-VP7 fusion gene.The prokaryotic expression plasmid pET28a-CTB-VP7 was constructed and transformed into E.coli(DE3),and the strain expressing the CTB-VP7 fusion protein was obtained.The molecular weight of the CTB-VP7 fusion protein was 49 kDa,which was consistent with the expected molecular weight.When the IPTG concentration was 0.08 mmol·L^(-1),the induction temperature was 37℃,the induction speed was 100 r.min^(-1),and the optimal induction time was 5.0 h,the expression of CTB-VP7 fusion protein was the maximum.The expression level of CTB-VP7 fusion protein was 2.45 mg·L^(-1).The CTB-VP7 fusion protein exists in the form of insoluble inclusion bodies in bacteria.When the fusion protein was purified,the concentration of imidazole in the elution buffer was 200 mmol·L^(-1),and the purity of the target protein was about 96%.In this study,CTB-VP7 fusion protein was obtained by gene synthesis and prokaryotic expression,which lays a foundation for further verifying whether the CTB-VP7 fusion protein can be used as a mucosal vaccine against GCRV.

关 键 词：草鱼呼肠孤病毒 VP7蛋白 CTB 粘膜疫苗 融合表达 

分 类 号：S96[农业科学—水产养殖]