猪链球菌2型重组suilysin的高效表达及其生物学特性

High-efficient expression of soluble recombinant suilysin of Streptococcus suis serotype 2 and its biological characteristics

根据猪链球菌2型(SS2)HA9801株溶血素(suilysin,SLY)基因全长序列设计引物,扩增SLY基因,与原核表达载体pET-28a连接,转入大肠杆菌Transetta(DE3)中诱导表达,收集表达菌体,超声破壁、镍柱纯化获得可溶性rSLY蛋白,电泳鉴定后测定rSLY的溶血活性及其溶血谱,系统研究温度、酸碱度、离子强度等理化因素对rSLY溶血活性的影响。结果表明,本试验建立的rSLY原核表达体系能高效可溶性表达具有溶血活性的rSLY,纯度达电泳纯,相对分子质量为54 000左右,100mL菌液诱导后能纯化出约8mg电泳纯的rSLY。rSLY具有高溶血活性,溶血比活为4 057HU/mg。rSLY对多种动物的红细胞有溶血作用,其中对猪、兔和豚鼠的作用最强。rSLY在4℃保存时溶血活性最稳定,保存48h后溶血活性不降低。在pH 6～7和离子强度为500mmol/L时溶血活性最高。本试验获得了高表达、可溶性、高溶血活性的rSLY,首次系统研究了不同理化因素对rSLY生物学活性的影响,为SLY致病机理深入研究及SS2亚单位疫苗和诊断试剂的研制奠定了基础。

A pair of primers were designed to amplify complete SLY gene,according to the reported suilysin （SLY） nucleotide sequences of Streptococcus suis serotype 2 （SS2） HA9801 isolate,SLY gene was cloned into prokaryotic expression vector pET-28a and transformed into Escherichia coli Transetta （DE3） for expression of rSLY. The cultured and induced E. coli cells was collected by centrifugation and the bacterial pellet was lysed by ultrasonic treatment,and then rSLY from bac-terial cells was purified with a Ni-NTA affinity column. Hemolytic activity and hemolysis test of rSLY to some kinds of animals＇ erythrocytes were determinated. Finally effect of different temperature,pH and ionic strength on the hemolytic activity of rSLY were neasured. The results showed that high-efficiently expressed and high-yield soluble rSLY with bioactivity was finally obtained. SDS-PAGE suggested the purified rSLY presented a Single band with the molecular weight of 54 ku. Eight mg of pure rSLY was obtained from E. coli cells in 100 mL induced culture, rSLY had high hemolytic activity and its specific activity was 4 057 HU/mg,with hemolytic activity to manykinds of animals＇ erythrocytes especially to pig,rabbit,and guinea pig. rSLY maintained stable he lytic activity when preserved at 4C for 48 hours. The optimal pH and ionic for rSLY was pH 6-7 and 500 mmol/L respectively. In this study,we obtained strength condition high-efficiently ex- pressed soluble rSLY with bioactivity, and firstly reported effect of temperature, pH and ionic strength on the hemolytic activity of rSLY,these results will lay a foundation for deeply research of pathogenic machinery of SLY and preparation of subunit vaccine and diagnosis reagent for SS2.