幽门螺杆菌疫苗候选抗原lpp20基因的克隆表达及其纯化研究

Cloning,expression and purification of lpp20 gene of Helicobacter pylori

目的克隆表达幽门螺杆菌(Helicobacter pylori,Hp)lpp20基因,为筛选预防或治疗幽门螺杆菌感染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lpp20基因片段,将目的基因插入表达载体pET22b中,重组载体pET22b-Lpp20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。采用镍离子亲和层析纯化蛋白,并用SDS-PAGE和W estern b lot鉴定。结果PCR扩增的lpp20基因长度为528bp,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99%,SDS-PAGE显示,经IPTG诱导目的蛋白占总蛋白的19.7%,纯化后,蛋白纯度达80%以上。W estern b lot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lpp20表达载体pET22b-Lpp20,并在大肠杆菌中表达。

Objective To perform cloning and expression of the lpp20 gene of Helicobacter pylori in order to construct a basis for vaccine to prevent and treat H. pylori infection. Methods The lpp20 gene of clinical isolates was amplified from H. pylori chromosomal DNA by PCR. The PCR product was inserted into the expression vector pET-22b. The recombinant vector pET22b-Lpp20 was identified by DNA sequencing and transformed to E. coli.. BL21 （ DE3 ） for expression under IPTG induction. The protein was purified by Chelating chromatography and identified by SDS-PAGE and Western blotting. Results The PCR product was about 528 bp. The gene segment inserted into the recombinant vector was proven 99% identical to the sequence in Gen-Bank, and expressed the target protein up to 19.7% of total somatic protein by IPTG induction. The protein purity reached more than 80% after purification. The protein could be recognized by rabbit anti-Hp blood serum. Conclusion The expression vector pET22b-Lpp20 was successfully constructed and the protein can be expressed in E. coli.