摘要
目的将改良人源杀菌肽LL-37(rLL-37)用两种方法构建,并分别在原核细胞中融合表达,以寻找较好的制备方法。方法①将编码rLL-37基因序列放入载体pET-28a(+),构建表达质粒pET-28a(+)-rLL-37,并于工程菌BL21(DE3)中融合表达、纯化;②将编码rLL-37基因序列中的部分原核细菌稀有密码子替换为原核细菌偏爱密码子,并在其N端加入一段编码带负电荷的承载蛋白(carrierproteinmolecule,CPM)基因序列,构建表达质粒pET-30a(+)-CPM-rLL-37,并于工程菌BL21Star(DE3)中融合表达、纯化。对比上述2种方法rLL-37的制备率,并初步研究其抗菌性。结果通过Touch-DownPCR法成功获得rLL-37多肽的DNA序列,质粒pET-28a(+)-rLL-37于杆菌BL21(DE3)中表达,融合蛋白约占全菌蛋白的20%,并成功由强阳离子交换柱芯Macro-PrepHighS纯化;设计了一段含28个氨基酸残基的CPM(pHi2.7、pH7.4时电荷为-6.0),成功构建表达质粒pET-30a(+)-CPM-rLL-37,并于杆菌BL21star(DE3)中表达,融合蛋白约占全菌蛋白的35%,并被TALON亲和柱芯有效纯化。经抑菌圈实验证明两种方法所获得的rLL-37多肽对G+、G-菌都具有较好的杀菌力。结论rLL-37在原核细胞中的高效表达,为深入研究rLL-37的杀菌活性奠定了基础。
Objective To employ 2 approaches to construct and express reconstructed LL-37 （rLL-37） in procaryotic system, and to explore a better preparation method. Methods The first method： the rLL-37 was inserted into vector pET-28a （ ＋ ） , then was induced to express in E. coll. BL21 （ DE3 ） and purified by chromatography; the second method： the rare codons in the rLL-37 gene sequence were substituted by the preferred codons of procaryotic cell, and a fragment of carrier protein molecule （CPM） was added to the N termination of the objective sequence to construct expression plasmid pET-30a（ ＋ ）-CPM-rLL-37, then the rLL-37 was expressed in E. coli. BL21 Star （ DE3 ） and purified by chromatography. The productive rates of the 2 methods were compared and the antimicrobial effects of obtained rLL-37 was studied. Results The first method： the DNA sequence of rLL-37 was obtained successively by Touch-Down PCR. The expression plasmid pET-30a （＋）-CPM-rLL-37 was expressed with fusion protein in E. coli BL21 （DE3）. The expression rate accounted for 20% of total bacterio-protein, then the expressed product was purified by using high positive ion exchange column Macro-Prep High S; The second method： a fragment of carrier protein molecule was designed that contained 28 amino-acid residue and its pHi was 2.7, net charge was - 6.0 at pH 7.4. After the expression plasmid pET-30a（ ＋ ）-CPM-rLL-37 was constructed successively, it was expressed in E. coli BL21 Star （DE3）. The expressed fusion protein accounted for 35% of total bacterio-protein, then the expressed product was purified by using affinity binding chromatography with TALON resins successfully. The obtained 2 kinds of rLL-37 were able to kill both Gram-negative and-positive bacteria by the means of inhibitory zone. Conclusion It＇s feasible to prepare efficiently rLL-37 in procaryotic system, which founds the basis for the further research on bactericidal activity of rLL-37.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第20期2020-2023,共4页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目(30400426)
西南医院创新基金资助项目(2002)~~
关键词
LL-37
蛋白质融合表达
承载蛋白
human cathelicidin LL-37
expression of fusion protein
carrier protein molecule
