幽门螺杆菌尿素酶B亚单位表位融合肽在大肠杆菌中的表达与纯化  

Expression and Purification of Helicobacter pylori Urease B Epitope Fusion Peptide in Escherichia coli

作　　者：赵文锋[1] 徐旭东[1] 吴梧桐[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《药物生物技术》2007年第4期235-240,共6页Pharmaceutical Biotechnology

摘　　要：构建霍乱毒素B亚单位(CTB)与幽门螺杆菌(Helicobacter pylori)尿素酶B亚单位(Ure B)表位的融合肽表达载体pET-CtUBE,在E.coliBL21(DE3)中表达融合肽CtUBE。以霍乱弧菌基因组DNA为模板,PCR扩增CTB编码序列,克隆到表达载体pET-28a,获得新质粒pET-CTB;化学合成Ure B表位编码序列,克隆到pET-CTB获得CtUBE表达载体pET-CtUBE,并转化E.coliBL21(DE3),1 mmol/L IPTG诱导融合肽表达,阴离子交换色谱柱纯化融合肽CtUBE,ELISA分析CtUBE复性结果。结果获得了表达融合肽CtUBE的工程菌,诱导后融合肽表达量占菌体总蛋白34.7%,CtUBE纯化后纯度为95.6%,复性后融合肽可与GM1神经节苷脂和抗Ure B血清结合。实验成功构建了表达Ure B表位融合肽的工程菌,融合肽CtUBE保持了CTB生物活性和反应原性,纯度符合疫苗抗原要求,可以用于抗HP感染的疫苗研制。To construct expression vector pET-CtUBE of fusion peptide of Urease B（Ure B） epitope and cholera toxin B subunit（CTB） and to transform it into E. coli BL21（DE3）, the coding sequence of CTB was amplified with PCR from the Vibrio cholerae genome DNA and cloned into pET-28a, so that a new plasmid pET-CTB was gained. Ure B epitope coding sequence which was synthesized by chemical methods was cloned into pET-CTB behind CTB coding sequence and transformed into E. coli BL21（DE3）. The expression of fusion epitope peptide CtUBE was induced with 1 mmol/L IPTG, purified by anion exchange chromatography and analyzed by ELISA. Results showed that Fusion epitope peptide CtUBE was expressed in the engineering bacteria, which accumucated about 34. 7% of the bacterial total pro tein. After being purified, the purity of fusion epitope peptide CtUBE was 95.6%. Renatured CtUBE can be recognized by GM1 ganglioside and Ure B antiserum. So it can be used in the research of Helicobacter pylori vaccine.

关 键 词：幽门螺杆菌 尿素酶B亚单位 CTB 融合表位肽 

分 类 号：Q7[生物学—分子生物学]