产肠毒素性大肠杆菌K99菌毛蛋白抗原基因的克隆与表达

应用PCR从产肠毒素性大肠杆菌（ETEC）中扩增出不含信号肽序列的K99菌毛蛋白基因片段，克隆测序后，将该片段连接到E.coli表达载体pET28a( )中，转化E.coli表达菌株BL21（DE3），筛选得到可诱导表达K99抗原的工程菌株。经IPTG诱导，分离纯化K99重组蛋白，以其免疫新西兰大白兔，获得重组蛋白的兔抗血清；免疫印迹分析表明，此重组蛋白制备的抗血清能与标准的K99强毒株姓明显的抗原抗体反应。

Cloning and Expression of the K99 Fimbriai Antigen Gene of Enterotoxingenic Escherichia coli

A K99fimbrial subunit gene without signal peptide sequence was amplified by PCR from enterotoxigenic E.coli K99strain.After cloning and sequencing,the fragment was inserted into an E.coli expression vector p ET2 8a( ) ,then the recombinant plasmid was transferred into E.coli BL2 1 ( DE3 ) ,and E.coli BL2 1 ( DE3 ) ( K99) obtained.Cultured and induced with IPTG,the over-ex- pressed recombinantprotein in the solubleprotein of E.coli BL 2 1 ( DE3 ) ( K99) wasdetected and pu- rified using preparation electrophoresis.The antiserum againstthe recombinantprotein was raised us- ing a rabbit.With theimmunological analysisthe anti-serum showed a specific immunological response to standard antigen of enterotoxingenic E.coli strain.This work provides a new method for preparing genetic engineering vaccine to prevent diarrheal disease in piglets.