Generation Of A Recombinant Lactobacillus Plantarum Expressing The ORF2 Gene Of Porcine Circovirus Type 2 And Its Immunogenicity In Mice

Porcine circovirus type 2(PCV2)is the main and primary causative agent of post-weaning multisystemic wasting syndrome(PMWS),and which is characterized by serious immunosuppression in the porcine host.Meanwhile,PCV2 is also closely associated with other diseases,such as congenital tremors,porcine dermatitis and nephropathy syndrome,respiratory disease complex,reproductive failure,and so on.In a word,porcine circovirus-associated diseases are a series of the floorboard of the related diseases affected by PCV2,a major problem influencing the productivity of the swine industry,causing considerable losses worldwide.At present,prevention and control of PCV2 mainly relies on vaccination with commercial PCV2 vaccine,therefore,thus there is an increasing demand of vaccines worldwide.However,the low drop of PCV2 virus in cell proliferation limited the development and utilization of traditional attenuated vaccine and inactivated vaccine.The capsid protein(Cap)of PCV2 encoded by ORF2 was identified as the major immunogenic protein of the virus,stimulating the body to produce corresponding PCV2 specific neutralizing antibody.Hence,modified a PCV2 subunit vaccine by genetic engineering technology has become the hot spot in the genetic engineering vaccine.Thus the cap protein of PCV2 produced in this study has the potential to be applied as an effective and novel subunit vaccine against PCV2.Lactobacillus plantarum(L.plantarum)is the normal flora in the intestinal,which has incomparable advantages as the expression of foreign proteins.L.plantarum as a probiotic can resistant to acid and bile salts in the digestive tract.L.plantarum can adhere strongly to mucosa,stimulating the body to induce mucosal immune response.Exogenous antigens located on the mucosal surface can induce specific cellular and humoral immune responses through the membrane mechanism and throughout the systemic immune system.In this study,we constructed recombinant L.plantarum expression vector expressing PC V2 Cap protein,and verified the biological activity of the genetically engineered recombinant L.plantarum,to provide a theoretical reference for the development of porcine circovirus virus oral vaccine.The whole genome of PCV2 was amplified using DNA of the positive material and then cloned to p MD19-T vector to sequence,to obtain PCV2 b subtypes strains.A pair of primers were designed and synthesized to amplify the PCV2 b O RF2 gene,which were purified and cloned into L.plantarum expression vector p SCPSP containing M6 precursor protein signal peptide SP and driven by super constitutive promoter SCP.The recombinant vector was transformed into LP1 by electroporation and positive transformants was screened by PCR named p SCPSP-Cap-LP1.Afterwards,the protein expressed in 24 h supernatant was detected by SDS-PAGE and Western blotting.Sequentially,8-week-old SPF BALB/c mice were randomly divided into lavage group,group of drinking water,vaccine group and control group,in which,lavage group and group of drinking water were set up different concentration gradients respectively.After 14 d,28 d,42 d and 56 d consecutive days,mice serum was collected to evaluate the level of specific antibody.Eventually,our experiments results proved that the expression vector p SCPSP-Cap was successfully constructed and successfully transferred to L.plantarum and the positive transformants p SCPSP-Cap-LP1 were screened;The espression of Cap protein of PCV2 was successfully expressed in Lactobacillus plantarum LP1 was verified by SDS-PAGE and Western blotting,expression of protein molecular weight was 28 k Da and with good antigenicity;In addition,the results of ELISA showed that higher levels of specific antibody in the serum of mice were observed by oral administration with L.plantarum expressing the PCV2 Cap antigen(P<0.05).There was no visual difference between the contol group and the immunization with L.plantarum L1 group and the empty vector transforma nts group compared with mental state pathological anatomical,indicating that L.plantarum and protein secreted by L.plantarum had no toxic side effects on mice.In conclusion,the Cap protein encoded by ORF2 gene of PCV2 b was successfully expressed in L.plantarum.The recombinant strain was successfully constructed and its immunological effect was evaluated and analyzed.Specific antibody was stimulated to produce by orally irrigated mice with genetically engineered bacteria.As a result,our research provides a theoretical reference for the development of porcine circovirus oral vaccine.