| Foot and mouth disease (FMD) is an important disease of cloven-hoofed animals. It is caused by a virus of the family Picornaviridae, genus Aphthovirus, of which there are seven serotypes (O, A, C, SAT1, SAT2, SAT3, and Asia1).My project includes two parts: one is producing recombinant vaccine against foot and mouth disease virus; the other is the construction and expression of the coagulation factor X.1 producing recombinant vaccine against foot and mouth disease virus,1.1 Research of the VP1-SFTepi protein vaccine1.1.1The construction of the expression vector of the protein vaccine According to the sequence of the gene of the VP1 protein and the T cell epitope of the swine fever virus, we designed series of PCR primers and synthesized the gene fragment of the VP1-SFTepi (a gene consisting of three two epitopes VP1 and one epitope from swine fever virus) by PCR. The gene was cloned into pET28a plasmid to create a pET28a-VP1-SFTepi recombinant plasmid.1.1.2 The expression and purification of the VP1-SFTepi protein in the bacteria. pET28a-VP1-SFTepi plasmid was transformed into the BL21(DE3)bacteria. The recombinant protein was expressed in host bacterial cell under the induction of IPTG .The target protein was expressed efficiently in the BL21 (DE3) bacteria seen from SDS-PAGE. The protein was purified by Ni2+-affinity chromatography. There are three forms of the purified protein which includes the monomer, dimer and polymer in SDS-PAGE.1.1.3 The function of the VP1-SFTepi recombinant protein against the virusAfter the VP1-SFTepi recombinant protein immune the pig, we acquire the immune serum. We The result display that the VP1-SFTepi recombinant proteins do not have the function of against the type O FMDV. That is because do not stimulate the body to produce the antibody, or the B cell epitope of the VP1 is covered. In order to play the function of the B cell epitole, we construct the poly-VP1epi recombinant protein.1.2 The research of the poly-VP1epi recombinant protein 1.2.1 The construction of the expression vector of the poly-VP1epi protein vaccine According to the amino acid sequence of the VP1 protein, ,we design 10 PCR primers and synthesized the gene fragement of the poly-VP1epi(a gene consisting of one kind of B cell epitope and two kinds of T cell epitope of the VP1 protein)by PCR and clone technology. The gene was cloned into pET28a plasmid to create a pET28a- poly-VP1epi recombinant plasmid.1.2.2 The expression and purification of the poly-VP1epi protein in the E st.After the recombinant plasmid transform the BL21(DE3), IPTG induce the recombinant protein to expressed. The goal protein have expressed in the BL21 (DE3) by SDS-PAGE display. The protein was purified by Ni2+-affinity chromatography. 1.2.3 The function of the poly-VP1epi recombinant protein against the virus After the poly-VP1epi recombinant immune the The result display that the poly-VP1epi recombinant protein have part function of against the type O FMDV .It is display that many epitope can make the B cell epitope have chance to expose on the surface of the protein. We must construct the more efficiency protein vaccine on the base of the optioned protein structure.2 The construction and expression of the recombinant coagulation factor X fusion protein The goal of the expression of recombinant coagulation factor X is acquire the tool enzyme in the lab. We synthesized gene fragment of the active site of the coagulation factor X by RT-PCR from the liver of the mice. We construct the expression vector pET28-HSP65-FXa by connect gene fragment with the HSP65gene fragment. The HSP65-FXa fusing protein is expressed in the E .In order to keep the active of the enzyme, we hope the expression form of protein is involved through fusing protein. But the expression form of the HSP65-FXa fusing protein is including body in the BL21 (DE3), it make the protein purification very difficult in the later. The work is not continue.Above all, we construct two kinds of recombine protein. The po...
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