摘要
目的改良人源杀菌肽LL-37的氨基酸序列,提高其杀菌力,并在原核细菌中表达。方法应用蛋白质分析软件(Anthepro5.0、SWISS-MODEL等),对人源杀菌肽LL-37的氨基酸序列进行二维、三维结构及理化特性分析,将其中部分氨基酸残基进行替换,提高其正电荷,并以人源杀菌肽LL-37的基因序列为模板,进行相应的DNA序列替换,转入表达载体pET-28a(+)于杆菌BL21(DE3)中表达、纯化,并初步研究其抗菌性。结果在维持LL-37空间构象不变的基础上,将Glu16、Asp26、Glu36分别替换为Gln16、Asn26、Gln36,其静电荷由pH7.4时的+5.8提高到+9.0,并且其多肽N端疏水、C端亲水的特性不变。通过Touch-Down PCR法,成功获得改良LL-37多肽的DNA序列,并将重组质粒pET-28a(+)-rLL-37在杆菌BL21(DE3)中表达,改良LL-37多肽成功由强离子交换柱芯Macro-Prep High S纯化,并对G+、G-菌具有一定的抗菌力。结论将杀菌肽LL-37多肽进行氨基酸残基替换并以原核细胞进行融合型表达是可行的,为改良LL-37的大量制备及杀菌活性研究奠定了基础。
Objective To reconstruct the proteinic sequence of human cathelicidin LL-37 to increase the bactericidal activity of LL-37 and to express the reconstructed LL-37 （rLL-37） in bacterium. Methods The two dimensional structure, three dimensional structure and chemical characteristic of LL-37 were analyzed by Soft Ware Anthepro 5.0 and SWISS-MODEL. Without the three dimensional changes of LL-37, some negative amino acids of human cathelicidin LL-37 were replaced by positive amino acids and the positive charge of LL-37 was increased. According to the proteinic sequence changes of rLL-37, the DNA sequence of rLL-37 was reconstructed by Touch-Down PCR and recombined with vector pET-28a （ ＋ ）, thus rLL-37 was expressed in E. coli. BL21 （DE3） by the induction of IPTG and was purified by chromatography. Results Glu^16, Asp^26, Glu^36 of LL-37 were replaced by Gin^16,Asn^26, Gin^36 and the static charge of LL-37 was increased from ＋ 5.8 to ＋ 9.0 at pH 7.4. The DNA sequence of rLL-37 was reconstructed and inserted into vector pET-28a （ ＋ ）, the rLL-37 was expressed in E. coli. BL21 （DE3） and purified by strong cation exchange supports Macro-Prep High S successfully. The rLL-37 was proved by the means of inhibitory zone to be able to kill Gram-negative bacteria and Gram-positive bacteria. Conclusion It is feasible to reconstruct human cathelicidin LL-37 and express the protein in bacteria by fusion, which make it possible to produce more rLL-37 and study its biological function deeply.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第7期636-639,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
人源杀菌肽LL-37
蛋白质融合表达
强离子交换层析
hurrah cathelicidin LL-37
confluent expression of protein
strong cation exchange chromatography
