Construction Of Recombinant Lactic Acid Bacteria Active Delivery Vector Expressing IBDV-VP2 And Evaluation Of Immune Effect

Lactic acid bacteria(LAB)is a kind of gram-positive bacteria which metabolic properties,physiological characteristics and shape are not exactly same.Its main characteristic is to produce lactic acid through the fermentation of carbohydrates,and most of species benefit animal and plant.In the early it has been widely used in food fermentation engineering and gained a huge success.With the further studies of the physiological characteristics and molecular genetics features of LAB,and researchers realized that LAB can appear as a live carrier to deliver heterologous antigen,which has great application prospect of the prevention and treatment of various diseases.Especially the systemic and mucosal responses were stimulated by these recombinant LAB as live vector vaccine.It has not any biological safety problems,but it hasn't high efficiency for expressing and delivering antibody protein to induce body strong immune response.The main purpose of this study is constructing recombinant LAB active delivery system,to improve the efficiency of antigen delivery,activate the body's immune response efficiency,then lay a foundation for the study of new type LAB live vaccine vector.In this study,completely food-grade gene engineering Lactococcus lactis NZ3900 and matching pNZ8149,the introduction of adhesive proteins,the fusion expression of internalin and IBDV-VP2.Firstly,PagN protein(support Salmonella enterica serovar Typhimurium adhesion to and invasion of epithelial cells)is introduced,constructed two recombination strains(L.Lactis-VP2-PagN and L.Lactis-PagN-VP2);secondly,the fusion expression of Omp(only 32 amino acids,the ligand of M cell receptor,mediated antigen uptake and immune stimulation)and IBDV-VP2,constructing recombinant strain L.Lactis-VP2-Omp.In vitro test results show that:L.Lactis-VP2-PagN expressed fusion protein correctly,whose size is73kDa and consistent with the expected.And the fusion protein is high stability,gains the ability of adhering to HT-29 cell;L.Lactis-VP2-Omp also expressed correctly and the size(50.46 kDa)is consistent with the expected by western bolt,stay stable under room temperature,and the content of fusion protein is 53.17 ug/mL.Animal experimental results of L.Lactis-VP2-PagN:(1)we have designed two experimental groups(oral immunization group and inject immunization group)for L.Lactis-VP2-PagN,each group of immune dose 1×10~9 CFU/feather;moreover,two control group(PBS control and blank strain control);the chickens were immunized at 7 days and 14 days,and conducted challenge experiment by HLJ0504virulent at 28 days.We didn't monitored IBDV-specific IgG;oropharynx mucosal sIgA of oral group was not significantly different from the control,intestinal mucosal sIgA was significant higher than the control(P<0.05);the survival rates of two experimental groups is 0.Our results indicate that L.Lactis-VP2-PagN failed to induce specific immune response,and didn't protect body against IBDV strain HLJ0504.Animal experimental results of L.Lactis-VP2-Omp:two experimental groups(oral immunization group and inject immunization group),two control(non-immunized challenged group and free handle group);the chickens of two experimental groups were immunized by L.Lactis-VP2-Omp lysates at 14days and 21days;and were challenged at 28 days.The results show that:(1)the survival rates of inject immunization group and oral immunization group were 100%and 60%respectively;(2)the IgG levels of two experimental groups are 0,but specific neutralizing antibody was detested(1:8 to 1:32);(3)compared with the control group,intestinal mucosal sIgA of oral immunization group was obviously increasing,and the difference is significant(P<0.05);(4)bursals of oral immunization group live chickens have suffered different degree of 100%damage,bursals of inject immunization group live chickens have suffered 80%damage.The above results indicate that L.Lactis-VP2-Omp lysates can induce body to produce specific immune response after injected and oral immunization.This research used two kinds of strategies to construct LAB live vector system which active delivery IBDV VP2,L.Lactis-VP2-Omp can induce specific response effectively after evaluation in vivo and in vitro,laid a solid foundation of the research of new type of LAB live vector.