Construction Of Recombinant Lactococcus Lactis Expressing VP7 Protein Of PoRV (G9 Type) And Preliminary Evaluation Of Its Immunogenicity

Porcine Rotavirus（Po RV）is a pathogen of Rotavirus genus,reovirus family,which can cause viral diarrhea of piglets.After infection,the clinical manifestations are vomiting,diarrhea,anorexia and other symptoms,resulting in increased mortality or growth delay of piglets,causing huge economic losses to the pig industry.Po RV can be divided into 7 groups A～G,and most of the infected Por Vs are group A.The molecular epidemiological investigation shows that the Po RV strains in China since 2011 are mainly G9 genotype of Group A,and the commercially available Po RV vaccine is mainly targeted at G5 genotype,and the protective effect of the vaccine is not ideal.The development of A new vaccine is of great significance for the prevention and control of the disease.The disease is mainly transmitted through the fecal-oral route and has the characteristics of mucosal infection.Therefore,it is possible to develop an effective new vaccine that can stimulate the mucosal immune system to produce a local immune response,and then trigger the systemic immune response.VP7 protein plays a decisive role in the immunogenicity of Po RV and stimulates the production of specific SIg A,which is the main antibody for mucosal immunity of newborn piglets.The oral vaccine of recombinant lactic acid bacteria constructed with lactic acid bacteria as expression vector can play a better effect on the prevention of Po RV infection,which has the advantages of simple inoculation,low immunization cost and good safety.Lactic acid bacteria not only has a very strong adhesion effect on intestinal mucosa,but also has prebiotic function,which can improve the body’s immunity.In this study,food-grade engineering carrier Lactococcus lactis NZ3900 was used as A live vector to present antigens,and recombinant Lactococcus lactis expressing VP7 protein of Po RV group A G9 strain was constructed.Piglets were injected with Nisin-induced recombinant Lactococcus lactis by oral immunization to detect its related immune indexes.To explore the immune effect of Po RV oral vaccine on piglets.First,the plasmid p ET-30a-VP7 containing group A G9 Po RV VP7 gene 6403bp stored in the laboratory was used as the template.After being digested by restriction endonuclidenase Sac I and Eco R I,the recovered target fragment VP7 was connected to plasmid p NZ8149.The Lactococcus lactis NZ3900 receptive cells were electrotransferred by electroconversion method,and the positive bacteria were screened by Elliker medium mixed with bromocresol violet.The recombinant plasmid p NZ8149-VP7 was successfully transferred into Lactococcus lactis NZ3900 after PCR,double enzyme digestion and sent to a biological company for sequencing.The constructed recombinant Lactococcus lactis was named p NZ8149-VP7/NZ3900.The recombinant Lactococcus lactis was induced by nisin inducer and identified by SDS-PAGE.Obvious bands appeared at about 37 KDa,and the protein size was as expected.Western-blot analysis showed that p NZ8149-VP7/NZ3900 successfully expressed 37 KDa of target protein.To further investigate the immunogenicity of recombinant Lactococcus lactis p NZ8149-VP7/NZ3900,piglets were used as experimental animals for immune efficacy analysis.The immunization procedure was as follows:12 weaned piglets were randomly divided into four groups on average.The first group was the triple vaccine group,and Houhai point was injected with the triple live vaccine of transmissible gastroenteritis of swine,porcine epidemic diarrhea and porcine rotavirus（TGEV-PEDV Po RV）for one time.Group 2:Recombinant bacteria experimental group,with two gradients of nisin used to induce p NZ8149-VP7/NZ3900,at 2 ng/m L and 4 ng/m L,respectively.After induction,piglet bacterial solution was administered orally 3×10¹¹ CFU/animal,orally immunized on day 0,day 7,and day 14,each time continuously administered for 3 days.The third group:the combined vaccine group,where both groups of immunization programs were administered simultaneously.Group 4:PBS group,administered with PBS on days 0,7,and 14.The anterior vena cava venous blood of 12 piglets were collected on days 0,7,14 and 21 to determine the specific Ig G,SIg A antibody levels and cytokines IL-4,IFN-γHorizontal.The data shows that from the 7th to 21st day after the first immunization,all three groups except for the PBS group can stimulate piglets to produce highly significant levels of specific Ig G antibodies compared to the PBS group（p<0.01）,and the Ig G antibody levels in the triple vaccine group are slightly higher than those in the recombinant bacterial experimental group（p>0.05）;From the 7th day to the 21st day after the first immunization,the Po RV specific SIg A antibody levels in the other three groups,except for the PBS group,showed an upward trend.Among them,the SIg A secretion in the combination group was significantly higher than that in the triple vaccine group from the 14th day to the 21st day（p<0.05）,and the recombinant bacterial experimental group was slightly higher than that in the triple vaccine group（p>0.05）;In IL-4,IFN-γOn the one hand,the combined use group stimulates the production of IL-4 and IFN in piglets-γThe secretion level of the triple vaccine group was the highest,and the secretion level was slightly higher than that of the recombinant bacterial experimental group,but the difference was not significant（p>0.05）.Animal experiments have found that p NZ8149-VP7/NZ3900,induced by nisin,can stimulate piglets to produce a certain level of humoral,mucosal,and cellular immune responses.The immune effect is second only to the commercially available TGEV-PEDV-Po RV triple live vaccine.In conclusion,the recombinant Lactococcus lactis p NZ8149-VP7/NZ3900 of group A G9Po RV VP7 protein was successfully constructed in this study,which can increase the secretion of Ig G,SIg A,IL-4 and IFN-γafter oral immunization of piglets,laying a foundation for further research on novel and efficient oral Po RV vaccine.