马铃薯卷叶病毒CP基因水溶性原核表达的研究

采用两种策略增强了马铃薯卷叶病毒(PLRV)CP基因的水溶性原核表达。首先,用5种可表达出分子伴侣的质粒分别转化重组菌TOP10(p BAD-LRCP),获得了既含p BAD-LRCP又含分子伴侣质粒的转化子;诱导转化子中PLRVCP基因与分子伴侣基因同时表达,SDS-PAGE结果表明,从转化子提取的水溶性蛋白中有明显的PLRV重组CP条带,即分子伴侣增进了重组CP的水溶性表达。其次,将PLRV-CP基因定向插入p ColdⅠ中构建了该基因的冷激诱导原核表达载体p Cold-LRCP,15℃、IPTG诱导24h,SDS-PAGE分析表明,从重组菌BL21(p Cold-LRCP)提取的上清蛋白中有明显的PLRV重组CP条带。试验结果表明,分子伴侣与冷激蛋白基因csp A的启动子均可提高PLRV-CP基因的水溶性表达,且后者的表达水平高于前者。

Studies on Water-soluble Prokaryotic Expression of Potato leafroll virus CP Gene

Two strategies were adopted to enhance the water-soluble prokaryotic expression of potato leafloll virus CP gene. Firstly， 5 chaperone-expression plasmids were transformed into the recombinant E. coli strain TOP10（pBAD-LRCP）， respectively. Restriction endonuclease digestion identification indicated that the yielding trans-formants contained both pBAD-LRCP and chaperone plasmid. After induction with L-arabinose or L-arabinose and tetracycline， PLRV-CP gene and chaperone gene were both expressed， and recombinant PLRV-CP band was observed in SDS-PAGE pattern of the supernatant protein from the trans-formanat， water-soluble expression of PLRV-CP gene was enhanced by the presence of chaperone protein. Secondly， PLRV-CP gene was inserted into prokaryotic expression vector pCold Ⅰ in direct orientation and cold-shock expression vector of PLRV-CP gene named pCold-LRCP was constructed. The recombinant strain BL21（ pCold-LRCP） was cultured at 15 ℃ for 24 hours to induce the expression of PLRV-CP gene. Obvious PLRV-CP band was found in SDSPAGE analysis of water-soluble protein of the recombinant strain. The results showed that chaperone and coldshock protein gene（ cspA） promoter both enhanced water-soluble expression of PLRV-CP gene， and the solubleexpression level of cspA promoter was higher than that of the chaperones.