牛病毒性腹泻病毒NS3基因的生物信息学分析、蛋白纯化及免疫原性分析  

Bioinformatics Analysis,Protein Purification and Immunogenicity Analysis of NS3 Gene of Bovine viral diarrhea virus

作　　者：杨宁宁 徐明国 张江伟 易继海 陈创夫[1,3] YANG Ningning;XU Mingguo;ZHANG Jiangwei;YI Jihai;CHEN Chuangfu(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;Intelligent Breeding of Livestock and Poultry,Tiemenguan Vocational and Technical College,Tiemenguan 836000,China;Co-Innovation Center for Zoontic Infectious Disesses in the Western Region,Shihezi 832003,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]铁门关职业技术学院畜禽智能养殖,新疆铁门关836000 [3]西部地区高发人兽共患传染性疾病防治协同创新中心,新疆石河子832003

出　　处：《华北农学报》2023年第4期205-212,共8页Acta Agriculturae Boreali-Sinica

基　　金：河北省重点研发计划(21322912D)。

摘　　要：为了探讨牛病毒性腹泻病毒(BVDV)非结构蛋白NS3(P80),并获得具有较高免疫原性的NS3重组蛋白,利用生物信息学研究方法对NS3蛋白氨基酸进行序列分析;经PCR技术扩增NS3基因序列;通过无缝克隆技术构建pET-22b(+)-NS3重组表达质粒,将其转化至大肠杆菌BL21(DE3)感受态细胞中,诱导其表达NS3重组蛋白并提纯;通过Western Blotting方法检测其反应原性;将获得的较高纯度的NS3重组蛋白免疫BABL/c小鼠,收集血清,ELISA方法分别检测血清中IgG、IgG1和IgG2a抗体水平;最后通过病毒中和试验检测其中和病毒的能力。结果表明:NS3蛋白氨基酸序列中无信号肽和跨膜区,二级结构主要以无规则卷曲为主,且NS3氨基酸序列中含有优势抗原表位;利用PCR和无缝克隆技术成功构建了pET-22b(+)-NS3重组表达载体,经诱导表达获得了较高纯度的NS3重组蛋白,大小约为75 ku,与理论大小相符;Western Blotting结果表明,NS3重组蛋白具有较高的反应原性;ELISA检测NS3重组蛋白免疫后的小鼠能够产生高水平的IgG、IgG1和IgG2a抗体,结果显示,免疫组小鼠产生的抗体水平极显著高于PBS阴性对照组(P<0.01);血清中和抗体水平也极显著高于PBS阴性对照组(P<0.01)。总之,成功获得了纯度高且具有免疫原性的NS3重组蛋白。To explore the non-structural protein NS3(P80)of Bovine viral diarrhea virus(BVDV),and to obtain high immunogenic NS3 recombinant protein,the amino acid sequence of NS3 protein was analyzed by bioinformatics software.The sequence of NS3 gene was amplified by PCR,and the recombinant expression vector pET-22b(+)-NS3 was constructed by seamless cloning technology.The recombinant expression vector was transformed into competent cells of E.coli BL21(DE3)and induced to express NS3 recombinant protein.The reactivity of NS3 recombinant protein was detected by Western Blotting.BABL/c mice were immunized with the obtained high purity NS3 recombinant protein,and the serum was collected.The levels of IgG,IgG1 and IgG2a antibodies in serum were detected by ELISA,and the ability of neutralizing virus was detected by virus neutralization test.The results showed that there was no signal peptide and transmembrane region in the amino acid sequence of NS3 protein,the secondary structure was mainly random coil,and the NS3 amino acid sequence contained dominant antigen epitopes.The recombinant expression vector pET-22b(+)-NS3 was successfully constructed by PCR and seamless cloning techniques,and the recombinant protein of NS3 with high purity was obtained by inducing expression,with the size of about 75 ku,which was consistent with the theoretical size.Western Blotting results showed that the NS3 recombinant protein had high reactivity.ELISA test showed that mice immunized with NS3 recombinant protein could produce high levels of IgG,IgG1 and IgG2a antibodies.The antibody levels of mice immunized with NS3 recombinant protein were extremely significantly higher than those of PBS negative control group(P<0.01),and neutralizing antibody level was also significantly higher than that of PBS negative control group(P<0.01).In conclusion,this study successfully obtained NS3 recombinant protein with high purity and immunogenicity.

关 键 词：牛病毒性腹泻病毒 NS3 生物信息学 原核表达 免疫原性 

分 类 号：S823[农业科学—畜牧学]