摘要
目的:制备天然和重组HBHA蛋白,探讨其在结核杆菌感染诊断中的应用价值。方法用PCR方法从结核分枝杆菌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,将其转化到大肠杆菌(E.coli)BL-21中,再经IPTG诱导表达HBHA蛋白,表达蛋白经SDS-PAGE及Western blot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(nHBHA)。观察抗体对不同浓度nHBHA和rHBHA诱导BCG聚集效应的影响。结果所得天然和重组蛋白均有诱导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) protein,and investigate the activity the vale of HBHA in clinical diagnosis.Methods he HBHA gene was amplified by PCR with specific primers from genomic DNA of M.tuberculosis H37 Rv strain,and was inserted into the cloning and expression vector PQE80 L.After sequenced,it was transfected into the host strain E.coli BL-21,and then induced by IPTG to express HBHA protein.After the expressed protein was analyzed by SDS-PAGE and Western blot,then it was purified by affinity chromatography.Then the polyclonal antibody against rHBHA was prepared by immunizing rabbit.After growing BCG to the stationary status in the 7H9 liquid medium,the native HBHA protein(nHBHA) was obtained by CL-6B column chromatography.And the antibody" s activity was determined in suppressing BCG aggregation induced by nHBHA and rHBHA.Conclusion The native and recombinant HBHA were successfully obtained.It was proved that the rHBHA could induce the aggregation of BCG similar as nHBHA,And polyclonal antibody against rHBHA could be also suppress the activity of nHBHA.It suggest that rHBHA could be further used in clinical diagnosis and vaccination.
引文
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