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

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

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Ni2+-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1 086 bp,编码361个氨基酸,与基因库公布的其他Hp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39 000,经Ni2+-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∶160 000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。

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 BL21 DE3 by molecular cloning method. The protein was expressed and characterized via SDS-PAGE method after induced by IPTG. The target protein Cagl was purified and collected by Ni^2＋ -NTA agarose. The puried protein was used to immunize rabbit to prepare polyclonal antibody serum. Results： The hp0540 has been cloned successfully. The sequence analysis for hp0540 showed that it shared 98% homology with other strains of Hp in GenBank. The molecular mass of the purified protein was 39 000. The potency of the poly- clonal antibody serum was 1 ： 160 000. Conclusion： We constructed the prokaryotic expression system and prepared the polyclonal antibody serum successfully, which established a foundation for the function investigation of the hp0540.