革胡子鲶生长激素成熟肽cDNA克隆及其在大肠杆菌中的表达

目的]构建革胡子鲶生长激素基因原核表达体系。方法]采用PCR方法,扩增得到了革胡子鲶生长激素基因成熟肽的cDNA序列,将该序列克隆至原核表达载体pRSET-A,构建重组质粒pRSET-mGH,并在大肠杆菌BL21(DE3)pLysS中进行表达。结果]革胡子鲶生长激素成熟肽cDNA序列全长534 bp,编码1个由178个氨基酸残基组成的生长激素成熟肽,分子量为20.28 kDa,等电点为5.93。SDS-PAGE和Western blot分析表明,表达的目的融合蛋白分子量为27.41 kDa,主要以非可溶性的包涵体形式存在于菌体沉淀中。目的融合蛋白的表达量高达细菌沉淀蛋白总量的36.8%。结论]该研究为革胡子鲶生长激素的产业化开发和应用奠定了基础。

CDNA Cloning of Mature Peptide of Growth Hormone from Clarias lazera and Its Expression in Escherichia coli

Objective]The aim was to construct the prokaryotic expression system of the growth hormone gene from Clarias lazera.Method] The cDNA sequence of mature peptide of growth hormone gene from C.lazera was amplified by PCR,then cloned into the prokaryotic expression vector pRSET-A to construct the recombinant plasmid pRSET-mGH and expressed in the Escherichia coli BL21(DE3)pLysS.Result]The full length of the cDNA sequence of mature peptide of growth hormone gene from C.lazera was 534 bp,encoding a mature peptide of growth hormone composed by 178 amino acid residues.Its molecular weight was 20.28 kDa and the isoelectric point was 5.93.Analysis of SDS-PAGE and western blot indicated that the expressed fusion protein had molecular weight of 27.41 kDa and the protein existed mainly in the deposition of bacterial in the form of undissolved inclusion body.The expression of the target fusion protein accounted for 36.8% of total cellular protein sediments.Conclusion]The research laid the foundation for the industrialization development and application of growth hormone from C.lazera.