Construction Of Multi-type FMDV VP1 Epitopes Embedded S-layer Protein Vector And Its Identification In Lactobacillus Fermentum

Foot-and-mouth disease is a highly infectious and economically devastating disease of livestock. Although vaccines, available since the early 1900s, have been instrumental in eradicating FMD from parts of the world, the disease still affects millions of animals around the globe and remains the main sanitary barrier to the commerce of animals and animal products. At present, inactivated vaccination is the most effective measure for prevention of FMD. However, inactivated vaccine had a lot of limitations:fail to induce long-term protective immunity; potential virus escape from the production facility; short shelf life of formulated products and requirement of dozens of antigens to address viral antigenic diversity. Therefore, this study attempts to design novel vaccine to content new requires for production of FMD vaccines.In this study, multi-type FMDV VP1 epitopes gene which was predicted by biological software was embedded into the specific site of Lactobacillus fermentum S-layer protein by using recombinant gene technology. The fusion gene was named SLPMultiVPl after lactic acid bacteria source of signal peptide gene-Usp45 was introduced on the 5’side and Staphylococcus protein source of LPXTG gene with cell membrane location abilities was introduced on the 3’side. In order to detect its expression level, we constructed the Escherichia coli expression Vector:pGEX-SLPMultiVPl at the first stage, and the expression of recombinant fusion protein vGST-SLPMultiVPl in Escherichia coli was also identified. And then, the SLPMultiVPl gene was cloned into the shuttle expression vector pMG36e and the Lactobacillus expression vector pMG-SLPMultiVPl was constructed. After identified the target gene sequence and correctness of the reading frame with double enzyme digestion, PCR and gene sequencing, the recombinant vector was transformed into Lactobacillus fermentum. Positive clones were non-induced after it was screened out by using PCR and expression products were identified by SDS-PAGE, Western blotting and IFAT. The results are as follows:1. SLPMultiVPl was successfully expressed in E. coli expression system. After identified by SDS-PAGE and Western blotting, GST-SLPMultiVPl was found in the bacterial Lysis and its molecular weight was 61KDa. Molecular weight of the GST tag protein was 26KDa, which was consistent with the theoretical value.2. SLPMultiVPl was successfully expressed in Lactobacillus expression system. After identified by SDS-PAGE and Western blotting, it was not only found 35kDa, a specific band with the theoretical result, but also found a suspicious band about 70kDa which was more than twice as much as target gene from the corresponding location.3.The recombinant Lactobacillus fermentum (including pMG-SLPMultiVP1) and controlled Lactobacillus fermentum (including pMG36e) were analyzed by IFAT using mixed bovine source serum A, O and Asia I of FMDV vaccine and high immunity mouse source of FMDV serum respectively. The results showed that the recombinant Lactobacillus were inspired by the strong green fluorescence, and the control lactobacillus did not showed fluorescence. It was also showed that the recombinant Lactobacillus fermentum had different adhesional capability while the control group had no such ability, which may associated with the adhesion property of the S-layer protein.In this study, multi-type FMDV VP1 gene epitopes was analyzed and live bacterial vaccine vector was constructed by the biological software and molecular biology techniques. The research were laid the basis of previous work for further study of the security and effective FMD vaccine.