人β-防御素-3基因定点突变,原核表达载体构建和融合蛋白表达

Site-directed mutation and prokaryotic expression vector construction and fusion protein expression of human beta-defensin 3

目的:利用PCR技术对人β防御素3基因进行定点突变,将突变后的基因连接入pGEX4T1表达载体.方法:采用PCR体外定点突变技术,设计两对引物,引入一个突变点,通过重叠延伸法进行两次PCR扩增,使人β防御素3基因第27位密码子由GAG突变为CGA,将突变后的片段克隆入pGEX4T1质粒中,转化至E.coliBL21,对鉴定含有目的片段的克隆进行测序.结果:获得预期大小的突变产物,经序列鉴定证实为突变的人β防御素3基因.将诱导后的菌体进行SDSPAGE电泳,在Mr31000处可见明显的高表达带.结论:获得突变型人β防御素3基因,构建了表达载体,融合蛋白得到表达,为基因工程制备肽类抗生素奠定了基础.

AIM： Site-directed mutation of human β-defensin-3 gene was conducted by PCR protocol and the mutated gene was subcloned into prokaryotic expression vector. METHODS： A two-step polymerase chain reaction （PCR） was used for the site-directed mutagenesis. Two sets of primers （P1, P2, P3, P4） were designed according to human β-defensin-3 gene sequence and the mismatch was introduced into P2 and P3. Mutagenesis was performed in a two-step PCR and the amplified fragments from the second PCR, which contain the mutation site, were subcloned into the vector pGEX-4T-I. Recombinant pGEX-4T-1 vectors were transformed into compotent cell BL21. Restriction analysis and PCR were performed to identify the recombinant plasmids containing the DNA fragment of interest, followed by sequencing. RESULTS： We obtained a 138 bp DNA fragment which was identical to human β-defensin-3 mutant. SDS-PAGE profile showed a clear protein band with a relative molecular weight of 31 000. CONCLUSION： Human β-defensin-3 gene is successfully mutated and expressed, which will help the preparation of antimicrobial peptide.