用PCR定点突变法构建人抗菌肽FALL-39基因突变体及其功能的研究

Construction of a point-directed mutant of human antimicrobial peptide FALL-39 and its functional study

目的 用PCR定点突变法构建人抗菌肽FALL 3 9基因突变体 ,并比较研究FALL 3 9及其突变肽的功能。方法 采用PCR体外定点突变技术 (PCR SDM ) ,设计一对方向相反的引物 ,其中一个引物引入一个突变点 ,应用高保真的PolybestDNA多聚酶进行含FALL 3 9肽的PGEXλ1T质粒的PCR扩增 ,使FALL 3 9第 2 4位密码子由CAG变为AAG ,将扩增片段自身连接后克隆入工程菌JM 10 9中表达其突变肽 (FALL 3 9 lys2 4) ,纯化后与FALL 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FALL 3 9 lys2 4抗菌活性增强 ,在抗菌剂量时对红细胞的溶解作用未见明显增加 ,较大剂量时对红细胞的溶解作用略有增加。结论 用高保真的PolybestDNA多聚酶进行PCR定点突变技术简单、有效 ,获得一个FALL 3 9 Lys2 4突变子 ;其表达分子的抗菌活性明显增强 ,对红细胞的溶解作用在抗菌剂量时未见明显增加 ,较大剂量时略有增加 ,这些结果提示在FALL 3 9分子中增加碱性氨基酸可以增强其抗菌活性 ,但必须注意避免溶血的副作用。

Objective To construct a mutant of FALL 39 and prokariotic expressive vector, and to compare its antibacterial activity with that of Fall 39. Methods A one step polymerase chain reaction (PCR) was used for the point directed mutation. The primers (P1, P2) were designed according to the sequence encoding mature FALL 39, and mismatch was introduced into P2 with AAG for CAG substitution at position 24. Mutation was performed in a one step PCR, the amplified fragment encircled linked and transductioned into the E.coli . Mutation was verified by DNA sequence analysis. Antibacterial activities of the E.coli based product against E.coli and P.aeruginosa were detected by MIC and MBC value. The haemolysis activity of the peptides was tested against human red blood cells (hRBC). Results A mutant of FALL 39, whose CAG at position 24 was substituted by AAG, was obtained by the one step PCR using Polybest DNA polymerase. The antibacterial activities of the E.coli based product of the recombinant mutant Fall 39 Lys24 against E.coli ML 35p and P.aeruginosa ATCC27853 were much stronger than that of FALL 39. MIC and MBC of the FALL 39 against E.coli were 18 75 and 50μg/ml, whereas FALL 39 Lys24 were 9 38 and 25μg/ml respectively. The haemolysis activity of the peptide FALL 39 Lys22 was increased only at the dose of more than antibacterial activities . Conclusion A point directed FALL 39 Lys24 mutant was obtained by the one step PCR using Polybest DNA polynerase. Its antibacterial activity was increased, but when compared to parent FALL 39, certain effect of the mutant on hemolysis was observed, which suggested that increasement of the cationic character of FALL 39 mutant might enhance its antibacterial activity, but the hemolytic effect would be a matter of concern.