| Chickens necrotic enteritis (NE) also called intestinal toxemia, caused by Clostridium perfringens type A or C, is an acute infectious diseases with the characteristics of small intestine thickening and severe necrosis lesions, It gives rise to significant economic impacts on the global poultry production. In this study, two key pathogenic factor of alpha toxin/phospholipase C (plc) and necrotic enteritis B-like toxin (NetB) C. perfringens were selected as antigen to construct recombinant Lactobacillus plantarum (L. plantarum) as a live oral bacterial vaccines. L. plantaruu has prominent characteristics of ability to colonize in intestine, express and transfer exogenous antigen, and induce local mucosal immunity. In the last, the new recombinant vaccine was used to immunize chickens for fully illustration of its protection mechanism, and provide theoretical basis for NE vaccine development.With the specific shuttle vector pSIP409for L. plantarum, C. perfringens genes of plc and NetB were subcloned and got the recombinant plasmid pSIP409-plc and pSIP409-NetB. After double enzyme digestion and sequencing, they were transformed into E. coli and induced by SppIP. Western blot proved that the recombinant plc and NetB protein was successfully expressed, and mainly existed in inclusion form with molecular weight of40kDa and36kDa. Then the recombinant plasmid pSIP409-plc and pSIP409-NetB were transformed to L. plantarum NC8. After the same identification procedure as above, Western blot and indirect immunofluorescence assay demonstrated that pSIP409-plc/L. plantarum and pSIP409-NetB/L. plantarum have ability to secrete plc and NetB proteins after induction by SppIP.In order to study the immune protection mechanism of recombinant L. plantarum secreting C. perfringens plc and NetB protein, chickens were used as experimental animals for immunization and challenge experiments. Experiment procedure was as followed:chickens in immune groups were orally given2kinds of108cfu/d recombinant plc/L. plantarum and NetB/L. plantarum differently from7d to28d consecutively, and the L. plantarum with empty vector and PBS group as negative control. Finally, all chickens were challenged with C. perfringens ATCC13124(109cfu/d) at42d for5days consecutively. After a period of time of the pathological assay, no observable tissue damage showed in recombinant Lactobacillus immunized chickens. During immunization and challenge period, the specific antibody titer against plc and NetB in serum, secreted IgA (sIgA) content in chicken feces, intestinal lavage fluid, the lysate of bronchial alveolar fluid and bile were measured. ELISA results showed that serum antibody to plc and NetB increased significantly and very significantly compared with those of the control chickens (p<0.05, p<0.01). The sIgA content in chicken feces extract, intestinal lavage fluid, bronchial alveolar fluid and bile was detected to keep arising trend with the experiment day went on, and continuely to increase even a period of time after the end of immunization. Furthermore, nearly all the test time, there were remarked difference between the immunized group and the contols. After challenge, all these antibody items had tmepratory decrease and turned ascend rapidly above the level before. This data illustrated that the recombinant L. plantarum could induce the specific humoral immune response, and particularly did induce solid immune response in local mucosal system. Clinical symptoms observation, pathological lesion examination, intestinal lesion scores and bacterial counting experiment proved that pSIP409-plc/L. plantarum and pSIP409-NetB/L. plantarum could be used to protect immunized chicken challenge from C. periferings.In conclusions, it is safe and effective of the oral immunization with pSIP409-plc/L. plantarum and pSIP409-NetB/L. plantarum in chicken. It provide theoretical and practical basis for the development recombinant L. plantarum of vaccine for chicken NE. |
