Study On Suicidal DNA Vaccine Encoding Glycoprotein C (gC) Of Pseudorabies Virus And Immunoenhancement Effect Of VP22 Protein Transduction On The Vaccine

Pseudorabies, also designated as Aujeszky's disease (AD), is a serious illness of swine and causes significant financial losses in the pig industry. To control AD and to reduce economic losses in the pig industry, active immunization with modified live or inactivated vaccines has been performed widely. Although these vaccines can provide protection against clinical signs of AD in swine to some degrees, they have certain defects, especially limited protective efficacy and the danger of reversion to a more virulent strain. Therefore, it is urgent to develop safer and more effective vaccines to control this disease.More recently, suicidal DNA vaccines, a new generation of vaccine based on the self-replicating RNA vaccines and conventional DNA vaccines, have been developed. The suicidal DNA vaccines not only have the advantages of biosafety and enhanced immunogenicity of self-replicating RNA vaccines, but also combines the advantages of much lower production, storage, and administration costs of conventional DNA vaccines. It has been demonstrated the suicidal DNA vaccine could break immunological tolerance by activating innate antiviral pathways. All these advantages indicate that suicidal DNA vaccines are an attractive vaccine delivery vehicle and an alternative strategy to conventional DNA vaccines and self-replicating RNA vaccine. Pseudorabies, as mentioned above, is one of the most economically important diseases of the global pork-producing community. The standard DNA vaccines against Pseudorabies have been studied widely. However, to date, the suicidal DNA vaccines of Pseudorabies have not been investigated.Protein transduction, a unique biology phenomena, was discovered in recent years. Proteins which have protein transduction property can penetrate the cell membrane and can be transported from the original expressing cell to surrounding nonexpressing cells. These findings not only are a challenge to conventional gene transfer techniques, but also open up the possibility for improvement of gene therapy and vaccination. To date, it has been demonstrated that the VP22 homologues from herpes simplex virus type 1 (HSV-1), bovine herpesvirus 1 (BHV-1), and Marek's disease virus serotype 1 (MDV-1) have the remarkable transport property and have been used as immunoadjuvant to enhance the immunogenicity of conventional DNA vaccines or self-replicating RNA vaccines. Pseudorabies virus (PrV), together with HSV-1, BHV-1, and MDV-1, is the member of alphaherpesvirus. However, whether VP22 homologue from PrV has the same unique protein transduction property is unknown. If the answer is yes, it is unclear whether there exist differences in protein transduction ability among these VP22 homologues. In addition, it is a question whether the VP22 can also be used as immunoadjuvant to enhance the immune response of suicidal DNA vaccines. To better understand these questions will enrich the protein transduction systems, widen the application of protein transduction technique, and develop more efficient delivery tools for gene therapy andvaccination.Based on the above mentioned research background, the present study investigated the immunogenicity and protective efficacy of suicidal DNA vaccine encoding the immunogenic PrV glycoprotein C (gC), evaluated the protein transduction property of PrV VP22, compared the ability of the four VP22 homologues to enhance the immunogenicity of DNA vaccines, and tested the immunoadjuvant effect of the chosen VP22 on suicidal DNA vaccine encoding model antigens or PrV gC. The main projects are:1. Cloning and sequence analysis of gC gene of PrV strain EaA 4.3kb DNA fragment containing the gC gene was isolated from the genomic DNA of PrV strain Ea and was identified by Southern blot. The intact gC gene was further cloned and its nucleotide sequence was determined. When compared the deduced amino acids of gC of PrV strain Ea with other PrV strains, especially the strain NIA-3 or the strain S-81, gC of PrV strain Ea was composed of 487 amino acids (AA), while NIA-3 or S-81 was 479 AA or 4...