[幽门螺杆菌黏附素基因保守区的克隆及免疫原性]

[Cloning and immunogenicity of conservative region of adhesin gene of Helicobacter pylori].

作者信息

Bai Yang, Zhang Ya-li, Wang Ji-de, Zhang Zhao-shan, Zhou Dian-yuan

机构信息

Institute of Digestive Medicine of the People's Liberation Army, First Military Medical University, Guangzhou 510515, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2003 May 10;83(9):736-9.

PMID:12899747

Abstract

OBJECTIVE

To construct a candidate strain of Helicobacter pylori (Hp) that expresses the proteins of the conservative region of 4 adhesins (BabA2, AlpA, AlpB, and HopZ) and study its immunogenicity.

METHODS

The DNA of Hp was extracted. Primers were designed according to the C-terminal structural gene sequence (called CB) of AlpA. The CB gene was amplified by PCR and inserted into the prokaryotic expression vector pET-22b (+) and expressed in BL21 (DE3) strain of Escherichia coli. The product of expression, CB, was purified by affinity chromatography and identified by Western blot analysis. ELISA assay was used to measure the CB-specific antibody in the specimens of serum of 55 Hp infected patients. Rapid urease test (RUT) was used on biopsy specimens collected by gastroscopy as parallel control.

RESULTS

A recombinant plasmid pET-22b (+)/CB was constructed with the conservative region of the 4 adhesins. DNA sequencing showed one open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The recombinant CB (rCB) protein, with a molecular weight of 22.5KD, amounted to 29% of the total bacterial protein. The purity of purified rCB was 96%. Western blot analysis showed that the rCB protein could be specifically recognized by the serum from Hp infected patients. The kappa coefficient was 0.76 for evaluation by ELISA and RUT results.

CONCLUSION

CB has the potential to be used as a vaccine against Hp infection.

摘要

目的

构建一株表达4种黏附素（BabA2、AlpA、AlpB和HopZ）保守区蛋白的幽门螺杆菌（Hp）候选菌株，并研究其免疫原性。

方法

提取Hp的DNA。根据AlpA的C端结构基因序列（称为CB）设计引物。通过PCR扩增CB基因，并将其插入原核表达载体pET-22b(+)中，在大肠杆菌BL21(DE3)菌株中表达。表达产物CB通过亲和层析纯化，并经蛋白质印迹分析鉴定。采用ELISA法检测55例Hp感染患者血清标本中的CB特异性抗体。对胃镜采集的活检标本进行快速尿素酶试验（RUT）作为平行对照。

结果

构建了含4种黏附素保守区的重组质粒pET-22b(+)/CB。DNA测序显示一个588 bp的开放阅读框，编码195个氨基酸的多肽。重组CB（rCB）蛋白分子量为22.5KD，占细菌总蛋白的29%。纯化后的rCB纯度为96%。蛋白质印迹分析表明，rCB蛋白能被Hp感染患者的血清特异性识别。ELISA与RUT结果评价的kappa系数为0.76。