Inclusion body purification, polyclonal antibody preparation and activity verification of Vibrio harveyi hemolysin
ZHAO Xiaoran, GUO Yi, DANG Huifeng, XING Zhenyu, HE Zhe, YE Shigen*
Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture and Rural Affairs, Dalian Key Laboratory of Marine Animal Disease Prevention and Control, Dalian Ocean University, Dalian 116023, China
Abstract: A prokaryotic expression vector of Vibrioharveyi hemolysin(VHH) was constructed based on the cloned vhh gene and a polyclonal antibody was prepared using the purified recombinant protein to study the biological function of VHH. The titer and specificity of the antibody were determined by DOT-ELISA and Western-blot, respectively, and the effect of the polyclonal antibody against the hemolytic activity of V.harveyi culture supernatant was evaluated by hemolysis test. The results showed that the recombinant protein was expressed as an inclusion body, and that the purified VHH was used to prepare the polyclonal antibody which was detected to have a titer of 1∶163 840, and recognized VHH protein specifically. Moreover, the polyclonal antibody had a significant inhibitory effect on the hemolytic activity of the supernatant of V.harveyi culture when the dilution factor was less than 1000 times. The findings show an important reference value for the preparation of VHH monoclonal antibody, the improvement of V.harveyi detection method and the research and development of V.harveyi vaccine.
2020, Vol. 35 
