幽门螺杆菌尿素酶A基因的克隆、表达及其抗原性的鉴定

Cloning, Expression and Antigenicity of UreaseA of Helicobacter pylori

目的构建幽门螺杆菌(Helicobacterpylori,Hp)标准株NCTC11639尿素酶A(UreaseA,UreA)编码基因的重组质粒,测定、分析其核酸序列,并在E.coli中表达,研究其抗原性。方法应用PCR技术从HpDNA染色体中扩增UreA编码基因片段,将其克隆至pMD18-T载体上进行序列测定,并与GenBank上公布的其它Hp菌株基因序列进行比较,再将目的基因克隆至表达载体pGEX-4T-1上进行表达和纯化,用Western-blot检测产物的抗原性并用于对24株抗Hp全菌小鼠单克隆抗体的鉴定。结果扩增的UreA基因全长675bp(登录号为DQ141577),与GenBank上公布的其它Hp菌株的UreA核酸序列的同源性为95%～98%,表达的UreA融合蛋白分子量为52000,表达产物可被病人血清和全菌免疫的小鼠血清识别,29株抗Hp小鼠单克隆抗体中有4株是针对UreA抗原的。结论重组UreA具有较好的抗原性,为Hp检测试剂和疫苗的研究奠定基础。

Objective Construction and expression of a recombinant plasmid containing UreaseA （UreA） of Helicobacter pylori （Hp） NCTC11639 strain. Methods The gene encoding UreA of Hp was amplified from lip chromosomal DNA by PCR. The sequence of UreA was determined using pMD18-T vector. Expression was achieved by cloning the gene into pGEX-4T-1 expression vector and expressed in E.coli. Immuno-reactivity of the recombinant protein to mouse monoelonal antibodies （mAbs） against Hp was determined by Western-blot. Results A gene fragment of 675 base pairs （DQ141577） was amplified. The sequence of Urea shared 95%～98% of nucleotide homology with the same gene of other stains of Hp in GenBank. Results from Western blotting analysis showed that the recombinant UreA protein （tool. wt. 52 kDa） reacted with the serum obtained from Hp-infected patients or the Hp immunized BA1B/c mice. In addition, UreA also reacted with 4 out of 29 anti-Hp mouse mAbs. Conclusion The recombinant UreA is immunogenie and it may further he developed for clinical diagnosis and the development of vaccine for Hp.