幽门螺杆菌CagA蛋白肽段的基因克隆、表达及抗原性检测

Gene Cloning of CagA Proteinic Peptide and its Expression and Antigenicity Determination of Helicobacter pylori

目的 克隆幽门螺杆菌 Cag A蛋白肽段的基因 ,确定 Cag A蛋白与相应抗体亲和力最强的肽段。 方法 根据幽门螺杆菌 cag A全基因序列以 DNAMAN软件设计 5对引物 ,采用 PCR方法扩增出 5条幽门螺杆菌cag A基因片段。将 PCR扩增产物插入原核表达载体 p GEX- 3X并转化至大肠杆菌 Top10中 ,形成 5种重组质粒。经 PCR、双酶切和测序鉴定后 ,以异丙硫半乳糖苷 (IPTG)诱导其表达 Cag A蛋白肽段。包涵体经 8mol/ L 尿素初步纯化后 ,运用 EL ISA方法检测其抗原性并确定相应抗体亲和力最强的 Cag A蛋白肽段。 结果 5条 cag A基因片段在大肠杆菌中都得到了表达 ,表达的蛋白均具有强弱不等的抗原性 ,其中 6 6 k D的蛋白肽段与抗 Cag A抗体亲和力最强。 结论 6 6 k D的蛋白肽段与抗 Cag

Objective\ To make the kit to diagnose the infection of Helicobacter pylori(Hp) with high toxigenicity, the Hp CagA proteinic peptide was expressed in E.coli Top10 with expression vector, and the antigenic protein fragment was determined.\ Methods\ According to the Hp cagA gene, five pairs of primers are designed with the DNAMAN software.\ Five cagA gene fragments have been amplified by PCR and inserted into the expression vector pGEX\|3X.\ The five reformed plasmids were transformed into E.coli Top10 by calcium\|chloride\|mediated transformation.\ By the means of PCR, endonucleases and gene\|sequence, the positive plasmids were selected.\ The selected plasmids are induced to express the \{CagA\} proteinic peptides using 1 mmol/L isoprogyl thiogalactoside(IPTG).\ The fusion proteins are primarily purified by 8 mol/L urea.\ ELISA is used to determine which proteinic peptide is the most antigenic.\ Results\ The CagA proteinic peptides were expressed in E.coli Top10, and the expressed proteinic peptides had antigenicity.\ Among these peptides, 66 kD fragment was the critical antigenic.\ \{Conclusion\}\ The 66 kD \{CagA\} proteinic peptide is the most antigenic.\;