Jiang Zheng, Huang Ai-Long, Tao Xiao-Hong, Wang Pi-Long
Department of Gastroenterology, the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China.
World J Gastroenterol. 2003 Aug;9(8):1756-61. doi: 10.3748/wjg.v9.i8.1756.
To construct a recombinant vector which can express outer membrane protein (OMP) with M(r)18,000 and heat shock protein A (HspA) from Helicobacter pylori (H. pylori) in E. coli BL21, and to exploit the possibility for obtaining the vaccine conferring protection from H. pylori infection.
The target gene of HspA was amplified from H. pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) by restrictive endonuclease enzyme kpn I, BamH I simultaneously. The recombinant vector was used to sequence, and then together with pET32a (+)/Omp(18), digested by restrictive endonuclease enzyme Hind III and BamH I simultaneously. pET32a(+)/HspA and Omp(18) were recovered from 1 % agarose gel by gel kit, and ligated with T(4) ligase by BamH I digested viscidity end. The recombinant plasmid of pET32a(+)/HspA/Omp(18) was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification, its antigenicity of the fusion protein was detected by Western blot.
Enzyme digestion analysis and sequencing showed that the target genes were inserted into the recombinant vector, composed of 891 base pairs, encoded objective polypeptides of 297 amino acid residues. Compared with GenBank reported by Tomb et al, there were 1.3 % and 1.4 % differences in obtained H. pylori nucleotide sequence and amino acid residues, respectively. SDS-PAGE analysis showed that relative molecule mass (M(r)) of the expressed product was M(r) 51,000, M(r) of protein expressed by pET32a (+) was about M(r) 20,000, and soluble expression product accounted for 18.96 % of total bacterial protein. After purification with Ni(+2)-NTA agarose resins, the purification of recombinant fusion protein was about 95 %. Western blot showed that recombinant fusion protein could be recognized by the patients' serum infected with H. pylori and anti-Omp(18) monoclone, suggesting that this protein had good antigenicity.
The gene coding for H. pylori M(r)18,000 OMP and HspA was cloned and expressed successfully. The results obtained lay the foundation for development of H. pylori protein vaccine and a quick diagnostic kit.
构建能在大肠杆菌BL21中表达幽门螺杆菌分子量为18000的外膜蛋白（OMP）和热休克蛋白A（HspA）的重组载体，探索获得预防幽门螺杆菌感染疫苗的可能性。
通过PCR从幽门螺杆菌染色体扩增HspA目的基因，然后用限制性内切酶Kpn I、BamH I同时将其插入原核表达载体pET32a(+)。对重组载体进行测序，再与pET32a(+)/Omp(18)一起用限制性内切酶Hind III和BamH I同时酶切。用凝胶试剂盒从1%琼脂糖凝胶中回收pET32a(+)/HspA和Omp(18)，通过BamH I酶切粘性末端用T(4)连接酶进行连接。将重组质粒pET32a(+)/HspA/Omp(18)转化至大肠杆菌BL21(DE3)中，在IPTG诱导下表达。纯化后，通过Western blot检测融合蛋白的抗原性。
酶切分析和测序表明目的基因插入到重组载体中，该载体由891个碱基对组成，编码297个氨基酸残基的目的多肽。与Tomb等人在GenBank报道的相比，获得的幽门螺杆菌核苷酸序列和氨基酸残基分别有1.3%和1.4%的差异。SDS-PAGE分析表明表达产物的相对分子量（M(r)）为51000，pET32a(+)表达的蛋白M(r)约为20000，可溶性表达产物占细菌总蛋白的18.96%。用Ni(+2)-NTA琼脂糖树脂纯化后，重组融合蛋白的纯度约为95%。Western blot表明重组融合蛋白能被幽门螺杆菌感染患者血清和抗Omp(18)单克隆抗体识别，提示该蛋白具有良好的抗原性。
成功克隆并表达了编码幽门螺杆菌分子量为18000的OMP和HspA的基因。所得结果为幽门螺杆菌蛋白疫苗和快速诊断试剂盒的研制奠定了基础。
