| The coccidiosis of Eimeria tenella which is caused by coccidia is a kind of harmly seriously parasitic disease in cell, it is one of the most common form of the disease on poultry breeding industry in the world.Seven kinds of Eimeria distinguishly are parasitic on different intestinal epithelial cells of chicken, causing different degrees of morbidity and mortality in chicken.The spores pathogenicity coccidia invade into the intestinal epithelium cells of chicken,after4～5days it can excret mature oocyst.when people can see mature oocyst through the microscope,it is the late of coccidiosis.And it has damaged the intestinal mucosa of chicken irreparably,affecting the normal digestion absorption of intestinal, reducing the feed utilization rate,hinderring, normal growth and development of the chicken,causing weight loss,the chick will dead when it infected seriously,the mortality rate as high as80%, or even the whole group of chicks are all dead.For a long time,we all depend on chemical drugs to prevent and control chicken coccidia.But due to the coccidia have drug resistance easily to chemical medicine,not only increased exposure leels, increasing the cost of control,but also causing drug residue,ecological environmental pollution and so on,so people have to find new methods to prevent chicken coccidia.In order to control and prevent chicken coccidiosis more preferably,it is urgent affairs that developed out chicken eimeria vaccine.In the genes of coccidia which have been reported, the antigen which is coded by SO7gene is located in refraction body of sporozoite of Eimeria tenella,which is a protective antigen,we use the expression product to immune chicken then we found it not only has certain defense effect to chicken coccidiosis, but also has some protection of other coccidiaThis study will put fusion DC-targeting peptide SO7gene into the shuttle expression vectors of lactobacillus which is pSIP-409,constructing restructured plasmid which is called pSIP-409-SO7-DCpep, then put it in lactobacillus to expressing,at the same time detect the immune results.The target gene is using the extractal technology of RNA extraction to extract out. According to the ORF of SO7gene which is published on Genbank designed a pair of specific primer. Put the producting of PCR amplification cloning into the vector of pMD18-T,transform it into competent cell DH5a,extract plasmid,then using the methods of enzyme digestion,PCR digestion,sequencing and analysis and so on to identify this gene,then intercomparison it to SO7gene which is published on Genbank,the homology is attained99%,aslo it succeed fusioning DCpep which contained36base pairs.This test was constructing restructured plasmid pSIP-409-SO7,then transformed into Lb.plantarum NC8,positive clones were screened,using western-blot analysis, the results showed that this recombinant strain was expressed fusion protein successfully at the position of23.87KDa,it conformed with expected results.we prepared restructuring lactobacillus pSIP-409-SO7-DCpep in the same method,then we can see a obvious protein bands at the position of25.04kDa.Feeding four different strains to1day age of broiler chick for two weeks,then use coccidio to infect,observating1weeks. The results showed that,compared with the control, the experimental group the lesion of cecum of chicken reduced significantly, pathogenesis symptoms reduced significantly,excret oocyst decreased,the difference was significant. The results of experiment showed that SO7-DCpep gene expressed in Lb.plantarum NC8successfully,so it planted the establish foundation for developing and restructuring probiotics preparations of resistance for chicken coccidia. |
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