| Coccidiosis is caused by one or more Eimeria(Eimeria coccidiosis) parasites in the intestinal epithelial cells. It hindering development of the poultry industry.Currently,prevention and treatment of coccidiosis in chickens immunized mainly in chemical-based drugs,but long-term drug prevention and treatment caused a series of food safety issues,While the existing commercial chicken coccidiosis vaccine in the prevention of coccidiosis in chickens and achieved some results,but its existence reversion of virulence and caused widespread infection risks.In recent years,live vector vaccine become an important direction for the development of vaccines,which can effectively deliver vaccine antigens to body, it makes antigen stimulation of the mucosal immune substances closer to the natural route of infection of the pathogen. Wherein the lactic acid bacteria as a carrier protein has many advantages, it is easy to culture,does not induce endotoxin, has strong colonization and Immune adjuvant effect, no need to be able to induce the expression of a large number of related immune stimulation and expression protein without purification, it has a simple extraction method. Making it a live vector vaccine research hotspot.With microbial surface display system deepening of the study, So that the foreign protein can be expressed in the cell surface in order to better stimulate the body to produce antibodies against the continued foreign protein. Because poly-gamma-glutamic acid synthetase A(Poly-gamma-Glutamic Acid Synthetase A, pgsA) can exogenous protein expressed on the surface of lactic acid bacteria,and has a good result. This experiment demonstrated the role of constructing a recombinant lactic acid bacteria having a surface will Eimeria(Eimeria tenella)protective antigen protein micro line 2(EtMIC2) in cell surface expression of the chicks were immunized in order to obtain good resistance to E.tenella effect.Specific contents and results are as follows:1.Construction and expression of the bacterial protein:Application of genetic engineering techniques to obtain EtMIC2 gene, Through pSIP409-pgsA carrier connected, Transferred into E. Coli amplified, Digestion and sequencing results verified correct, then pSIP409-pgsA- EtMIC2 recombinant plasmids were transformed into Lactobacillus NC8 by electroporation method. And verify protein expression by Western Blot, The results showed that the protein size of 90 KDa consistent with the expected results.And by immunofluorescence and flow cytometry to identify the target protein surfacedisplay case, The results showed that the protein can be well expressed in the cell surface, Flow cytometry relative expression level was 26.4%.2. Effects of recombinant bacterial to E.tenella infection chickens,Immune Level and Evaluation of protective immunity:This study use 1 to 25-day old male meat chickens for laboratory animals, and successfully built a EtMIC2 protein of pSIP409-pgsA-EtMIC2 recombinant Lactobacillus. We select plant lactobacillus NC8 pSIP409- pgsA recombinant lactic acid bacteria and PBS group as contrast.The similar weight and 125 healthy 1 day age meat chicks were randomly divided into A, B, C, D, E five groups, and 25 for each group of animals are housed in natural infection-free indoor.Chicks during 1 to 15 days, A and B groups pour PBS200 mu L before feeding every other day.C, D and E groups fill and take their corresponding strain2×108CFU/200μL every other day.The group C irrigation pSIP409- pgsA- EtMIC2 of recombinant strains,The group Dirrigation pSIP409-pgsA of recombinant strains and group E take plant lactobacillus NC8.Chicks at the age of 15 days,kill 10 in each group in peripheral blood lymphocyte proliferation ability,CD3~+, CD4~+and CD3~+CD8~+T lymphocytes content detection,antibody IgG content and SIgA in the chyme and IFN-γ and IL2 cytokine secretion detection.Apart from the group A, the other chicks remaining in the age of 15 inoculate spores of Eimeria tenella sacs 5×104.Insect attack after 8 days, kill all chickens at 25th-day old. In addition to testing the above indicators, we will calculate chicks’ relative growth rate, blood score, appendix lesions integral and oocyst value of the cecum contents, OPG and other datas.And observe the pathological section to draw anticoccidial index.(1)The effect of Recombinant bacteria on the immune level of the chicks that was infected with E.enella: Respectively evaluate the immune indexes in the groups of the ball insect infected chicks before and before the age of 15 days and after the age of 25 days age,and the result showes that after taking their corresponding lactic acid bacterium,the lymphocyte proliferation ability in the peripheral blood can be improved not only before the ifection but after the infection,Wherein the group C compared with A, B two groups were significantly different(P<0.05). And furthermore these bacterium can also Promote the peripheral blood of CD3~+,CD4~+and CD3 + CD8 + T lymphocytes whose aim is to improve the production of the peripheral in blood cytokines IL- 2 and IFN- γ,and we can Raise the level of chicks humoral immune by increasing peripheral blood IgG antibodies and intestinal contents SIgA antibody secretion.Amnog them, pSIP409-pgsA- EtMIC2 recombinant lactobacillus has the best effect,but there are few differences between Lactobacillus NC8 and pSIP409-pgsA recombinantlactobacillus(P<0.05).(2)Recombinant bacteria on chicken E. tenella’s protective efficacy evaluation : The results show that, before and after coccidia infection, the three groups chicks’ drenching probiotic average weight are higher than Group B and Group C and B’s difference is significant(P﹤0.05). Chick Group C, D and E’s blood score are lower than group B and Group C is the lowest.Chick Group B’s caecum lesion is most serious, and lesion scoring average is 3.75. It is significantly higher than other probiotic groups. Chick Group C, D, E taking probiotics the oocyst discharge were lower than group B and Group C is the lowest. Group C, D and E’s Fedding probiotics anti-coccidial index is 145.15, 115.57, 126.81.Group C is between 120~160.The anticoccidial effects is medium. |
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