猪伪狂犬病病毒LY株gD基因的克隆与核酸疫苗研究

Cloning of the glycoprotein D from pseudorabies virus LY strain and studies on its nucleic acid vaccine

将伪狂犬病病毒LY株的免疫糖蛋白基因(gD基因)分别克隆到核酸疫苗载体pIRESneo、pEGFP-C1的多克隆位点上,成功地构建了含有相应目的基因的重组质粒pIgD、pFgD和PRRSVLX-1株的E基因的双基因共表达二联核酸疫苗质粒pID-E。将构建的各核酸疫苗质粒,通过脂质体法分别转染到BHK-21细胞中,经ELISA检测所有目的蛋白均得到表达,表达产物也均具有一定的反应原性。小鼠免疫试验证实这些核酸疫苗质粒具有很好的免疫原性,能诱导小鼠产生特异性抗体,第3次免疫后,抗体阳性率为100%,pIgD抗体效价均不低于1∶160,最高可达1∶640。pIE与pIgD的联合应用时,不影响各自特异性抗体的产生。构建的含有PRRSVE基因和PRVgD基因的双基因共表达质粒pID-E,脂质体转染BHK-21细胞后,经ELISA检测,相应的PRRSV和PRV的目的蛋白均获得了瞬时表达;小鼠免疫试验表明E和gD2种糖蛋白基因能在小鼠体内共表达,且能诱导免疫小鼠产生针对PRRSV和PRV的相应中和抗体。上述研究为进一步探讨PRRSVE蛋白及PRVgD蛋白的免疫生物学特性,进而为开展PRRSV与PRV相关基因在哺乳动物细胞中的联合表达及基因免疫提供理论基础。

To develop a new type of vaccine against PRRSV and PRV infection, the following experiments have been carried out. Firstly, the gD gene of a PRV strain, LY strain, was obtained by PCR with the specific primers, and the nucleic acid sequence was analyzed. The results indicated that the ORF of the gD gene consisted of 1 203 nucleotides. The homology between LY, LA strains and the other PRV strains were 96.3 % - 99.0%. Then, the gD gene of PRV L Y strain was subeloned into the powerful expression vector pIRESneo and pEGFP-C1 to construct the eukaryotic expression plasmids, pIgD and pFgD. Meanwhile, a hi-combined nucleic acid vaccine plasmid, pID-E, was constructed by replacing the neo gene of pIRESneo with EcoR Ⅰ/Xba Ⅰ fragment reclaimed the gD from plasmid pUgD and inserting the E gene from PRRSV into the MCS of pIRESneo. All the recombinant plasmids were prepared in a large scale by a modified alkali lysis method. Thereafter 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 indicated that all the target proteins were expressed in BHK-21 cells,and specific reactivity to the PRV antiserum was confirmed. Thirdly, the capability of these recombinant plasmids expressing the gene products of the individual viral genes （E 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 0.5 g/L of plasmids pIE,pIgD, pIE＋ pIgD and pID-E for three times at 21-day intervals,respectively. Indirect ELISA, lymphocyte conversion and neutralization tests were applied to determine the immunological response. Results indicated that： （a） after the booster immunization,sero-conversion occurred in all groups excepting negative controls; （b） antibody level went up swift- ly 14 days after the third injection and the highest VN-titers were observed; （c） both immunological response can be detected when the plasmid plgD associated with plasmid piE, and the interference of immunological effect between the two plasmids did not occur,so the plasmid pIgD based on PRV can be used in company with piE based on PRRSV. （d） Both the immtmological effect of glycoproteins E and gD expressed by plD-E were detected, and the antibody level was similar with that induced by piE or pIgD alone. The results indicated that both the gD gene of PRV and the E gene of PRRSV can be expressed together in the eukaryotic vector pIRESneo, and the bi-combined nucleic acid vaccine plasmid pID-E was proved to be effective in stimulating specific lmmune response in mice.