Yang Yun-xia, Feng Yun, Wang Bo-yao, Wu Qi
Department of Pharmacology, West-China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Acta Pharmacol Sin. 2004 Feb;25(2):239-45.
To construct PGEX-1lambdaT-FALL-39 expression vector and its mutant vector, and study the relationship of function and structure.
A cDNA encoding mature FALL-39 was cloned from SPCA-1 cell mRNA and the prokaryotic expression vector PGEX-1lambdaT-FALL-39 was constructed. Two kinds of polymerase chain reaction (PCR) for the site-direction mutagenesis were used to construct FALL-39 mutant expression vector, FALL-39-Lys-32 and FALL-39-Lys-24. Minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration were used to assay the antibacterial activities of these peptides. Effects of different solution on the antibacterial activity of FALL-39 and FALL-39-Lys-32 were observed by CFU determination. The hemolytic effects of these peptides were also examined on human red blood cells.
Two site-specific mutants FALL-39-Lys-32 and FALL-39-Lys24 were obtained by PCR-induced mutagenesis. In comparison with two-step PCR which required two pairs of primers, one step PCR which required one pair of primers is a simple and efficient method for the PCR based site-specific mutagenesis. Using the prokaryotic expression system, the E coli-based products of recombinant FALL39 and its mutant peptides were also obtained. The antibacterial assay showed that FALL-39-Lys-32 and FALL-39-Lys24 were more potential in the antibacterial activity against E coli ML35p and Pseudomonas aeruginosa ATCC27853 than that of FALL-39, and no increase in hemolysis was observed at the antibacterial concentrations. The antibacterial activity of FALL-39-Lys-32 against E coli was more potent than that of FALL-39 in NaCl-containing LB medium, while its activity was almost the same as FALL-39 in SO4(2-) containing Medium E.
PCR-based mutagenesis is a useful model system for studying the structure and function relationship of antimicrobial peptides. Keeping a-helical conformation of FALL-39 and increasing net positive charge can increase the antibacterial activity of FALL-39 without increasing hemolysis at the antibacterial concentrations.
构建PGEX - 1λT - FALL - 39表达载体及其突变体载体，并研究其功能与结构的关系。
从SPCA - 1细胞mRNA中克隆编码成熟FALL - 39的cDNA，构建原核表达载体PGEX - 1λT - FALL - 39。采用两种用于定点诱变的聚合酶链反应（PCR）构建FALL - 39突变体表达载体FALL - 39 - Lys - 32和FALL - 39 - Lys - 24。用最小有效浓度、最小抑菌浓度和最小杀菌浓度测定这些肽的抗菌活性。通过菌落形成单位（CFU）测定观察不同溶液对FALL - 39和FALL - 39 - Lys - 32抗菌活性的影响。还检测了这些肽对人红细胞的溶血作用。
通过PCR诱导诱变获得了两个位点特异性突变体FALL - 39 - Lys - 32和FALL - 39 - Lys24。与需要两对引物的两步PCR相比，需要一对引物的一步PCR是基于PCR的定点诱变的一种简单有效的方法。利用原核表达系统，还获得了基于大肠杆菌的重组FALL39及其突变肽产物。抗菌试验表明，FALL - 39 - Lys - 32和FALL - 39 - Lys24对大肠杆菌ML35p和铜绿假单胞菌ATCC27853的抗菌活性比FALL - 39更强，且在抗菌浓度下未观察到溶血增加。在含NaCl的LB培养基中，FALL - 39 - Lys - 32对大肠杆菌的抗菌活性比FALL - 39更强，而在含SO4(2-)的培养基E中其活性与FALL - 39几乎相同。
基于PCR的诱变是研究抗菌肽结构与功能关系的有用模型系统。保持FALL - 39的α - 螺旋构象并增加净正电荷可在不增加抗菌浓度下溶血的情况下提高FALL - 39的抗菌活性。
