绿脓杆菌外毒素ArecA基因的融合及融合蛋白的表达  

Construction and Expression of PEA recA Fusion Gene of P aeruginosa

作　　者：刘晓明[1] 朱平[1] 刘子[1] 马从林 冯书章[1] 

机构地区：[1]解放军农牧大学军事兽医研究所

出　　处：《中国兽医学报》1997年第2期135-139,共5页Chinese Journal of Veterinary Science

摘　　要：将绿脓杆菌ｒｅｃＡ基因克隆到ｐＵＣ１８中，构建了中间载体ｐＵＣＲ１８；然后以正确的向位及阅读框架将ｒｅｃＡ基因亚克隆到毒性基因缺失的绿脓杆菌外毒素Ａ（ＰＥＡ）基因中，构建了ＰＥＡ－ｒｅｃＡ融合基因质粒ｐＥＲＡ；融合基因经ＢａｍＨＩ及ＥｃｏＲＩ酶切，以正确阅读框架插入带有Ｔ７表达启动子的质粒ｐＥＴ－１７ｂ，构建了可表达ＰＥＡ－ｒｅｃＡ融合基因的质粒ｐＥＲＡ－１７ｂ。经酶切分析及ＰＣＲ扩增检测证明，绿脓杆菌ｒｅｃＡ已插入ＰＥＡ毒性基因中。ｐＥＲＡ－１７ｂ转化到ＤＥ３溶原态大肠杆菌ＨＭＳ１７４中，经ＩＰＴＧ诱导，表达了ＰＥＡ－ＲｅｃＡ蛋白。ＳＤＳ－ＰＡＧＥ和凝胶薄层扫描ＰＥＡ－ＲｅｃＡ蛋白，表明ＰＥＡ－ＲｅｃＡ的分子量约为９００００，与理论推算相符，表达率占菌体总蛋白量３．４２９％，用ＰＥＡ抗血清和抗ＲｅｃＡ单克隆抗体作免疫印迹分析。For correct reading code frame, P aeruginosa recA gene NruⅠfragment was cloned into pUC18Sma Ⅰsite.Then isolated by digesting with Eco RI/Bam HI,the recA was inserted to PEA gene which deleted part of toxic gene by digesting SacⅡand XhoⅠ,DNA polymerase Klenow fragment and ligated by T 4DNA ligase to construct PEA recA chimera gene.Then took the PEA recA downstream of T 7 promoter in pET 17bplasmid to construct PEA recA expression plasmid identified by endonuclease enzymes digesting and PCR,the PEA recA gene was downstream of T 7 promoter.pERA 17bwas transformed to T 7 promoter expression host cell E recA BL21and HMS174series,induced by IPTG to expression PEA recA fusion protein,PEA recA gene didn′tbe expressed in BL21series host cell but expressed in HMS174series host cell,PEA recA expression rate was3.429%total cell protein,Mr of PEA recA was about90000,similar to that estimated by aminoacid number and could react with anti PEA serum and anti recA McAb in ELISA and immunoblotting.

关 键 词：绿脓杆菌外毒素A RECA 基因融合 融合蛋白 

分 类 号：Q78[生物学—分子生物学] R378.991[医药卫生—病原生物学]