改良人源LL-37杀菌肽的融合表达及其杀菌活性

Fusion expression and bactericidal activities of the reconstructed human cathelicidin LL-37

目的:将改良LL37多肽以融合肽形式表达、纯化,并初步研究其杀菌活性.方法:将编码改良的LL37多肽的DNA序列放入载体pET30a(+),转入工程菌BL21star(DE3)中,用IPTG诱导表达得到含6×His标记的改良融合蛋白LL37.再以TALON柱芯亲和层析、SDSPAGE和Westernblot鉴定后,用FXa裂解融合肽.将裂解后多肽进一步用强阳离子交换柱MacroPrepHighS纯化、收集各洗脱峰,脱盐、冻干.采用抑菌圈法检测改良LL37的抗菌活性.结果:改良的LL37多肽于载体pET30a(+)在杆菌BL21star(DE3)中高效融合表达,用凝胶扫描显示融合蛋白的表达量约占全菌蛋白的35%.融合蛋白经纯化后用SDSPAGE鉴定在Mr4000处见单一条带.经抑菌圈法检测改良的LL37多肽对革兰氏阴性菌和革兰氏阳性菌都具有很好的杀菌力.结论:改良的人源性LL37多肽以原核细胞进行融合表达是可行的,并具有较强的杀菌活性.

AIM： To express and purify the reconstructed LL-37 （rLL-37） and study its bactericidal activity. METHODS： The DNA sequence of rLL-37 was recombined with vector pET-30a （ ＋ ） , and expressed in E. coli BL21 star （DE3） by the induction of IPTG, thus the 6 x His tagged fusion protein rLL-37 was obtained. After the expressed product was purified by affinity binding chromatography with TALON resins, the product was certified by SDS- PAGE, Western blot and cleaved by thrombin lector Xa. Then, by using high positive ion exchange column Macro-Prep High S, we purified the cleaved product and collected the every chiffon peak. Finally, the bactericidal activity of the rLL-37 was determined by the means of inhibitory zone. RESULTS： The DNA sequence of rLL-37 was inserted into vector pET-30a （ ＋ ） and expressed in E. coli BL21 star（DE3）. Gel scanning analysis showed that fusion protein accounted for 35% of total bacterio-protein. After the fusion protein was purified and certified by SDS-PAGE, a single Mr 4000 strip may be found. Then, the rLL-37 was proved by the means of inhibitory zone to be able to kill both Gram-negative bacteria and Grampositive bacteria. CONCLUSION： It is possible that the rLL-37 is expressed in procaryotic cell by fusion and it possesses a strong bactericidal activity.