摘要
目的 构建表达幽门螺杆菌临床株粘附素 (HpaA)的重组质粒 ,在大肠杆菌中表达获得重组蛋白 ,探讨重组蛋白作为Hp抗原在感染诊断中的价值。 方法 用PCR方法从幽门螺杆菌临床株DNA中扩增HpaA基因片段。克隆及序列分析后在大肠杆菌中进行高效表达 ,表达产物经纯化和Westernblot鉴定后 ,作为Hp抗原 ,ELISA法对 10 0例临床标本进行相应抗体检测 ,并与细菌培养、组织学染色和快速尿素酶试验比较来评价其应用的可行性。结果 经酶切、测序分析插入的基因片段全长 783bp ,与基因文库中的粘附素基因同源性达 98 87%。表达蛋白经SDS -PAGE分析 ,相对分子量 (Mr)约为 300 0 0 ,可溶性表达占全菌的 4 1 6 7%以上 ,经亲和层析后可获得纯度为 90 %以上的重组蛋白。Westernblot证实了其免疫反应性。通过对临床病例的检测结果显示细菌培养、组织学染色、快速尿素酶试验和HpaA抗体检测的敏感性、特异性和准确性分别为 10 0 0 %和 80 9% ;10 0 0 % ;和 90 5 % ;90 5 %和 85 8% ;93 3%和 85 9% ,相差不多。结论 HpaA能在大肠杆菌中高效表达 ,具有较强的免疫反应性 ,作为检测试剂敏感性和特异性均较好 ,有望成为Hp新的非侵入性的检测方法。
To construct a recombinant plasmid expressing the abhesin gene HpaA from clinical strains of Heliobacter pylori(Hp),to express the recombinant proteins in E.coli,and to determine its diagnostic value,the gene encoding the adhesion subunit protein was cloned, sequenccd and expressed.The DNA fragment to the HapA gene from clinical strains of Hp was amplificed by PCR.After cloning and sequence analysis,it was expressed with high efficency in E.coli,and the expressed products after purification and identification with Western blotting could be used as the Hp antigen.The feasibility of its clinical applications with comparative evaluation was done by the antibody testing with ELISA,germ-cultures,histological staining and rapid urease test in 100 clinical specimens.It was found that the total length of gene cloned was 783 bp with a 98.8% sequence homology with adhesin genes in GenBank,and the molecular weight was found to be 30kDa as determined by SDS-PAGE.The percentage of soluble expression was about 41.67% of total cell proteins.After purification with affinity chromatography the purity of the recombinant protein was about 90%,and its immumnogenicity was confirmed by Western blotting.As judging with 100 clinical specimens,it was also found that the percentages of germ-culture,testing,histology staining test,rapid urease test,and the sensitivity,specificity and accuracy of HpaA antibody assay were 100%,80.9%,100%,90.5%,85.8%,93.3%,and 85.9% respectively.It conculudes that rHpaA is well expressed in E.coli and the hpaA protein has perfect immunogenicity.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2004年第4期275-278,共4页
Chinese Journal of Zoonoses
