重组大鼠溶菌酶N端多肽的原核表达及其对晚期糖基化终产物的结合作用  

Prokaryotic expression of recombinant rat lysozyme N-terminal fragment and its AGEs binding activity

作　　者：周翔[1] 莫宏波[1] 周羽竝[1] 刘誉[1] 

机构地区：[1]暨南大学医学院生物化学教研室,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2006年第6期784-790,共7页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：暨南大学科研基金的资助(51204004)

摘　　要：目的:探讨溶菌酶N端多肽片段基因的重组并在原核中表达,研究该片段与晚期糖基化终产物(advanced glycation end products,AGEs)的结合作用。方法:从SD大鼠外周血白细胞中抽提总RNA,经逆转录并扩增大鼠溶菌酶N端基因片段,以质粒pET-30 a(+)为载体构建表达质粒pLY77。含pLY77的工程菌Rosetta(DE3)经IPTG诱导,对该重组多肽进行高效表达。表达产物经N i+-NTA琼脂糖柱层析纯化后,得到的溶菌酶N端多肽片段LY77经SDS-PAGE和W estern印迹法鉴定,ELISA法分析体外结合活性。结果:LY77在Rosetta(DE3)中高效表达,主要以可溶性蛋白的形式存在,相对分子质量约为11 000;LY77对体外制备的晚期糖基化蛋白BSA-AGEs具有显著的结合活性。结论:LY77对AGEs具有很高的亲和力,为进一步探讨LY77可能应用于体内促进AGEs的清除和参与免疫调节作用奠定基础。Aim： The aim of the present study was to express the recombinant rat lysozyme N -terminal fragment gene in prokaryotes and to determine its binding activity with advanced glycation end products （AGEs）. Methods： The total RNA was extracted from leucocytes of Sprague - Dawley rats, and the cDNA for rat lysozyme N - terminal sequence was prepared using RT - PCR methods. The sequence, encoding for the N - terminal fragment of rat lysozyme, was inserted into an expression vector pET -30a（ ＋ ）. The recombinant expression vector, pLY77, was then transfected into E. coli Rosetta（DE3） for expression of the recombinant rat lysozyme nt, LY77, through induction of IPTG. The expression products were purified by using Ni^＋ - NTA agarose chromatography, and the prepared LY77 was identified by using SDS- PAGE and Western blotting techniques. ELISA analysis its AGEs binding activity in vitro. Result： These results showed that LY77 existed mainly in soluble forms in the expression system, with a molecular weight of about 11 kDa. ELISA analysis showed that LY77 exhibited a potent binding activity with in vitro prepared bovine serum albumin -AGEs. Conclusion： These results suggested a high affinity of the fragment for AGEs and implicated a possible application of LY77 in vivo to promote clearance of AGEs and to modulate the immune system.

关 键 词：溶菌酶 原核表达 重组多肽 晚期糖基化终产物(AGEs) 

分 类 号：R730.631[医药卫生—肿瘤]