肝素结合血凝素蛋白在结核病诊断中的应用研究

Research on the application of HBHA protein in the diagnosis of tuberculosis

目的制备天然和重组肝素结合血凝素(HBHA)蛋白,探讨其在结核杆菌感染诊断中的应用价值。方法用聚合酶链反应从结核分枝杆菌H37Rv基因组扩增出HBHA基因片段。结核分枝杆菌HBHA基因克隆至PQE80L克隆表达载体中,序列测定正确后,将其转化到大肠埃希菌BL-21中,再经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达HBHA蛋白,表达蛋白经SDS-PAGE及免疫印迹法分析后,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。同时将7H9液体培养基中的卡介苗(BCG)培养至稳定生长期,用CL-6B层析柱分离纯化HBHA蛋白。观察抗体对不同浓度天然HBHA和重组HBHA诱导BCG聚集效应的影响。结果所得天然和重组HBHA蛋白均有诱导BCG聚集的作用,并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的临床诊断价值提供了实验依据。

Objective To purify native and recombinant heparin-binding haemagglutinin（HBHA） proteins,and investigate the application value in clinical diagnosis of tuberculosis.Methods The HBHA gene was amplified by polymerase chain reaction with specific primers from genomic DNA of Mycobacterium tuberculosis H37Rv strain,and was inserted into the cloning and expression vector PQE80L.After sequencing,it was transferred into the host strain Escherichia coli BL-21,and then induced by isopropyl-β-D-1-thogal actopyranoside（IPTG） to express HBHA protein.After the expressed protein was analyzed by SDS-PAGE and Western blot,it was purified by affinity chromatography.The polyclonal antibody was prepared by immunizing New-Zealand rabbit.After growing bacille calmette-guerin（BCG） to the stationary status in the 7H9 liquid medium,the native HBHA protein was obtained by CL-6B column chromatography.The antibody′s activity was determined in inhibiting BCG aggregation induced by native HBHA and recombinant HBHA.Results The native and recombinant HBHA proteins could induce the BCG aggregation,which could be inhibited by both polyclonal antibodies.Conclusions The native and recombinant HBHA proteins are successfully obtained.It is proved that the recombinant HBHA protein could induce the aggregation of BCG as similar as native HBHA protein,and polyclonal antibody against recombinant HBHA protein could be also inhibit the activity of native HBHA protein.It suggests that HBHA could be further used in clinical diagnosis and research of HBHA.