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。
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.
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第12期113-118,共6页
Biotechnology Bulletin
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)
