文献类型：期刊文献

中文题名：红笛鲷T淋巴细胞酪氨酸激酶的原核表达及多克隆抗体制备

英文题名：Prokaryotic Expression and Polyclonal Antibody Preparation of Lymphocyte Cell Kinase in Red Snapper(Lutjanus sanguineus)

作者：黄瑜[1,2];张雪利[1,2];蔡佳[1,2];简纪常[1,2];吴灶和[2,3];鲁义善[1,2];樊云霞[1,2]

机构：[1]广东海洋大学水产学院,湛江524088;[2]广东省水产经济动物病原生物学及流行病学重点实验室广东省教育厅水产经济动物病害控制重点实验室,湛江524088;[3]仲恺农业工程学院,广州510225

年份：2013

卷号：29

期号：12

起止页码：113

中文期刊名：生物技术通报

外文期刊名：Biotechnology Bulletin

收录：CSTPCD、、北大核心2011、CSCD_E2013_2014、北大核心、CSCD

基金：国家自然科学基金项目(41240041);广东省科技厅国际合作项目(2012B050600029)

语种：中文

中文关键词：红笛鲷;lck基因;原核表达;Western;blot分析;抗原性分析

外文关键词：Lutjanus sanguineus Lck gene Prokaryotic expression Western blot analysis Antigenicity analysis

中文摘要：为研究红笛鲷(Lutjanus sanguineus)T淋巴细胞酪氨酸激酶(Lymphocyte cell kinase,LCK)蛋白在机体中的组织分布情况,克隆出红笛鲷lck(LS-lck)基因序列,经酶切、连接等步骤,构建重组质粒pET-28a-LS-LCK,再将其转入大肠杆菌BL21(DE3)菌株后进行IPTG诱导表达。通过表达条件的优化,重组蛋白在37℃、0.03 mmol/L IPTG条件下诱导4 h后获得最大表达量,且主要以包涵体形式存在。Western blot检测重组蛋白可与鼠抗His-tag单克隆抗体发生特异性结合,表明为目的蛋白。利用纯化后的rLS-LCK重组蛋白免疫小鼠,获得了1∶40 000高效价的抗血清,Western blot分析显示,融合蛋白能被小鼠的阳性血清识别,说明rLS-LCK融合蛋白具有较好的免疫反应性和免疫原性。

外文摘要：To investigate the distribution of Lymphocyte cell kinase（LCK）protein in the Lutjanus sanguineus tissues, the red snapper lck（LS-lck）gene sequences was cloned, and the recombinant plasmid pET-28a-LS-LCK was constructed, which was transferred into E. coli BL21（DE3）strain for translation into protein by IPTG induction. Through the optimization of expression conditions, the recombinant protein obtains the maximum expression level when the cells were induced at 37℃ in 0.1 mol/L of IPTG for 4 hours. The expected protein was mainly detected in the insoluble fraction of E. coli cell lysates. Western blot analysis showed that the recombinant protein could be combined with mouse anti-His-Tag Mab, so the expression protein was definitely confirmed as the target protein. Mice were immunized with rLS-LCK recombinant protein to produce antiserum whose titer is 1∶ 40 000. Western blot analysis showed that the fusion protein can be recognized by the positive serum of mice. The result illustrated that the rLS-LCK fusion protein has good immunoreactivity and immunogenicity.