黄喉拟水龟转铁蛋白重组表达及抗菌活性分析
RECOMBINANT EXPRESSION AND ANTIMICROBIAL ACTIVITY ANALYSIS OF TRANSFERRIN IN ASIAN YELLOW POND TURTLE
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摘要: 转铁蛋白具有多种生物学功能, 不仅参与铁的转运, 而且具有抗菌、免疫调节等功能。对黄喉拟水龟转铁蛋白(Mauremys mutica transferrin, MaTf)原核表达、重组蛋白纯化、抗菌活性检测及MaTf在蛋白水平的表达特征进行了分析研究。通过用RT-PCR技术扩增MaTf的编码序列, 克隆至载体pET-32 a (+); 转化BL21 (DE3), 构建了其原核表达载体; 优化表达条件(30℃, 4h, 0. 8 mmol/L IPTG), 使MaTf融合蛋白在BL21中高效表达; 采用His Bind柱亲和层析与浓缩的方法纯化目的蛋白, Western blot证实获得纯度较高的MaTf重组蛋白; 抑菌试验表明, MaTf重组蛋白对金黄色葡萄球菌、大肠杆菌和黏质沙雷氏菌均有明显的抑菌作用; 用MaTf重组蛋白制备多克隆抗血清, Western blot免疫印记法分析转铁蛋白在体内的表达特征, 结果显示其在肝脏、脾脏、肾脏、心脏4个组织中的表达量为：肝脏脾脏肾脏心脏, 与MaTf在RNA水平上的表达特征具有相似性。研究为探索转铁蛋白在黄喉拟水龟非特异性免疫反应中的作用提供了重要信息。Abstract: Transferrin has a variety of biological functions such as iron transportation, anti-microbial, immunoregulation, etc. In this paper, the expression pattern and the antibacterial activity of purified recombinant protein of Mauremys mutica transferrin (MaTf) were analyzed. The ORF of MaTf were obtained by RT-PCR, then ligated into pET-32 a (+) vector to construct recombinant vector for prokaryotic expression, then transformed into E. coli BL21 competent cells. The putative protein was successfully expressed with the induction of IPTG (0. 8 mmol/L) at 30℃ for 4 hours. The solubility of the expressed products was tested by PAGE electrophoresis of supernatant and precipitation separately resulted from smashed bacterial. The results indicated that the protein was in inclusion body and insoluable. The precipitation of smashed bacterial including the recombinant protein was washed with 1×binding buffer including 2M urea, resolved in 1×binding buffer including 6 M urea at 37℃ for 1h, purified with His bind column and then renatured by dialysis. The purity was tested by PAGE electrophoresis and western blot, which confirmed the high purity of the recombinant protein of MaTf obtained. The biological activity of anti-microbial was tested in vitro by assessing the effect on bacterial growth repression. According to the diameter of repressed bacteria circle on plate for equal dose of purified protein, the antibacterial effects to three kind of bacteria tested are different, which are Staphylococcus aureus Escherichia coli Serratia marcescens. The expression pattern in different tissues in M. mutica were quantified by western blot, which showed that the pattern at protein level is similar to the mRNA level, which is: liver spleen kidney heart. This research provides an important information for exploring the biological function of transferrin in non-specific immune response.
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Keywords:
- Asian Yellow Pond Turtle /
- Transferrin /
- Recombinant Expression /
- Antibacterial
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