| Lactic acid bacteria is a kind of the G+ bacteria.Its metabolic status,physiological characteristics and morphology are completely different from other bacteria.Its main function is to ferment carbohydrates to produce a large amount of lactic acid.Lactobacillus is a probiotic in the digestive tract.Its safety is recognized in the world(GRAS).Due to the regulation of lactic acid bacteria itself and other vaccines,many researchers in the world have begun to use lactic acid bacteria as a living carrier to express foreign sources.Protein,developed for oral and vaccination.Object: To construct a recombinant lactic acid bacteria IBDV-VP2 live vector vaccine,to improve the technical platform for the development of lactic acid bacteria live vector vaccine and to study its immune mechanism.Methods:(1)Construction of r-L.Lactis-USP45-VP2-RCK-Acm A2 and evaluation of immune effect: This study used NZ3900 as a live vector,targeting the VP2 gene of the IBDV strain.The VP2-RCK gene and Acm A2 gene were inserted into the food-grade vector p NZ8149 by homologous recombination technique,and electrotransferred into NZ3900 strain to obtain the recombinant strain r-L.Lactis-USP45-VP2-RCKAcm A2.The immune effects were evaluated using in vitro assays and animal experiments.Animal experiments were designed in two experimental groups(oral immunization group,injection immunization group)and two control groups(blank group,empty strain group).The experimental group had an immunization dose of 1×109 CFU/ml,and the first immunization was on the 7th,and the seconde immunization was the age of 14 days.They were all attacked at 28 days of age(1000 ELD50/feather).(2)Optimizing OmpH active presentation of recombinant lactic acid bacteria live carrier platform: M cells in the intestine are used as target cells for presenting antigen lactic acid bacteria(M cells are cells presenting and taking antigen in the intestine),with the purpose of enhancing antigenic protein by the body.The probability of ingestion and presentation.In this study,the lactic acid bacteria expression vector plasmids pNZ8149-VP2-2OmpH,p NZ8149-OmpH-VP2-OmpH,pNZ8149-2OmpH-VP2-2OmpH for the first time to achieve.The NICE expression system detects the expression of the fusion protein in vitro,analyzes the stability of the protein,and optimizes the induction conditions by orthogonal experiments.(3)Immune effect of r-L.Lactis-VP2-RCK under high level maternal antibody: This study used ELISA to detect maternal antibody of commercial chickens,and selected the highest peak period of maternal antibody to recombine through animal experiments.The r-L.Lactis-VP2-RCK and B87 were compared in the chickens with maternal antibodies,and evaluate the immune mechanism of the r-L.Lactis-VP2-RCK.Results :(1)Inducible expression by nisin,the protein VP2-RCK-Acm A2 was correctly expressed in recombinant lactic acid bacteria by Western Blot,and it had high stability,and its expression was further verified.By flow cytometry analysis,it was shown that nearly 20% of recombinant lactic acid bacteria were able to display protein on the cell wall.Quantitative analysis of the expressed protein by Western Blot assay and protein quantification kit revealed that the expression level of the fusion protein reached 12.4 μg / mL.Animal experiment results: Serum neutralizing antibody was negative in the injection and oral groups;compared with the control group,there was no significant difference in the s Ig A antibody titer of the oropharyngeal mucosa in the oral group;all the chickens in the oral and injection groups died,and the attack protection rate is 0.(2)The expression of r-L.Lactis-VP2-2OmpH,r-L.Lactis-OmpH-VP2-OmpH and r-L.Lactis-2OmpH-VP2-2OmpH recombinant protein was detected by Western Blot.Through protein stability experiments,it was proved that the proteins expressed by the three recombinant lactic acid bacteria could stably express for more than one week at 4 ℃.Quantitative analysis of the expressed protein by BCA and thin layer gel scanning experiments showed that the expression levels of the protein were 98.846μg /m L,411.8084ug/m L and 93.2552ug/m L,respectively.The expression levels of r-L.Lactis-OmpH-VP2-OmpH were the highest among the three recombinant lactic acid bacteria.(3)This study was the first to study the r-L.Lactis-VP2-RCK to immunize commercial chickens with high levels of maternal antibodies.By comparing with the live vaccine B87,the r-L.Lactis-VP2-RCK was found to be active.The marked vaccine antigen presentation mode and the immune response are not interfered with by maternal antibodies.By analyzing the serum neutralizing antibody,the r-L.Lactis-VP2-RCK produced neutralizing antibodies faster than B87,and the number of mature B cells in the blood was not reduced,and the bursa of Fabricius was not damaged and developed normally.Conclusion: The platform of the expression system of Lactococcus is further improved,and the construction of IBDV-VP2 protein and Salmonella RCK protein anchored on the cell wall of recombinant lactic acid bacteria is completed.This study revealed that the r-L.Lactis-VP2-RCK recombinant lactic acid bacteria live vector vaccine antigen presentation method and immune response are not interfered by maternal antibody,and also revealed that the recombinant lactic acid bacteria vaccine induces the immune mechanism different from the conventional vaccine immune response. |
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