Preparation And Identification Of Recombinant Pneumolysin (PlyLD) Protein Vaccine Antigen

This study is focused on the construction,fermentation as well as the purification of a recombinant engineering strain to provide a new candidate pneumococcal vaccine with lower hemolytic activity and better protective immunity.For the purpose of getting an attenuated vaccine as well as meeting the requirements of industrial production,molecular cloning technology,E.coli prokaryotic expression system and pET9 a expression plasmid which equipped with a T7 promote were used to establish the PlyLD engineering strain.And the producing haemolysin has a leucine instead of an aspartic acid at the carboxyl terminal.At the same time,the process parameters in fermentation were determined by condition experiments: The pre-inductive culture temperature was 34~37?;The inductive culture temperature was 27~30?;The OD value of subcultivation was 1.0~2.0;The OD value of inducement was 15~20;The pH value of fermentation was 7.1;The concentrations of feed was 1.0g/L;The concentrations of IPTG was 1.0mM;The cells were collected after induced of IPTG for 8~10 hours.Besides,the technical schemes in purification process were developed: The OD value of broken(two times of broken was necessary)was 100~125;The sample was precipitated by adding ammonium sulphate to a final concentration of 30%(g/ml).Column chromatography and ultrafiltration were also necessary to the purification process.In addition,abilities of mice immunized by PlyLD against incursions of pneumococcus WU2,TIGR4 and Sp6 A were improved remarkably.Cytotoxicity and serotype restriction of pneumococcal conjugate vaccine were settled in these studies which were very important for the industrial production of PlyLD vaccine.