ABSTRACT
Objective To construct the expression vector and express the recombinant human L-selectin in prokaryotic system, and purify the aimed protein for the preparation of rabbit anti-human L-selectin polyclonal antibody. Methods The partial cDNA of human L-selectin was amplified from the open reading frame (ORF) of N-terminus sequence of L-selectin containing 153 amino acids by PCR, then cloned into the prokaryotic expression vector pQE40 at restriction sites BamH Ⅰ and Hind Ⅲ. The recombinant expression plasmid pQE40-L-selectin was transformed into E. coli M15 for expression. The fusion protein including 6-His-tag was purified by Ni-NTA chromatographic column and analysed by SDS-PAGE. The purified protein was used to immune rabbit for preparing polyclonal antibody. Western blotting analysis and dot immunoblot assay (DIBA) were used to test the titer of the antiserum. Results The expression plasmid pQE40-L-selectin was constructed and confirmed with restriction enzyme digestion. The quantity of the purified protein from E. coli M15 was 600 mg ? L-1. By immuning the rabbit, the polyclonal antibody was successfully prepared. The results of dot immunoblot assay (DIBA) showed that the antiserum had the high titer (1 : 1 000). Conclusion The recombinant human L-seleclin prolein can express with high efficiency in E. coli M15. The prepared polyclonal antibody has a high titer.
ABSTRACT
Objective:To express and purify hemoglobin receptor(HgbA) and its partial fragment(HgbAF) from Haemophilus ducreyi and to develop a sandwich ELISA for the detection of H.ducreyi infection.Methods:The HgbA,a hemoglobin-binding outer membrane protein of H.ducreyi and its partial fragment(HgbAF) were expressed by cloning the genes of hgbA and its 705bp fragment into pET30a and pET28a respectively,and the expressing products were purified from E.coli BL21 with Ni-NTA-His affinity chromatography.The polyclonal antibodies were developed by immunizing rabbits with the rHgbA and rHgbAF.The anti-rHgbA IgG and anti-rHgbAF IgG were purified respectively by saturated amine sulphate precipitation,and their immunoactivity with rHgbA and rHgbAF was tested by Westen blot and ELISA analysis.A Sandwich ELISA was developed for the detection of chinical infection of H.ducreyi using the specific polyclonal antibodies.Results:The HgbA and its partial fragment,HgbAF of H.ducreyi,were expressed and purified successfully by cloning their genes respectively.The results obtained by Western blot analysis showed that each of the antibodies could react with both antigens,rHgbA and rHgbAF.The results of the ELISA analysis showed that H.ducreyi strain was strongly positive,and all other bacteria,including H.influenzae and the bacteria known to relate to genital ulcers were negative.The results of the ELISA analysis showed that the minimum amount of rHgbA detected was 1.56 ng/ml and the minimum number of CFU of H.ducreyi detected was 2?105 cfu/ml in buffer and 1?106 cfu/ml in pus.Conclusion:HgbA and its partial fragment,HgbAF of H.ducreyi are expressed and purified successfully.The polyclonal antibodies developed by immunizing rabbits using rHgbA and rHgbAF could react not only with rHgbA and rHgbAF,but also with H.ducreyi specifically.They do not react with other bacteria,including H.influenzae and the bacteria known to relate to genital ulcers.So the ELISA based on the polyclonal antibodies was specific for the detection of H.ducreyi.Although the level of sensitivity of the ELISA may not be sufficient to detect H.ducreyi in all clinical specimens,further work to increase the sensitivity could potentially make this assay a valuable tool in areas where chancroid is endemic.