摘要
目的 通过基因工程技术获得具有良好免疫原性的重组N-乙酰神经氨酰乳糖结合原纤维样血凝素（HpaA）。 方法 利用PCR技术从幽门螺杆菌（Helicobacter pylori，Hp）总DNA中钓取hpaA结构基因，克隆及序列分析后在大肠杆菌中进行高效表达，表达产物经纯化后用酶联免疫吸附法（ELISA）检测其抗原性。结果 序列分析证实hpaA结构基因长度为783 bp，编码261个氨基酸，SDS-PAGE显示表达产物相对分子质量约为29 000，可溶性表达占全菌的20%以上，经硫酸铵分级沉淀和阴离子交换层析后获得纯度为90%的重组蛋白。ELISA法显示该蛋白可被Hp全菌抗血清识别。结论 基因重组粘附素HpaA具有良好的抗原性，可望作为一种有效的蛋白疫苗用于Hp感染的防治。
Objective To obtain the antigen of recombinant N-acetylneuraminyllactose-binding fibrillar hemagglutinin (HpaA) by genetic engineering. Methods The gene encoding an adhesion subunit protein, HpaA, was cloned, sequenced and expressed. After purification, the antigenicity of recombinant HpaA was analyzed by ELISA. Results DNA sequence analysis identified 1 open reading frame, encoding the polypeptides of 261 amino acids with molecular weight of 29 kD as predicted. The HpaA gene was then cloned into pTrc99A and efficiently expressed in E. coli JM109. The level of soluble expression product was about 20% of total cell protein. After ammonium sulfate fractionation and anion-exchange chromatography, the purity of rHpaA was above 90%. Conclusions ELISA results showed recombinant HpaA could be recognized by anti-serum against HP, suggesting that this protein has perfect antigenicity and may be used as a protective antigen in preventing Helicobacter pylori (Hp) infection.
出处
《第一军医大学学报》
CSCD
2000年第3期210-213,共4页
Journal of First Military Medical University
基金
国家自然科学基金资助项目(39700007)
