TAT-BPI融合蛋白原核表达载体的构建

目的 探讨使BPI能高效率穿透细胞膜或多种屏障,中和不同解剖部位的LPS,构建TAT-BPI原核表达载体并鉴定的方法,为研制多肽抗生素打下基础.方法 人工合成编码TAT蛋白转导区的DNA片段,插入载体pUC57后得到pUC57-TAT重组质粒;提取正常人外周血多形核粒细胞总RNA, 采用RT-PCR技术扩增BPI N端cDNA片段,构建pUC57-TAT-BPI克隆载体;用酶切和双脱氧测序法进行鉴定.结果 获得含有约630 bp的TAT-BPI融合蛋白基因片段序列,测序分析与Genebank中该序列相符,同源性分析TAT序列中第10、30位核苷酸有突变,氨基酸同源性为93%,蛋白质同源性为90%,在BPI序列中234、454、495、556位核苷酸有突变,氨基酸同源性为98%,蛋白质同源性为95%.结论 成功构建了TAT-BPI融合蛋白的原核表达载体,为进一步研究BPI的抗菌作用奠定了基础.Abstract：Objective To enable BPI efficiently penetrate the cell membrane or a variety of barriers, and neutralize endotoxin in different anatomical parts, construct and identify a prokaryotic expression vector of TAT-BPI, in order to lay the foundation for new bio-antibiotic development. Methods A synthesized DNA fragment encoding TAT protein transduction domain was inserted into pUC57 vector. Then Total RNA was extracted from human polymorphonuclear neutrophils(PMN) and then the human BPI cDNA gene was amplified by RT-PCR. The PCR product was cloned into pUC57 plasmid and the sequence was confirmed by restriction enzyme digestion and dideoxy-mediated-chain termination. Results Restriction enzyme digestion and DNA sequence analysis revealed that bioactive N-terminal fragment of BPI were not only identical to those of the published human BPI sequence but also were contained in the recombinant vector. Conclusions pUC57-TAT-BPI is successfully constructed, which lays the foundation for studying the protein of BPI.

Construction of TAT-BPI fusion protein prokaryotic expression vector

TAT Bactericidal / perme-ability increasing protein(BPI) Fusion protein