| The three major structural proteins, E, M and N, of porcine reproductive and respiratory syndrome virus (PRRSV) play an important role in immune response. The glycoprotein D (gD) is one of the structural proteins of Pseudorabies virus (PRV). Both the E and the gD stimulate protective immune response after inoculation. To develop a new type of vaccine against PRRSV and PRV infection, the following experiments have been carried out.Firstly, a PRRSV strain, LX-1 strain, was isolated from internal organs of piglets and was characterized by IFA, VN and RT-PCR. The CPE it induces can be inhibited by the positive serum of American genotype. The physico-chemical characterization showed that the isolates was sensitive to chloroform, ether, formalin and 56℃. The spheriform virion with envelope was observed by negative stain. Four pairs of primers were designed, synthesized and RT-PCR was employed to amplify the full length cDNA of E, M and N gene (ORF5,6,7) of LX-1 isolate. Sequencing showed that the homology between LY-1 and LX-1 strains was 99.3% in ORF5-7. The homology of ORF5-7 between LX-1 and other reference strains (e.g. ATCC VR-2332, RespPRRS MLV, and BJ4) were higher than 99.5%. The conformability between LX-1 and LV were 66.6%. Compared to the N gene of LV, the homology were found to be 67.4%, and there was no difference between LX-1 strain and other American-derived strains. The results showed that the LX-1 strain was closely related to North American genotype.Secondly, a PRV strain, LY strain, was isolated from pigs in Liaoyang. The virus isolate was purified by plaque technique on CEF monolayer and the liter was 10-7.29 TCID50/ml. Virus neutralization(VN) test showed that the isolate could be neutralized by PRV positive serum. Envelope and peplomers were observed on the virion surface under electron microscope. At the same time, its efficacy and safety were evaluated. It was pathogenic to 3-day-old mice and rabbits, but was safe to pigs, and could provide a high level of protective immunity to the virulent field strain of PRV (LA strain). The above results indicated that the isolate might be an attenuated PRV with good protection against virulent strain of PRV. With the specific primers, a fragment about 1240bp was obtained by PCR and the nucleic acid sequence was analyzed. Then amino acid sequence comparison of gD gene was carried out. The results indicated that the gene gD of LY strains consisted of 1203 nucleotides. The homology between LY, LA strains and the other PRV strains were 96.3%-99.0%.Thirdly, the E and N genes from PRRSV and the gD gene from PRV were subcloned into the powerful expression vector pIRESneo and pEGFP-Cl to construct the eukaryotic expression plasmids, pIE, pIN, pGE, pGN, plgD and pFgD. In the mean time, a bi-combined nucleic acid vaccine plasmid, pID-E, was constructed by replacing the neo gene of pIRESneo with EcoRI/Xbal fragment reclaimed the gD from plasmid pUgD and inserting the E gene from PRRSV into the MCS of plRESneo. All the recombinant plasmids were prepared in a large scale by a modified alkali lysis method. Whereafter the recombinant plasmids were transferred into BHK-21 cells by lipofectamine respectively, and then the cell lysates were assayed by indirect ELISA for antigen presence after transfection. The results showed that all the target proteins were expressed in BHK-21 cells, and specific reactivity to the PRRSV or PRV antiserum was confirmed. Then the capability of these recombinant plasmids expressing the gene products of the individual viral genes (E, N and gD) in induction of an immune response in mice was investigated. Span-glycerol was chosen as adjuvant and mixed with the plasmids to form nucleic acid vaccine. Different groups of BALB/c mice (twenty mice per assay) were immunized with a dosage of 50ug/100u.l of plasmids pIE, pGE, pIN, pGN, pIgD, pIE+pIN, pIE+pIgD, pIN+pIgD and pID-E for three times at 21-day intervals, respectively. Indirect ELISA, lymphocyte conversion and neutralization tests were applied to determine the imm...
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