高表达牛气管防御素的真核重组载体的构建及其鉴定

Construction and Identification of Eukaryotic Expression Vector of Highly Expressing Bovine Tracheal Antimicrobial Peptide

根据GeneBank中牛TAP的序列设计了1对特异性引物,采用RT-PCR方法从牛气管组织扩增牛TAP基因,并克隆到pEASY-T3载体。测序结果表明,扩增的片段含有144 bp核苷酸,编码48个氨基酸,与已报道的bTAP有1个氨基酸不同。该基因与真核表达载体FG9重组,经过PCR,酶切和测序鉴定,筛选牛TAP基因重组真核表达载体。使用脂质体法将FG9-TAP重组质粒转染293T细胞,斑点ELISA检测结果表明,成功构建了高表达牛TAP基因的真核表达载体,为抗金黄色葡萄球菌转牛TAP基因奶牛培育研究奠定基础。

One pair of specific primers of bovine tracheal antimicrobial peptide（TAP） gene was designed and synthesized according to the published sequence in GeneBank.The mRNA of bovine TAP was cloned from tracheas of cow by RT-PCR and cloned into the vector pEASY-T3.The full length cDNA of bovine TAP consistes of 144 bp encoding 48 amino acid residues which there was one different amino acid compared with bovine TAP gene reported.The gene recombined into the eukaryotic expression vector FG9.After identification with restriction digestion,PCR and sequencing,recombinant bovine TAP expression vector was obtained.Recombinant plasmid FG9-TAP were transfected into 293T cells by the method of lipofectamine transfection.Dot-ELISA test results showed that the recombinant expression vector highly expressing TAP was successfully constructed.This work has laid the base for cultivating the bovine TAP gene transfer cows with resistantance to Staphylococcus aureus.