摘要
厚壳贻贝(Mytilus coruscus)黏附蛋白分子mcofp-3(M.coruscusfoot protein-3)主要分布于贻贝足丝盘,贻贝在水环境下的黏附过程中起到关键作用,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mcofp-3的真核表达体系,并获得足够的mcofp-3黏附蛋白进行后续研究,采用酵母表达体系对mcofp-3进行了重组表达。通过PCR方法克隆厚壳贻贝的mcofp-3基因,构建mcofp-3的酵母真核表达载体pVT102U/α/mcofp-3,鉴定结果表明,重组表达质粒pVT102U/α/mcofp-3由真核载体pVT102U/α和mcofp-3的成熟肽DNA片段组成,插入的mcofp-3成熟肽DNA片段与预期序列完全一致;采用LiAC转化法将重组表达质粒转化到S78酿酒酵母中,经过RT-PCR分析以及1.0%的琼脂糖凝胶电泳检测,结果表明,重组的mcofp-3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-Tricine-SDS-PAGE检测,结果表明,重组的厚壳贻贝黏附蛋白分子mcofp-3得到了成功表达,表达产物分子量约为6kD,与预期分子量一致。
M.coruscus foot protein-3 which locates mainly in plaques plays fundamental roles in underwater adhesion between the byssus and surrounding materials.The abundance of natural mytilus foot proteins（MFP）is extremely low in mussels and the foot proteins are practically insoluble in water that makes it difficult in the further study.In order to pursue follow-up researches,here we report the novel production of a recombinant M.coruscus foot protein-3 in eukaryotic expressing vector.The eukaryotic expression vector of mcofp-3 was constructed according to the mcofp-3 gene of Mytilus coruscus.Mcofp-3 gene was obtained from Mytilus coruscus＇s mcofp-3 cDNA by PCR.Vector pMD19-T/mcofp-3 was cloned and transformed into E.coli DH5α,and the recombinant pVT102U/α/mcofp-3 was constructed and identified.The result showed that 186 bp specific fragment was amplified and identitied.Sequencing result showed that the reading frame was the same with GenBank.The result of identification and sequencing of pVT102U/α/mcofp-3 plasmid was accordance with the expected length.The recombinant pVT102U/α/mcofp-3 which was transformed into Saccharomyces cerevisiae S78 by LiAC method was analyzed preliminarily by reverse translation PCR and 1.0% agarose gel electrophoresis after fermentation process.1.0% agarose gel electrophoresis for RT-PCR amplification fragments of mRNA extracted from fermented yeast cells obtained specific strip.The result suggests that the recombinant pVT102U/α/mcofp-3 was successfully transcribed.The yeast fermentation broth was purified by CM-sepharose,and C8 reverse phase HPLC and then analyzed by Tris-Tricine-SDS-PAGE suggested the relative molecular mass around 6 kD which was consistent with expected.
出处
《生物技术通报》
CAS
CSCD
北大核心
2010年第12期148-153,共6页
Biotechnology Bulletin
基金
浙江省科技厅面上科研农业项目(2008C22026)
浙江省教育厅重点项目(20070430)
