The Screening Of Major Antigenic Proteins Of Clostridium Perfringens Type A And Their Immunoprotective Effect

Clostridium perfringens(C. perfringens) was initially found by William Hery Welch, so it also was known as Clostridium welchii, it is a Gram-positive anaerobic bacillus. C. perfringens can produce a variety of external toxins and enzymes, and most of them have lethal effect. The main lethal toxins include ɑ, β, ε and ι toxin. According to the major four lethal toxins produced, C. perfringens can be divided into five types of A to E. C. perfringens type A can cause chicken necrotic enteristis. This disease had a serious economic effect on the global poultry industry, so the prevention and control of necrotic enteristis is always the research hotspot.Since C. perfringens was found in 1891, the study on the pathogenesis and effective preventive measures has never been stopped. But it still lacks adequate information about the interaction between bacteria and host and the effective vaccines to control the bacteria infection. In this study, the whole cell protein sample was prepared with the standard strains ATCC13124 of C. perfringens, two dimensiomal gel eletrophresis was applied to separate the bacterial protein, then polyclonal antibody against C. perfringens was used to screen the novel immune proteins with Western-Blot. After analysis, we selected 16 dots to do the MALDI-TOF-MS and PMF analysis in duplicate. Finally, 12 diffrent proteins were identified as newly found immunogenic proteins except the pyruvate-flavodoxin oxidoreductase(PFO) and hypothetical protein(HP). And most of them located in the bacterial cytoplasm, and involved in cell growth and metabolism.Four immunogenic proteins, including of cysteine synthase(Cys), putative transketolase(PTC), alcohol dehydrogenase(ADH), purine nucleoside phosphorylase(PNP), were selected to construct their prokaryotic expression plasmid to express these recombinant proteins, then prepared four kinds of polyclonal antibody with purified proteins. Western blot analysis showed they all had good reactive ability with natural bacterial proteins of C. perfringens. At last, we constructed the eukaryotic plasmids of these four protein genes. After positive reaction of highly expression of respective proteins with transient transfection in vitro, they were used to study immune protective effect via the animal experiment. The results showed that four recombinant eukaryotic plasmids could resist the challenge of C. perfringens infection in varying degrees, reduced intestinal lesion scores of chickens and could induce good immune response. In which, Cys and PNP, these two kinds of proteins could provide better immune protection against bacterial challenge. In summary, this study provided the material basis for the vaccine reserach of C. perfringens, and have important significance for the clinical control of necrotic enteritis.