Purification And Immunogenicity Of A Recombinant Protein Consisting Of B And T Cell Epitopes Derived From Type O Swine Foot And Mouth Disease Virus

Foot-and-mouth disease (FMD) is a highly contagious disease with a blister for the main symptom, which is occurred cloven-hoofed animals such as cattle, sheep and pig and caused by Foot-and-mouth disease virus(FMDV). The disease causes explosive epidemics and heavy losses in the economic, agriculture, and trade worldwide. The FMDV is divided into seven kinds in serotype, the type of O and AsiaⅠare the most popular in mainland China. The major virus antigenic site is the GH loop (amino acid 140-160) which is prominent in the surface of virus of the structural protein VP1. On the top of GH loop is arginine-glycine-aspartic acid (RGD) tripeptide. The RGD motif is highly conserved in field strains of FMDV. It is a ligand of mediating cell surface receptor attachment by binding to the virus, and it is a main site of binding the antibody. Until now, to prevent this disease, people have been woking hard. So various vaccines have been appeared. But the inter-serotype almost has no cross-immunity, bringing a great difficult in developing the vaccine.Currently, the major strategy for controlling FMD is using chemically inactivated FMDV vaccine. Because these vaccines exist some dangerous factors, developing new vaccines becomes an inevitable trend. These vaccines include subunit vaccines, peptide-based vaccines, recombinant viral vector vaccines, DNA vaccines, transgenic plant vaccines and recombinant protein vaccines.The most recombinant protein vaccines are expressed in the form of inclusion bodies in E.coli. The methods of refolding and purifying are important to get the protein wit high stability and high activity. Currently, people have done a lot of work on the refolding and purification of the recombinant protein. The methods of refolding the recombinant protein include refolding by diluting, refolding by ultrafiltrating, refolding by dialying and refolding in the column, which is the most popular method. When we choose a method to purify the recombinant VP1 protein, we based on the characteristics of the protein. At the same time, in the process of purifying the protein, we are required to avoid losing the activity of the protein.The recombinant protein consisting of B and T cell epitopes derived from type O swine foot and mouth disease virus has been constructed and expressed by our laboratory. Our major work are to purify, refold the protein and detect the immune activity.This study was composed of following parts:1 Lysising the inclusion bodies of the recombinant VP1 proteinTo lysis the inclusion bodies effectively, we design three conditions: the ratio of the weight of inclusion bodies and the volume of lysis buffer (W/V) is 1: 5, the time of mixing is 2.5h or 4h and the W/Vis 1:7, the time is 4h. Through analyzing the strip by SDS-PAGE, finally determining the condition is W/V is 1:5, the time is 4h.2 The conditions of purification and refolding of the recombinant VP1 proteinAfter lysising the inclusion bodies, we detect the conditions of purifying and refolding of the recombinant VP1 protein. we choose the methods including refolding by diluting and refolding in the column(refolding in the column of GFC, refolding in the column of IMAC) and we also use the different desalting buffers, these are 20mmol/L Na2HPO4, 0.9%NaCl, 1mol/L urea,pH 7.8, 20mmol/L Tris,50mmol/L KCl,1mol/L urea,pH 7.0 and 10mmol/L Na2HPO4,137 mmol/L NaCl,2.7mmol/L KCl,KH2PO4,1mol/L urea, pH7.8. The concentration of urea is 1mol/L in the final solution of the recombinant protein VP1. By contrast of these methods, we determine the method is refolding in the column of IMAC, and the desalting buffer is 20mmol/L Na2HPO4, 0.9%NaCl, 1mol/L urea,pH 7.8.3 Purification of the recombinant VP1 proteinWe purify the recombinant VP1 protein after determining the method and the desalting buffer and the concentration of the protein is 5171μg/ml, the purity is 95% and we detect the immune activity of the recombinant VP1 protein.4 Detecting the immune activity of the recombinant VP1 proteinIn order to detect the activity of the recombinant VP1 protein, firstly, we immune the swines secondly using the protein and at 28 days, 3 months and 6 months after the second immuned, we let them infect the virus. At the same time, we collect the serum for detecting the antibody using the methods of suckling mice protective assay and cell protective assay. We can see the antibody reaches the peak at 28 days after the second immuned and the protein can protect the swine well.We determine the methods of refolding and purifying the recombinant VP1 protein. Through the methods we get the protein with more purity. To detect the immune activity, we immune the swines using the protein and let them infect the FMDV and detect the ratio of protecting the swine and the level of the antibody.