作　　者： ; ; ; ; ; ; (郭保党）;

机构地区： 南阳师范学院

出　　处： 《河南农业科学》 2014年第12期140-143,共4页

摘　　要： 为获得具有生物学活性的鸡胸腺肽β4、β15,根据大肠杆菌密码子偏爱性,设计合成鸡胸腺肽Tβ4、Tβ15基因片段,采用重叠PCR获得完整的Tβ4、Tβ15基因,将其连接到pET-28a(+)载体上,转化至大肠杆菌BL21(DE3)进行诱导表达,经SDS-PAGE鉴定,应用镍柱亲和层析纯化蛋白质,采用E玫瑰花环试验测定其生物学活性。结果显示,成功构建大肠杆菌表达载体pET28a-Tβ4和pET28a-Tβ15,目的蛋白在大肠杆菌中大量可溶性表达,蛋白质分子量大小均为6kD,镍柱亲和层析获得了纯化的蛋白质。E玫瑰花环试验结果显示,重组Tβ4的活力为35.5%,Tβ15的活力为18.7%,均能够明显增强T淋巴细胞的活性。表明Tβ4和Tβ15基因得到成功表达和纯化,并具有良好的生物学活性。 This study aimed to get chicken thymosinβ4andβ15.According to E.coli codon preference,chicken thymosinβ4andβ15cDNA sequences were designed and synthesized.Two full length genes were amplified by overlap PCR technique.Then the Tβ4、Tβ15 genes were cloned into vector pET-28a（＋）respectively.The recombinant plasmids were transferred into E.coli BL21（DE3）for expression.The two proteins were purified by Ni 2＋affinity chromatography after analysis by SDS-PAGE,and then their activities were detected by E rosette test.The results showed that two expression plasmids pET28a-Tβ4 and pET28a-Tβ15 were constructed successfully and proteins were expressed efficiently in soluble form.The molecular of two proteins were all 6kD.The two purified proteins were obtained by Ni 2＋affinity chromatography.The E rosette test demonstrated that the activity of Tβ4was 35.5%,and which of Tβ15was 18.7%.These results indicated that the protein Tβ4and Tβ15could significantly enhance the activity of T cells.In conclusion,the soluble proteins Tβ4and Tβ15 were highly expressed in E.coli and displayed high level of biological activity.

关 键 词： 胸腺肽 基因 胸腺肽 基因 表达 纯化 玫瑰花环试验

领　　域： [农业科学] [农业科学] [农业科学]