炭疽芽孢杆菌PA-LF1融合蛋白的原核表达及其反应原性研究  

Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis

作　　者：吕双飞 马勋[1] 王静[1] 孔翠莲 寇丽君 刘彩霞 史唯地 康立超[2] 任慧杰 LYU Shuangfei;MA Xun;WANG Jing;KONG Cuilian;KOU Lijun;LIU Caixia;SHI Weidi;KANG Lichao;REN Huijie(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China;Institute of Agricultural Quality Standards and Testing Technology,Xinjiang Academy of Agricultural Sciences,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院,石河子832000 [2]新疆农垦科学院农业质量标准与检测技术研究所,石河子832000

出　　处：《中国畜牧兽医》2023年第2期674-683,共10页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31860712、32160834)。

摘　　要：【目的】构建炭疽芽孢杆菌弱毒株C40-202 PA-LF 1融合基因表达载体并对其进行原核表达和反应原性检测,为炭疽亚单位疫苗制备、炭疽诊断和疫苗免疫效果检测提供依据。【方法】根据GenBank中登录的炭疽芽孢杆菌PA和LF基因序列设计2对引物,利用PCR方法从炭疽芽孢杆菌弱毒株C40-202中分别扩增出PA和LF 1基因片段,经XhoⅠ限制性内切酶消化后,通过黏性末端进行连接,获得PA-LF 1融合基因片段,利用SWISS-MODEL分析软件分别对PA、LF1和PA-LF1融合蛋白进行三级结构预测;将融合基因片段克隆至pET32a(+)质粒中,构建重组质粒pET32a-PA-LF1,并将重组质粒pET32a-PA-LF1转化大肠杆菌BL21(DE3)感受态细胞进行诱导表达,SDS-PAGE分析重组蛋白表达,Western blotting鉴定融合蛋白并分析其反应原性。【结果】从炭疽芽孢杆菌弱毒C40-202株成功扩增出PA、LF 1和PA-LF 1融合基因;成功构建了炭疽芽孢杆菌PA-LF 1融合基因表达质粒pET32a-PA-LF1,融合蛋白三级结构预测可折叠为正确的空间构象;构建的重组质粒经诱导表达、纯化和SDS-PAGE,获得分子质量为96 ku的融合蛋白,以包涵体形式表达;经Western blotting验证,纯化后的重组蛋白与经Ⅱ号炭疽芽孢苗免疫后的绵羊血清发生特异性结合,表明其具有良好的反应原性。【结论】PA-LF1融合蛋白具有良好的反应原性,可为炭疽诊断和疫苗免疫效果检测提供理论依据,同时也可作为一种炭疽亚单位疫苗的候选组分。【Objective】The purpose of this experiment was to construct the PA-LF 1 fusion gene expression vector of Bacillus anthracis(B.anthracis)attenuated C40-202 strain and to detect its prokaryotic expression and regenicity,so as to provide the basis for the preparation of anthrax subunit vaccine,the diagnosis of anthrax and the detection of vaccine immune effect.【Method】Two pairs of primers were designed based on the sequences of B.anthracis PA and LF genes in GenBank,and the PA and LF1 gene fragments were amplified from B.anthracis weakly virulent strain C40-202 by PCR.After digestion by XhoⅠrestriction endonuclease,the PA-LF 1 fusion gene fragment was obtained by connecting the sticky ends.SWISS-MODEL analysis software was used to predict the tertiary structures of PA,LF1 and PA-LF1 fusion proteins respectively.The fusion fragments were cloned into pET32a(+)plasmid to construct recombinant plasmid pET32a-PA-LF1,and the recombinant plasmid pET32a-PA-LF1 was transferred into E.coli BL21(DE3)competent cells for induced expression.SDS-PAGE was used to analyze the expression of the recombinant protein,and Western blotting was used to identify the fusion protein and analyze its reactogenicity.【Result】PA,LF 1 and PA-LF 1 fusion genes were successfully amplified from B.anthracis weakly virulent strain C40-202.The B.anthracis PA-LF 1 fusion gene expression plasmid pET32a-PA-LF1 was successfully constructed,and the tertiary structure of the fusion protein was predicted to fold to the correct spatial conformation.The recombinant plasmid was induced,purified and detected by SDS-PAGE,and the fusion protein with molecular weight of 96 ku was obtained.Western blotting showed that the purified recombinant protein had specific binding with the sheep serum immunized with anthrax spore vaccineⅡ,indicating that it had good reactivity.【Conclusion】The PA-LF1 fusion protein had good reactogenicity and could provide a theoretical basis for anthrax diagnosis and vaccine immunity testing,and also could be used as a candida

关 键 词：炭疽芽孢杆菌 PA-LF 1基因 克隆 原核表达 

分 类 号：S852.61[农业科学—基础兽医学]