2种泥鳅生长激素基因cDNA的克隆和原核表达研究

从脑垂体中提取总RNA,用RT-PCR方法扩增并克隆到了泥鳅和大鳞副泥鳅的生长激素(GH)基因cDNA,分析了其核苷酸序列和推测的氨基酸序列.结果表明:2种泥鳅生长激素基因的开放阅读框(ORF)均包括633个核苷酸,编码210个氨基酸,其中包括22个氨基酸的信号肽和188个氨基酸的成熟肽.在cDNA序列上2者有11个核苷酸的差异;在蛋白质水平上,2者的差异仅为1个氨基酸,位于信号肽中,在成熟多肽中,2者的氨基酸序列相同.把GH成熟肽的cDNA克隆入表达载体pET-28 a,用IPTG诱导重组蛋白的表达,其表达量超过细胞蛋白总量的50%,主要为不溶性的包含体.细菌裂解液沉淀溶于浓度为8mol/L的尿素后,用固定化金属配体亲和层析纯化,获得了分子量为24 kDa的单一蛋白带.

GH cDNA Clone and Pronucleus Expression of Misgurnus Anguillicaudatus and Paramisgurnus Dabryanus

GH cDNA was amplified and cloned from total RNA isolated from pituitary gland of Misgurnus anguillicaudatus and Paramisgurnus dabryanus by RT-PCR.The accession numbers in GenBank were DQ350433 and DQ350432 respectively.There were 11 different sites in cDNA.Only one different site was found between their peptides,which presented in signal peptide.Each sequence included an ORF of 633 bp which encoded a precursor of 210 aa comprising a 22 aa signal peptide and a 188 aa mature protein.The identity of the mature GH between Misgurnus anguillicaudatus and Paramisgurnus dabryanus was 100 %.The cDNA encoding the mature peptide of GH was subcloned into expression vector pET-28a and expressed in E.coli BL21(DE3) as fusion polypeptide containing a His6 at the N-terminus.By the induction of IPTG,the recombinant protein was expressed and composed of more than 50 % of the total bacterial proteins and accumulated as inclusion bodies.The inclusion bodies were solubilized in 8M urea and further purified by immobilized metal affinity chromatography(IMAC) under denaturing condition due to the fused N-terminal His-Tag.The purified GH fusion polypeptide migrated as a single band of 24 kD on SDS-PAGE.