牙鲆淋巴囊肿病毒主要衣壳蛋白基因片段真核表达载体的构建、表达与免疫效果评价

Construction of DNA vaccine against lymphocystis disease virus （LCDV） in flounders and evaluation of its immune efficacy

用淋巴囊肿病毒LCDV-cn感染牙鲆鳃细胞系FG-9307,提取细胞总RNA,用RTPCR法获得了LCDV-cn主要衣壳蛋白（MCP）0.6kb基因片段.将该LCDV-cn-MCP0.6kb片断克隆入真核表达载体pEGFP-N2,得到重组质粒pEGFP-N2-LCDV-cn-MCP 0.6.采用脂质体法将重组质粒转染入牙鲆鳃细胞系FEC,并进行瞬时表达.通过荧光显微镜观察和特异性RT-PCR检测,证实重组质粒pEGFP-N2-LCDV-cn-MCP 0.6kb已成功转染到FEC细胞,并得到了初步表达.将重组质粒肌注入牙鲆体内,检测牙鲆外周血、肠、脾脏、前肾和淋巴细胞的增殖反应、呼吸爆发活性及抗体产生水平.结果表明,构建的核酸疫苗pEGFP-N2-LCDV-cn-MCP 0.6可诱导牙鲆特异性体液免疫和细胞免疫,具有明显的免疫保护作用.

Total RNA was extracted from a gill-derived cell strain FG-9307 of the Japanese flounder, and a fragment of major capsid protein encoding gene （approximately 0.6 kb in length） was amplified using RT-PCR and cloned into an eukaryotic expression vector pEGFP-N2, yielding a recombinant plasmid pEGFP-N2-LCDV0.6 kb. This plasmid successfully achieved immediate expression after being transferred by liposome into the eukaryotic cell strain FEC. pEGFP-N2- LCDV0.6 kb was injected into Paralichthys olivaceus intramuscularly and T- lymphopoiesis, respiratory burst in peripheral blood, hind-intestines, spleen, head-kidney and antibodies against LCDV were evaluated. The results indicated that the plasmid of pEGFP-N2-LCDV0.6 kb could induce a unique humoral and cell-mediated immune response and possess the function of immune protection. Therefore, further studies are necessary for the development and application of a DNA vaccine.