人抗菌肽FALL-39基因定点突变体的构建及其大肠杆菌表达产物的抗菌活性研究  

Construction of a Site-directed Mutant of FALL-39 and Antibacterial Activity of Its E.coli-based Product

作　　者：杨云霞[1] 冯云[2] 王伯瑶[2] 

机构地区：[1]四川大学华西基础医学与法医学院药理学教研室,成都610041 [2]四川大学华西基础医学与法医学院感染与免疫研究室

出　　处：《四川大学学报（医学版）》2003年第3期390-394,共5页Journal of Sichuan University(Medical Sciences)

基　　金：美国纽约中华医学基金 ( 98-681)资助

摘　　要：目的　构建人抗菌肽 FAL L - 3 9定点突变子及研究其抗菌活性。方法　采用 PCR体外定点突变技术( PCR- SDM) ,设计两对引物 ,引入一个突变点 ,通过重叠延伸法两次 PCR扩增 ,使 FAL L - 3 9第 3 2位密码子由AAT突变为 AAG,将扩增片段克隆入 PGEXλ1T质粒中 ,转化大肠杆菌 JM10 9,并用 IPTG诱导 ,表达的 FAL L -3 9- lys3 2经纯化后与 FAL L - 3 9进行抗菌活性比较。结果　 DNA测序结果表明在预期位点发生突变 ,突变后的FAL L - 3 9- lys3 2蛋白对大肠杆菌 ML - 3 5 p的抗菌活性明显增强 ,其最低抑菌浓度 ( MIC)和最低杀菌浓度 ( MBC)分别由 FAL L - 3 9蛋白的 18.75和 3 7.5 0μg/ m l降低为 FAL L - 3 9- L ys- 3 2的 10 .94和 2 1.88μg/ m l,对绿脓杆菌ATCC2 785 3的 MIC和 MBC值分别为 FAL L - 3 9蛋白的 3 1.2 5和 3 7.5 0μg/ m l降低为 FAL L - 3 9- L ys- 3 2的 18.75和 3 1.2 5μg/ ml。两种蛋白均在含 Medium E的 L B培养基中表现出较高活性 ,在 L B培养基中抗菌活性最低 ,但在同一种培养基中 FAL L - 3 9- L ys- 3 2蛋白抗菌活性要高于 FAL L - 3 9蛋白。结论　用两步 PCR定点突变技术获得一个 FAL L - 3 9- L ys3 2突变子 ,其表达分子的抗菌活性明显增强 ,提示在 FAL L - 3 9分子中增加碱性?Objective Constructing a point-directed mutant of FALL-39 and prokaryotic expressive vector, and determining its antibacterial activity. Methods A two-step polymerase chain reaction (PCR) was used for the site-directed mutagenesis. Two sets of primers (P 1, P 2, P 3, P 4) were designed according to FALL-39 gene sequence and mismatch was introduced into P2 for a substitution of AAG for AAT at position 32. Mutagenesis was performed in a two-step PCR, the amplified fragments from the second PCR, which contain the mutation site, were subcloned into the vector PGEXλ1T and verified by sequencing analysis. Antibacterial activities of the E.coli-based product against E.coli and P.aeruginosa were detected by using minimal inhibitory concentration(MIC), minimal effective concentration(MEC) and minimal bactericidal concentration(MBC). Results A mutant of FALL-39, whose AAT at position 32 is substituted by AAG, was obtained by the two-step PCR. The antibacterial activities of the E.coli-based product of the recombinant FALL-39-Lys32 against E.coli ML-35p and P.aeruginosa ATCC27853 were much stronger than those of FALL-39. MIC and MBC of the FALL-39 against E.coli were 18 75 μg/ml and 37.50 μg/ml, whereas those of FALL-39-Lys32, 10.94 μg/ml and 21.88 μg/ml respectively. Conclusion A point-directed FALL-39-Lys32 mutant was obtained by using two-step PCR and its antibacterial activity was increased, suggesting that increased cationic FALL-39 mutant might enhance its antibacterial activity.

关 键 词：人抗菌肽 FALL-39 基因 定点突变 大肠杆菌 表达 抗菌活性 研究 

分 类 号：R346[医药卫生—基础医学]