Optimization Of The Capsid VP1 Gene Of Enterovirus 71 (EV71) And Research Of Its' Immunogenicity

PartⅠ: Optimization of the VP1`gene of enterovirus 71Object: To get a codon-optimized VP1 gene according to the preferred codons of mammalian cells to promote its'expression and to synthesis the codon-optimized VP1 gene.Methods: The VP1 gene of EV71 BJ4211 strains (Genbank access number EU024958.1) was selected and analyzed by gene software MacVector 7.2. The preference of codon usage of the widetype EV71 VP1 gene was compared with that of mammalian cells. Then the VP1 gene was adjusted and changed with the preferred codon usage of mammalian cells to allow a higher expression of the resulting VP1 protein in mammalian expression system. Sequence optimization was also performed to make the mRNA more stable and to make the gene more favorable for transcriptional and translational processes. Despite such DNA level sequence changes, the final codon optimized VP1 gene sequence still produce the same VP1 amino acid sequences as in the original VP1 of EV71. The codon optimized VP1 gene was chemically synthesized containing two enzyme sites (PstⅠand BamHⅠ). Then the codon optimized VP1 gene was inserted into vector pGA4, that's, recombinant plasmid pGA4/VP1-opt. pGA4/VP1-opt was tansformed into E.coli HB101 and scratch three times on LB solid medium to get bacteria strain.Result and conclusion: By enzyme digestion and sequencing, we got the codon optimized VP1 gene. Compared with widetype VP1 gene, the codon optimized VP1 has more mammalian cells preferred codons and would be expressed more easily in the mammalian cells. Object: To promote the expression, secretion and immunogenicity of VP1 protein, DNA vaccine was selected as a tool to design several typies of VP1 DNA vaccine based on the optimized VP1 gene. At the same time, the immunogenicity of these VP1 DNA vaccines was compared to get a potential camdidate vaccine of EV71.Methods: The pGA4/VP1-opt plasmid was digested by PstⅠand BamHⅠand the optimized VP1 gene were got by gel purification. Then the optimized VP1 gene was cloned into eukaryotic expression vector pJW4303. The recombinant DNA vaccine was named pJW4303/VP1 (VP1-wt). To promote secretion of expressed VP1 protein, VP1 gene contained two restriction sites, BamHⅠand NheⅠ, was cloned by PCR and inserted into the downstream of tPA leader sequence of pJW4303, which can guide the downstream protein to extracellular. The newly constructed DNA vaccine was pJW4303/tPA-VP1 (tPA-VP1). It was reported that the VP1 gene can self-associate to form dimer and to constitute special icosahedron. This maybe a good idea to design a dimer VP1 DNA vaccine, that's, two VP1 gene named insert1 (contained NheⅠand KpnⅠ) and insert2 (contained KpnⅠand BamHⅠ) were amplified and cloned into the downstream of tPA leader sequence of pJW4303 at the same time. That's pJW4303/tPA-VP1-dimer (VP1-dimer). As we all known, IgG Fc piece (Fcγ) has great advantages in immune response. So the human Fcγand mouse Fcγwere amplified (both contained KpnⅠand BamHⅠ). The two Fcγgenes were connected with insret1 in the vector pJW4303 respectively to construct pJW4303/tPA-VP1-Fcγ(VP1-Fcγ). Five types of VP1 DNA vaccine were transformed into E.coli HB101 and positive clones were selected and identified with enzyme digestion and sequencing to get bacteria strains.Five recombinant VP1 DNA vaccines were mega extracted and transfected into 293T cells. The supernatant and cell lysis were collected for the detection of expressed VP1 protein by enzyme-linked immunosorbent Assay (ELISA), indirect fluorescent assay (IFA) and Western blot. Extracted plasmids were also used to immunize New Zealand white rabbits by intramuscular injection followed by electroporation. Rabbits were immunized at weeks 0, 2, 4 and 8. Sera were collected prior to the first immunization and 2 weeks after each immunization. VP1-specific antibody responses were evaluated by ELISA, Western blot and IFA.Results and conclusion: Five types of VP1 DNA vaccine were successfully constructed and all can be expressed in 293T cells. It was really that tPA leader sequence can significantly increase the expression and secretion level of VP1 protein. Although the dimer form and VP1-huFc DNA vaccines have really increased expression and secretion level, the immunogenicity of these two DNA vaccines were not ideal for un-known reason and the antibody responses were lower than that of tPA-VP1.