改良人源LL_医学论文.docVIP

改良人源LL_医学论文 改良人源LL_医学论文 作者：杨艳丽,葛晓冬,刘友生,邹佳 【关键词】 人源杀菌肽LL 　　Fusion expression and bactericidal activities of the reconstructed human cathelicidin LL37 　　【Abstract】 AIM: To express and purify the reconstructed LL37 (rLL37) and study its bactericidal activity. METHODS: The DNA sequence of rLL37 was recombined with vector pET30a（+）, and expressed in E.coli BL21 star (DE3) by the induction of IPTG, thus the 6×His tagged fusion protein rLL37 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 fector Xa. Then, by using high positive ion exchange column MacroPrep High S, we purified the cleaved product and collected the every elution peak. Finally, the bactericidal activity of the rLL37 was determined by the means of inhibitory zone. RESULTS: The DNA sequence of rLL37 was inserted into vector pET30a (+) and expressed in E.coli BL21 star(DE3). Gel scanning analysis showed that fusion protein accounted for 35% of total bacterioprotein. After the fusion protein was purified and certified by SDSPAGE, a single Mr 4000 strip may be found. Then, the rLL37 was proved by the means of inhibitory zone to be able to kill both Gramnegative bacteria and Grampositive bacteria. CONCLUSION: It is possible that the rLL37 is expressed in procaryotic cell by fusion and it possesses a strong bactericidal activity. 　　【Keywords】 human cathelicidin LL37 fusion expression of protein bactericidal activity 　　【摘要】 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BL21 star(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Western blot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MacroPrep High S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BL21 star(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Mr 4000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. 　　【关键词】 人源杀菌肽LL37；蛋白质融合