维氏气单胞菌AcrV基因的克隆与原核表达

Cloning and prokaryotic expression of AcrV gene of Aeromonas veronii

【目的】克隆维氏气单胞菌(Aeromonasveronii)低钙反应蛋白(AcrV)并进行原核表达,为进一步研究AcrV蛋白的结构功能及AcrV基因工程亚单位疫苗的制备奠定基础。【方法】克隆维氏气单胞菌TH0426菌株AcrV基因,通过生物信息学软件分析其编码蛋白的理化性质、疏水性、信号肽、跨膜区、二级结构及三级结构。将AcrV基因克隆到pET-30a(+)原核表达载体上,构建原核重组表达质粒pET-30a(+)-AcrV,将其转化至大肠杆菌BL21(DE3)中,进行EcoRⅤ和XhoⅠ双酶切鉴定和测序鉴定。用IPTG对pET-30a(+)-AcrV进行诱导表达,对IPTG浓度(0.2,0.4,0.6和0.8mmol/L)和诱导时间(2,3,4和5h)进行优化。在最佳条件下诱导表达重组蛋白AcrV,经镍柱纯化且尿素梯度复性后免疫BALB/c小鼠,制备多克隆抗体,进行SDS-PAGE及Westernblot分析,对目的蛋白的免疫原性进行检测。【结果】成功获得了1086bp的AcrV全基因,编码361个氨基酸。AcrV蛋白是无跨膜结构且不编码信号肽的蛋白,有2个高度保守的结构功能域,属于全α型蛋白。成功构建了原核重组表达质粒pET-30a(+)-AcrV,其最佳诱导表达条件为IPTG0.8mmol/L诱导5h。SDS-PAGE及Westernblot分析结果显示,AcrV蛋白能被鼠抗阳性血清识别,表明其具有一定的反应原性。【结论】成功克隆了1086bp的AcrV基因,分析了其编码蛋白的生物信息;获得了免疫原性良好的重组AcrV蛋白。

【Objective】The AcrV gene of Aeromonas veroni(A.veronii)was cloned and expressed in prokaryotic cells to provide basis for further study on structural function of AcrV protein and preparation of AcrV genetic engineering subunit vaccine.【Method】The AcrV gene of A.veronii strain TH0426 was cloned,and the physicochemical properties,hydrophobicity,signal peptide,transmembrane region,secondary structure and tertiary structure of the encoded protein were analyzed by bioinformatics software.The AcrV gene was cloned into the prokaryotic expression vector of pET-30a,and the prokaryotic recombinant expression plasmid pET-30a(+)-AcrV was constructed and transformed into E.coli BL21(DE3)for double restriction enzyme digestion with Eco RⅤand XhoⅠand sequencing identification.The expression of pET-30a(+)-AcrV was induced by IPTG,and IPTG concentration(0.2,0.4,0.6 and 0.8 mmol/L)and induction time(2,3,4 and 5 h)were optimized.Under the optimal conditions,the recombinant protein AcrV was induced to express.After purified by Ni-NTA and urea gradient renaturation,the BALB/c mice were immunized to prepare polyclonal antibodies.Then SDS-PAGE and Western blot analysis were performed to detect the immunogenicity of the target protein.【Result】The length of AcrV gene was 1 086 bp,encoding 361 amino acid residues.The AcrV protein is a protein with no transmembrane structure and encoding no signal peptide.There were two highly conserved structural functional domains,belonging to allα-type protein.The prokaryotic expression plasmid pET-30a(+)-AcrV was successfully constructed,and the optimal expression condition was IPTG 0.8 mmol/L and induced for 5 h.SDS-PAGE and Western blot analysis showed that AcrV protein could be recognized by mouse anti-positive serum,indicating that it has certain reactogenicity.【Conclusion】The 1 086 bp AcrV gene was successfully cloned,and the biological information of the encoded protein was analyzed.The recombinant protein with good immunogenicity was obtained after induction of expression.