哈维氏弧菌glyA基因的克隆及原核表达分析

Cloning and Prokaryotic Expression Analysis of glyA Gene of Vibrio harveyi

【目的】哈维氏弧菌(Vibrio harveyi)是存在于海水中的正常菌群,但近几年大密度养殖使它成为海水鱼致病甚至死亡的原因之一。对哈维氏弧菌ZJ0603株中的glyA基因进行克隆及原核表达分析,以期为下一步研制亚单位疫苗奠定基础。【方法】应用PCR技术克隆哈维氏弧菌菌株ZJ0603的glyA基因,并对其编码的丝氨酸羟甲基转移酶(serine hydroxylmethyltransferase,SHMT)进行理化性质、信号肽、亚细胞定位及高级结构分析。将克隆获得的glyA基因与表达载体pET-28a连接构建重组质粒pET-28a-glyA,然后构建BL21-pET-28a-glyA重组菌株,测序后选取正确的菌株用IPTG诱导并对重组蛋白进行Western blot分析鉴定。对表达重组菌株BL21-pET-28a-glyA的表达条件进行优化以获得大量蛋白。【结果】生物信息学分析结果表明,glyA基因全长为1296 bp,共编码431个氨基酸,SHMT的理论等电点为6.18,不稳定系数为28.66,总平均亲水性为-0.200,整体表现为亲水且分布在细胞质中,预期蛋白分子量为46.60 ku。成功构建表达重组质粒pET-28a-glyA并转入表达菌株中,经IPTG诱导后进行Western blot分析,结果表明成功获得了SHMT重组蛋白。【结论】用控制变量法对表达条件优化后发现,IPTG最佳诱导时间、浓度以及温度分别为4 h、0.4 mmol/L和37℃。

【Objective】Vibrio harveyi is a normal flora present in seawater,however,it has been one of the reasons for the disease and even death of seawater fish in recent years.The main purpose of this study was to clone and analyze the prokaryotic expression of glyA gene in V.harveyi ZJ0603 strain in order to lay a foundation for the development of subunit vaccine.【Method】The PCR technique was used to clone the glyA gene of V.harveyi strain ZJ0603,and the analysis on the physicochemical properties,signal peptides,subcellular localization and advanced structural of its encoded serine hydroxylmethyltransferase(SHMT)were performed.The obtained glyA gene was connected to the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-glyA,and then the recombinant strain BL21-pET-28aglyA was constructed.After sequencing,the proper strains were selected and induced with IPTG and Western blot analysis and identification of the recombinant protein were conducted.And the expression conditions of the recombinant strain BL21-pET-28a-glyA were optimized to obtain a large number of proteins.【Results】Bioinformatics analysis showed that the glyA gene had a total length of 1296 bp,a total of 431 amino acids,a theoretical isoelectric point of 6.18,an instability coefficient of 28.66,and a total average hydrophilicity of-0.200.Generally,the glyA gene was hydrophilic and distributed in the cytoplasm,and the expected molecular weight of the protein was 46.60 ku.The recombinant plasmid pET-28a-glyA was successfully constructed and transferred into the expression strain.After induction by IPTG,Western blot results showed that the SHMT recombinant protein was successfully obtained.【Conclusion】The optimal induction time,concentration and temperature of IPTG were 4 h,0.4 mmol/L and 37℃after optimizing the expression conditions by the variable-controlling method.