重组幽门螺杆菌AhpC基因的克隆表达及其初步纯化

Cloning,expression and purifyication of AhpC gene of Helicobacter pylori

目的克隆表达幽门螺杆菌(helicobacter pylori)AhpC基因,为筛选预防或治疗幽门螺杆菌的疫苗保护性抗原奠定基础。方法用PCR方法从临床分离株9806的染色体DNA中扩增出AhpC基因片段,将目的基因插入表达载体pET-11c中,重组载体pET-AhpC经DNA测序鉴定后,将重组质粒转化大肠杆菌E.coli BL21(DE3),IPTG诱导表达。采用阴离子交换层析纯化蛋白,并用SDS-PAGE和Western blot鉴定。结果PCR扩增的AhpC基因长度为597bp,经酶切和测序分析,插入到载体的基因片段与GenBank公布的序列相似性达98%,SDS-PAGE显示,经IPTG诱导目的蛋白占总蛋白的28.7%,经纯化后,蛋白纯度达70%以上。Western blot检测该蛋白与兔抗Hp的多克隆抗血清发生结合反应。结论成功构建了AhpC表达载体pET-AhpC,并在大肠杆菌中高效表达。

Objective To perform cloning and expression of the AhpC gene of Helicobacter pylori in order to construct a basis for the investigation work of vaccine to prevent and treat Helicobacter pylori infection. Methoods The AhpC gene of clinical isolate was amplified from Hp chromosomal DNA by PCR techniques. The PCR product was inserted into the expression vector pET-11c. The recombinant vector pET-AhpC was identified by DNA sequencing and transformed to E. coil BL21 （DE3） for expression under inducing of IPTG. The protein was purified by Ion-Exchange chromatography and identified by SDS-PAGE and Western blot. Results The PCR product was shown consisting of about 597 base pairs. It was determined using restriction enzyme digestion and sequencing methods that the gene segment inserted into the recombinant vector was identified to GeneBank show 97% identity. SDSPAGE was shown that expressed up to 28.7% .of total somatic protein by IPTG induction. The purity of protein reached more than 70% after purification. The protein could be recognized by rabbit anti-Hp blood serum. Conclusions The express vector pET-AhpC was successfully constructed and the protein can be highly expressed in E. coli.