Purification and activity determination of native and recombinant HBHA protein
10.3760/cma.j.issn.1009-9158.2010.03.019
- VernacularTitle:重组和天然HBHA蛋白制备及其活性研究
- Author:
Shan ZHOU
;
Yueyun MA
;
Jiayun LIU
;
Mingquan SU
;
Hai ZHANG
;
Changhong SHI
;
Lihua WANG
;
Xuguang GUO
;
Xiaoke HAO
- Publication Type:Journal Article
- Keywords:
Mycobacterium tuberculosis;
Lectins;
Antibodies
- From:
Chinese Journal of Laboratory Medicine
2010;33(3):271-275
- CountryChina
- Language:Chinese
-
Abstract:
Objective To purify native and recombinant heparin-binding hemagglutinin(HBHA)protein,and investigate the activity of HBHA polyclonal antibody against aggregation of Bacillus CalmetteGuerin(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 gene 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 0.2μg/ml,BCG could be induced to aggregate,while the rHBHA concentration is 2μg/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.
