Cloning And Expression Of Alpha-toxin Gene Of Clostridium Perfringens Type A And Its Immunological Study

Alpha-toxin of Clostridium perfringens type A(CPA) is the major factor leading to the disease of gas gangrene and one of the most important biological toxins. In order to find an effecient way to defense or treat this disease, in this study the alpha-toxin genes of CPA were cloned and highly expressed hi E.coli DH5 a .After having been purified , the c-terminal domain's primary immunological protective function was studied and it's Egg Yolk Immunoglobulin (IgY) was prepared.By routine polymerase chain reaction, the alpha-toxin totall gene (cpa!229 gene) and the c-terminal gene (cpa408 gene) were amplified. After having been cloned into the pMD18-T Vector and analysed , the two fragments then were connected with the expression vector pBV220 and transferred into the host cell E.coli DH5 a .Induced by 42 癈, the cpa408 gene was highly expressed ,which expression amount was to the 43.75 percent of the totall bacterial protein. After having been pelleted by 80%(NH4)2SO4 and dialysised, the expressed protein was isolated and purified by the gel filtration choromatography. Then according to an amount of l.Omg/Kg, the hens(SPF degree and aged 30 weeks) and the Kunming Mice(body weighted 18g) were immuned with the purified protein individualy by multiple muscle injection and intraperitoneal inoculation. One week after the first enhanced immunization, the Kunming Mice were attacked with an amount of 1 .OMLD alpha-toxin, in which the eight mice immuned all survive and the control group all died . During the period of immunization, the titre of the mouse's serum antibody and the Egg Yolk Immunoglobulin(IgY) was measured by ELISA. One week after the first immunization, the titre of the mice's serum antibody was l:800,but that of one week after the first enhanced immunization reached to 1:6400. From one week to elevenweeks after the first immunization, the titre of IgY rose from 1:800 to 1:25600. By ELISA and double immunological diffusion , the IgY could react well with cpa408 protein and alpha-toxin, which assumed that the IgY could be applied to the examination of the alpha-toxin.The immunological protective function produced with cpa408 protein in mice suggests that the cpa408 gene product is a good antigen and it's antibody can protect the mice against the alpha-toxin. Therefore, the cpa408 gene can be considered as one of the protective antigen genes. This will supply not only an ideal material but also an theoretical support to the next research for the vaccine and the anti alpha-toxin.