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

生物技术通报 ›› 2013, Vol. 0 ›› Issue (12): 113-118.

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

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}

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

收稿日期:2013-06-25 出版日期:2013-12-20 发布日期:2013-12-20
作者简介:黄瑜，男，硕士，研究方向：水产动物病害防治；E-mail：472604742@qq.com
基金资助:
国家自然科学基金项目（41240041），广东省科技厅国际合作项目（2012B050600029）

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

Huang Yu^{1, 2}, Zhang Xueli^{1, 2}, Cai Jia^{1, 2}, Jian Jichang^{1, 2}, Wu Zaohe^{2, 3}, Lu Yishan^{1, 2}, Fan Yunxia^{1, 2}

1. Fisheries College of Guangdong Ocean University，Zhanjiang 524088
2. Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions，Zhanjiang 524088
3. Zhongkai University of Agriculture and Engineering，Guangzhou 510225

Received:2013-06-25 Published:2013-12-20 Online:2013-12-20

摘要/Abstract

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。

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

Abstract:

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.

Key words: Lutjanus sanguineus, Lck gene, Prokaryotic expression, Western blot analysis, Antigenicity analysis

黄瑜, 张雪利, 蔡佳, 简纪常, 吴灶和，鲁义善, 樊云霞. 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[J]. 生物技术通报, 2013, 0(12): 113-118.

Huang Yu, Zhang Xueli, Cai Jia, Jian Jichang, Wu Zaohe, Lu Yishan, Fan Yunxia. Prokaryotic Expression and Polyclonal Antibody Preparation of Lymphocyte Cell Kinase in Red Snapper（Lutjanus sanguineus ）[J]. Biotechnology Bulletin, 2013, 0(12): 113-118.

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