重组和天然HBHA蛋白制备及其活性研究  

Purification and activity determination of native and recombinant HBHA protein

作　　者：周珊[1] 马越云[1] 刘家云[1] 苏明权[1] 张海[2] 师长宏[1] 王丽华[1] 郭旭光[1] 郝晓柯[1] 

机构地区：[1]第四军医大学西京医院检验科,西安710032 [2]第四军医大学西京医院实验动物中心

出　　处：《中华检验医学杂志》2010年第3期271-275,共5页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目（30872358）

摘　　要：目的制备天然和重组HBHA蛋白，并比较其诱导产生的抗体对其聚集BCG活性的抑制效应。方法将7H9液体培养基中的BCG培养至稳定生长期，用CL-6B层析柱分离纯化HBHA蛋白（nHBHA）。同时，克隆HBHA基因片段，表达于大肠杆菌BL-21中，经亲和层析法纯化蛋白，并以此免疫新西兰兔制备多克隆抗体。在BCG的液体培养基中分别加入不同浓度的HBHA蛋白，观察菌体的聚集情况。同时，用抗HBHA抗体与HBHA蛋白孵育后，加入BCG培养基中，观察菌体聚集情况。结果获得天然HBHA蛋白纯度为99％，浓度为1．016mg／mL重组蛋白表达量约占菌体蛋白总量的36％，纯化得到纯度为97．1％，浓度为10．98mg／m]的重组HBHA蛋白。所得天然和重组蛋白均有诱导BCG聚集的作用，当nHBHA浓度为0．2μg/ml时BCG出现少量聚集，而rHBHA浓度达到2μg／ml时BCG才出现聚集。并且此聚集现象均可被多克隆抗体抑制。结论成功获得天然HBHA和重组HBHA蛋白；证实了两者均具有促进BCG聚集的功能，并可被由重组HBHA诱导产生的多克隆抗体所抑制。从而为进一步研究HBHA的临床诊断和疫苗价值提供了实验依据。Objective To purify native and recombinant heparin-binding hemagglutinin （HBHA） protein, and investigate the activity of HBHA polyclonal antibody against aggregation of Bacillus Calmette- Guerin （BCG） induced by HBHA. Methods After growing BCG to the stationary phase in the 7H9 liquid medium, the native HBHA protein （nHBHA） was obtained by CL-6B column chromatography. At the same time, the HBHA geue fragment was cloned and expressed by transforming Escherichia coli BL-21. Then the polyclonal antibody against rHBHA was prepared by immunizing rabbit. Different comcentration of the HBHA protein was added to the BCG liquid medium, and the aggregation of the BCG was observed. Then, add the HBHA protein that incubated with anti-HBHA antibodies to the BCG culture medium and observe the aggregation of BCG. Results The purity of native HBHA was 99% and the concentration was 1. 016 mg/ml. The expressed product contained 36% of total somtic protein. After purified, the purity of the recombinant HBHA protein was 97.1% and the concentration was 10. 98 mg/ml. Both the rHBHA and nHBHA could induce the aggregation of BCG. When then concentration of nHBHA is 0. 2 μg/ml, BCG could be induced to aggregate,while the rHBHA concentration is 2 μg/ml could induce the aggregation. Both aggregations could be suppressed by the polyclonal antibody against rHBHA. Conclusions The native and recombinant HBHA are successfully obtained. It is proved that the rHBHA could induce the aggregation of BCG similar as nHBHA, and polyclonal antibody against rHBHA could also suppress the activity of nHBHA. It suggested that rHBHA could be further used in clinical diagnosis and vaccination.

关 键 词：分枝杆菌 结核 外源凝集素类 抗体 

分 类 号：R392[医药卫生—免疫学]