牛乳铁蛋白基因N-lobe的cDNA克隆及毕赤酵母表达载体的构建

cDNA Cloning of Bovine Lactoferrin N-lobe Gene and Construction of Pichia Pastoris Expression Vector

以牛乳腺组织总RAN为模板,经RT-PCR得到牛乳铁蛋白基因的N-lobe片段并将其克隆到pGM-T克隆载体上,最后将其连入表达载体pPICZαA中,构建牛乳铁蛋白基因N-lobe的酵母表达载体。结果表明:克隆片段大小为1028bp,与Gen Bank中登录的序列的相应部位相比,所获牛乳铁蛋白N-lobe cDNA序列的同源性为99.7%;重组表达质粒经酶切和PCR鉴定构建正确,为酵母表达乳铁蛋白基因N-lobe奠定了基础。

Using the total RAN from bovine mammary grand as a template to construct Pichia pastoris Expression Vector of bovine Lactoferrin N-lobe Gene. the N- lobe gene of bovine lactoferrin have been amplified with RT-PCR method. The obtained DNA fragment was then cloned into pGM-T vector. Pos - itive recombinant plasmid was digested with EcoRl and XbaI and the N-lobe fragment was reco- rered. The Pichia pastoris expression Vector was constructed by inserting the N-lobe fragment into vector of pPICZα A The sequencing results of recombinant cloning vector demonstrated the obtained N-lobe gene has 1028bp and 99.7 % homology with it＇s gene in GenBank. The sequencing results of recombinant Expression vector proved by restriction enzymes and sequencing that our construction is correct. It provides bases for Expression of N-lobe in Pichia pastoris.