幽门螺杆菌Cag致病岛hp0540基因的克隆表达及其多克隆抗体的制备

目的：构建幽门螺杆菌（Helicobacter pylori，Hp）Cag致病岛（Cag—pathogenicity island，Cag—PAI）编码的hp0540基因的原核表达系统，并制备其多克隆抗体。方法：应用PCR技术从Hp11637基因组DNA中扩增hp0540基因片段，克隆至pMD18-2T载体后，进行序列测定，并对其序列进行生物信息学分析；构建pET-28a—hp0540原核表达载体，转化表达宿主菌BL21DE3；经IPTG诱导表达后，SDS—PAGE法鉴定目的蛋白的表达，并以Ni^2＋-NTA柱分离纯化目的蛋白；将纯化蛋白免疫新西兰大白兔制备多克隆抗体并测定抗体效价。结果：成功克隆了hp0540基因，全长1086bp，编码361个氨基酸，与基因库公布的其他坳菌株基因序列的核苷酸同源性为98％。工程菌诱导后SDS—PAGE显示新生表达蛋白带，相对分子质量约为39000，经Ni^2＋NTA柱纯化后可获得重组蛋白，重组蛋白免疫新西兰大白兔后获得效价为1：160000的多克隆抗体。结论：成功构建了hp0540基因的原核表达系统并制备了其多克隆抗体，为研究该基因的功能奠定了基础。

Construction of hp0540 expression system and the preparation of the polyclonal antibody in Helicobacter pylori Cag-PAI

Objective: To construct the expression system of hp0540 from strain NCTC11637 of Helicobacter pylori(Hp) and prepare the polyclonal antibody serum of CagI.Methods: The hp0540 gene was amplified by PCR with the genomic DNA of Hp NCTC 11637 as template.The plasmid pMD18-2T-hp0540 was constructed via T-A clone method and sequenced.The sequence of hp0540 was analyzed through bioinformatics approach.The expression vector pET-28a-hp0540 was constructed and transformed into E.coli BL21DE3 by molecular cloning meth...