幽门螺旋杆菌尿素酶B亚单位的重组表达及在临床诊断中的应用

Recombinant Expression of the Urease B Subunit of Helicobacter Pylori and its Application in Clinical Diagnosis

自幽门螺旋杆菌(Helicobacter pylori,H.Pylori)被科学家发现以来,已经明确其与多种上消化道疾病密切相关,准确地检测H.Pylori是对疾病进行规范化治疗的前提。血清抗体检测法可以反映一段时间内H.Pylori感染状况,是唯一不受近期用药和胃内局部病变影响的检测方法。本研究制备了高纯度、具有生物活性的尿素酶B亚单位(Urease B,UreB)蛋白,并对该蛋白进行分析鉴定,建立一种间接ELISA法,用于检测患者由于感染幽门螺旋杆菌而产生的UreB抗体。利用PCR方法扩增出H.Pylori-UreB基因,然后进行双酶切、连接构建重组表达载体UreB-pTrcHis2C,转化至大肠杆菌Rosetta(DE3)中,经IPTG诱导表达,利用金属离子螯合磁珠结合离子交换层析进行纯化,纯化后的蛋白通过Western Blot进行抗原反应性鉴定。将验证后的蛋白包被于酶标板中,建立间接ELISA法,用于检测患者血清中的UreB抗体。该蛋白在25℃条件下,以1 mmol/L IPTG,诱导4 h蛋白表达量最高,蛋白相对分子质量为69000,纯化后蛋白纯度为97.3%,Western Blot结果显示,该蛋白与H.Pylori阳性血清可以发生特异性结合。通过棋盘滴定法确定了UreB蛋白的包被浓度和血清的稀释倍数,建立了间接ELISA法,并利用该方法对43例待测血清进行检测,间接ELISA结果与临床检测结果比较,准确度为88.4%,灵敏度为100%,特异性为82%。试验成功表达并纯化出高纯度的重组UreB蛋白,并利用该蛋白建立了一种检测患者血清中UreB抗体的间接ELISA法。

Since the discovery of Helicobacter pylori(H.Pylori) by scientists, it has been clear that H.Pylori is closely related to a variety of upper gastrointestinal diseases.Accurate detection of H.Pylori is a prerequisite for standardized treatment of diseases.Serum antibody detection can reflect the status of H.Pylori infection in a period of time, and it is the only detection method not affected by recent medication and local lesions in the stomach.In this study, a highly pure and bioactive urease B subunit(UreB) protein was prepared, analyzed and identified.An indirect ELISA method was established to detect UreB antibodies produced by patients infected with H.Pylori.The recombinant expression vector UreB-pTrcHis2 C was constructed by double enzyme digestion and ligation.The recombinant expression vector UreB-pTrcHis2 C was transformed into E.coli Rosetta(DE3).The recombinant expression vector UreB-pTrcHis2 C was induced by IPTG and purified by metal ion chelating magnetic beads combined with ion exchange chromatography.Western Blot was utilized to detect the antigen reactivity of the purified protein.An indirect ELISA method to detect UreB antibodies in patients′ serums by coating the verification protein in an ELISA plate was created.The protein expression reached the highest point under the situation of25 °C and the final concentration of 1 mmol/L IPTG induced for 4 h.The molecular weight of the protein is 69 000,and the purity of the purified protein is 97.3%.Western Blot results demonstrate that the protein can specifically bind to H.Pylori-positive serum.The authors established an indirect ELISA method using this protein to detect UreB antibodies, and used this method to detect 43 serums.The ELISA results obtained when compared to those of the clinical results, gave an accuracy, sensitivity and specificity of 88.4%,100% and 82% respectively.The authors successfully expressed and purified high-purity recombinant UreB protein, and established an indirect ELISA method for detecting UreB antibodies in patient serum by using this protein.