厚壳贻贝足丝盘黏附蛋白mcofp-3的重组真核表达.pdfVIP

生物技术通报 BIO TE CHN OLOG Y B ULLETIN 2010 12 m cofp3 李楠楠 王智平 鲁涛 廖智 ( , 316004) : 厚壳贻贝 (M y tilu s coruscus) 黏附蛋白分子 mcofp3 (M . coruscus foot protein3)主要分布于贻贝足丝盘, 贻贝在 水环境下的黏附 程中起到关键作用,但因其难溶于水且在贻贝足丝盘中含量极低, 故妨碍了对其进行深入研究为建立厚 壳贻贝足丝蛋白 m cofp3的真核表达体系, 并获得足够的 m cofp3黏附蛋白进行后续研究, 采用酵母表达体系对m cofp3进行 了重组表达通 PCR方法克隆厚壳贻贝的 m cofp3基因, 构建 m cofp3的酵母真核表达载体 pVT 102U //m cofp3, 鉴定结果 表明, 重组表达质粒 pVT 102U //m cofp3由真核载体 pVT 102U /和 m cofp3的成熟肽 DNA 片段组成,插入的 m cofp3 成熟肽 DNA片段与预期序列完全一致; 采用 Ai C转化法将重组表达质粒转化到 S78酿酒酵母中, 经 RTPCR 分析以及 10% 的琼 脂糖凝胶电泳检测, 结果表明, 重组的 mcofp3得到了成功的转录; 发酵菌液经阳离子交换柱及高效液相色谱分离, 以及 Tris T ricineSDSPAGE检测, 结果表明,重组的厚壳贻贝黏附蛋白分子m cofp3得到了成功表达, 表达产物分子量约为6 kD, 与预期 分子量一致 : 厚壳贻贝 足丝蛋白 m cofp3 PCR 真核表达载体 酿酒酵母 S78 R ecom binant Expression of m cofp3 from My tilus coruscus P laque iNannan W ang Zhip ing u Tao iao Zhi (L aboratory of M ar ineB iolog ical Source and M olecular E ng ineering, Colleg e of M arine Sciences, Zhej iang O cean University, Zhoushan 316004 A bstrac:t M. coruscus foot protein3w hich locatesm ainly in plaques p lays fundamental roles in underw ater adhesion betw een the byssus and surround ing m aterials. The abundance of naturalmytilus foot proteins (MFP) is extrem ely low in mussels and the foot pro teins are practically insoluble inwater that m akes it d ifficu lt in the further study. In order to pursue followup researches, here we report the novel production of a recom binantM . coruscus foot protein3 in eukaryotic expressing vector. The eukaryotic expression vector of m cofp3 was constructed according to them cofp3 gene ofM y tilus coruscus. M cofp3 gene was obtained fromM y tilus coru scus sm cofp 3 cDNA by PCR. Vector pMD19T /m cofp3w as cloned and transform ed intoE. coli DH 5, and the recomb inant pVT 102U /