The Expression And Immunogenicity Of The Main Immunogenic Genes Of Lawsonia Intracellularis In Lactic Acid Bacteria

Porcine proliferative enteritis(PPE)is a contact infectious disease caused by Lawsonia intracellularis(LI).It has a high degree of hyperplasia of the colon and caecum near the ileum and the blind node as a feature and infected pigs show diarrhea and growth inhibition,which can cause sudden death.At present,antibiotics are mainly used to prevent the disease.Restricted use of antibiotics due to the increasingly stringent regulations on antibiotic use and the emergence of antibiotic resistance in the meat industry.Given the above factors,effective LI vaccine is an effective measure to prevent PPE.An lintracel lularis modifed-live vaccine and shows efcacy in reducing the severity of clinical symptoms in infected pigs,but it is expensive and it is necessary to study the corresponding subunit vaccine.Lactic acid bacteria live carrier oral microecological preparations can be directly taken orally,it is widely used because it is easy to administer and safe and non-toxic.This study successfully constructed three recombinant Lactobacillus plantarum,Named as D-p LP3-Pslp A-Opt A-L1 、D-p LP3-Pslp A-Opt B-L1、D-p LP3-Pslp A-Lhly-L1.Using this system to express the intracellular Lawsonella immunogenic proteins Opt A,Opt B,Fli C and Lhly,studying the immune effect of recombinant Lactobacillus plantarum provides a new way for the development of a new vaccine against Lawsonia intracellulare and the control of Lawsonia intracellulare.The contents of this study are as follows:First,DNA extraction from suspected PPE,and design primers according to the sequence of NCBI,the gene sequences of the Lawsonella immunogenic proteins Opt A,Opt B and Lhly were amplified,and the target gene was cloned into a Lactobacillus plantarum expression vector D-p LP3-Pslp A,it containing a constitutive promoter S protein and secretion signal peptide,Obtaining recombinant Lactobacillus plantarum expression vectors D-p LP3-Pslp A-Opt A,D-p LP3-Pslp A-Opt B,D-p LP3-Pslp A-Lhly.The recombinant Lactobacillus plantarum expression vector was electrotransformed into the expression host LP1,and recombinant positive transformants were screened out by PCR identification.Afterwards,the protein expressed in 12 h supernatant was detected by SDS-PAGE and Wes tern blotting,results showed specific bands at 13 KD,38 KD,and 34 KD,these four proteins of interest were successfully expressed in Lactobacillus plantarum LP1.Next,prokaryotic expression and identification of three immunogenic proteins of Lawsonella were performed.Opt A,Opt B,and Lhly proteins are immunogenic proteins of Lawsonia sclerotiorum.Based on the sequence design and PCR,three gene fragments of300 bp,954bp and 753 bp were amplified and cloned into p ET-28a(+)In the expression vector.After PCR identification and sequencing,the obtained positive recombinant plasmid was transferred into E.coli BL21(DE3),after induction with IPTG,the target protein can be obtained.The recombinant protein is soluble or renatured and purified by affinity chromatography,the protein is identified by Western blot electrophoresis.The reaction was carried out,indicating that the target protein was successfully purified.Last,in order to explore the immune effect of recombinant plant lactobacillus,this experiment was divided into 6 groups.PBS is blank control,D-p LP3-Pslp A-LP1 is empty v ector control,and D-p LP3-Pslp A-Opt A-LP1,D-p LP3-Pslp A-Opt B-LP1,D-p LP3-Pslp A-Lh ly-LP1 test group and 3 recombinant protein mixed bacteria group.Seven-week-old Balb/c mice were immunized by drinking water and continuously immunized for 30 days.Five mice were randomly selected from each group at 0 days,7 days,14 days,21 days,and 30 days,and blood was collected from the orbital vein.In order to detect the level of anti bodies produced by the protein of interest in mice.Detection of sera produced by immuniz d mice by an indirect ELISA method using a protein expressed by E.coli as a coating ant igen.The experimental results show that Opt A,Opt B,and Lhly all have good immunoge nicity,and when the mixed bacteria of Opt A,Opt B,and Lhly were used to immunize mice together,the titer of antibody production was higher than that of single bacteria.In conclusion,lawsonella immunogenic proteins were successfully expressed in lact obacillus plantarum,oral recombinant plant lactic acid can stimulate the production of spe cific antibodies in mice,which provides a theoretical reference for the development and research of oral vaccines for porcine proliferative enteritis.