大鲵虹彩病毒主衣壳蛋白MCP基因DNA疫苗的构建及其免疫效果

Construction and immune efficacy of an MCP-containing DNA vaccine for Chinese giant salamander iridovirus

根据大鲵虹彩病毒(Chinese giant salamander iridovirus,GSIV)主衣壳蛋白(major capsid protein,MCP)基因序列设计特异性引物,经PCR扩增得到MCP基因编码框1392 bp全长序列,将其定向克隆到真核表达载体pc DNA3.1(+)中,构建重组质粒并命名为pc DNA-MCP。将pc DNA-MCP质粒转染大鲵肌肉细胞系(GS-M),间接荧光免疫染色结果显示,MCP蛋白可在GS-M细胞中表达,且转染后72 h的表达量显著高于转染后48 h;收集转染后72 h的GS-M细胞,经Western blot检测,可检测到MCP蛋白的特异性表达。将真核表达质粒pc DNA-MCP以20μg/尾的剂量经背部肌肉注射免疫健康大鲵(Andrias davidianus),分别在免疫后第1、3、5、7、14、21、28、35天随机从实验组与对照组中采样,尾静脉采血进行外周血血细胞计数、白细胞分类计数及测定血清中和抗体效价。结果表明,免疫大鲵体内红细胞、中性粒细胞和单核细胞数量明显增加,红细胞数在第5天极显著高于对照组(P<0.01);中性粒细胞(neutrophil)分类百分比从第3天开始升高,第5天达到峰值(26.33±1.04)%,极显著高于对照组(P<0.01);单核细胞(monocyte)的变化趋势和中性粒细胞相似,第7天达到峰值(15.83±0.76)%,极显著高于对照组(P<0.01)。随后淋巴细胞大量增殖,第28天淋巴细胞分类百分比达到峰值(68.33±1.53)%,极显著高于对照组(P<0.01)。血清中和试验结果表明,免疫大鲵体内产生了抗MCP蛋白的抗体,免疫后第28天抗体效价最高[1︰(370.01±31.55)]。真核质粒pc DNA-MCP在免疫大鲵的肌肉、肝、脾和肾的组织表达检测结果显示,免疫接种后第1、3、5、7、14、21、28天大鲵的肌肉、肝、脾和肾组织中均存在真核质粒的分布;RT-PCR结果显示,免疫后第7天和28天,在大鲵的上述组织中均有目的基因的表达。攻毒感染试验结果显示,免疫组大鲵相对免疫保护率可达73.3%。本研究为真核表达质粒pc DNA-MCP作为潜在候选疫苗应用于大鲵虹彩病毒病的预防和控制奠定了前期基础。

Based on major capsid protein （MCP） gene sequences of Chinese giant salamander iridovirus （GSIV） in GenBank, specific primers were designed, and the full-length MCP sequence （1392 bp） was amplified by PCR. Then, MCP was cloned into the eukaryotic expression vector pcDNA3.1 （＋） to construct the recombinant expression vector pcDNA-MCP. Giant salamander （Andrias davidianus） muscle （GS-M） cells were transfected with the recombinant ex-pression vector, pcDNA-MCP. At 48 h and 72 h post-transfection, MCP protein expressions in GS-M cells were de-tected by indirect immunofluorescence assay. The results showed that the protein expression level at 72 h post-transfection was significantly higher than that at 48 h post-transfection;western blot assay also confirmed the spe-cific expression of MCP in GS-M cells at 72 h post-transfection. The eukaryotic plasmid pcDNA-MCP was used as a DNA vaccine to immunize Chinese giant salamanders by injection in dorsal muscle at a dose of 20 μg/ind;then, pe-ripheral blood from Chinese giant salamanders in both tested and control groups was collected on day 1, day 3, day 7, day 14, day 21, day 28, and day 35 post-immunization for hemocyte count, classification, and serum-neutralizing anti-body titration. The red and white blood cell counts showed significant increase in numbers of erythrocytes and leuko-cytes in the peripheral blood of immunized Chinese giant salamanders on day 5 and day 7 post-immunization （P〈0.01）. Additionally, the differential leukocyte counts of neutrophils and monocytes were （26.33±1.04）%and （15.83±0.76）%, respectively, at day 5 and day 7 post-immunization, and both significantly changed compared with the control group （P〈0.01）. The percentage of lymphocytes was （68.33±1.53）%at day 28 （P〈0.01）. The serum neutralization assay dem-onstrated that the antibody titer peaked on day 28 post-immunization [1︰（370.01±31.55）]. PCR results revealed that pcDNA-MCP was distributed in the muscle, liver, spleen, and kidney from day 1 to day 28 post-vaccination. RT-PCR results revealed that MCP was expressed in all of the above tissues at day 7 and day 28 post-vaccination. A challenge test was conducted at day 28 post-immunization and produced a relative survival of 73.3%. This study provides a fun-damental basis for the application of the pcDNA-MCP plasmid as a potential DNA vaccine to prevent and control GSIV infection in Chinese giant salamanders in the future.