CAJ | 학술논문

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pcDNA3.1(+)中,构建重组质粒并命名为pcDNA-MCP。将pcDNA-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pcDNA-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pcDNA-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 pcDNA-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。根據大鯢虹綵病毒(Chinese giant salamander iridovirus, GSIV)主衣殼蛋白(major capsid protein, MCP)基因序列設計特異性引物,經 PCR 擴增得到 MCP 基因編碼框1392 bp 全長序列,將其定嚮剋隆到真覈錶達載體pcDNA3.1(+)中,構建重組質粒併命名為pcDNA-MCP。將pcDNA-MCP質粒轉染大鯢肌肉細胞繫(GS-M),間接熒光免疫染色結果顯示, MCP蛋白可在GS-M細胞中錶達,且轉染後72 h的錶達量顯著高于轉染後48 h；收集轉染後72 h的GS-M細胞,經Western blot檢測,可檢測到MCP蛋白的特異性錶達。將真覈錶達質粒pcDNA-MCP以20μg/尾的劑量經揹部肌肉註射免疫健康大鯢(Andrias davidianus),分彆在免疫後第1、3、5、7、14、21、28、35天隨機從實驗組與對照組中採樣,尾靜脈採血進行外週血血細胞計數、白細胞分類計數及測定血清中和抗體效價。結果錶明,免疫大鯢體內紅細胞、中性粒細胞和單覈細胞數量明顯增加,紅細胞數在第5天極顯著高于對照組(P<0.01)；中性粒細胞(neutrophil)分類百分比從第3天開始升高,第5天達到峰值(26.33±1.04)%,極顯著高于對照組(P<0.01)；單覈細胞(monocyte)的變化趨勢和中性粒細胞相似,第7天達到峰值(15.83±0.76)%,極顯著高于對照組(P<0.01)。隨後淋巴細胞大量增殖,第28天淋巴細胞分類百分比達到峰值(68.33±1.53)%,極顯著高于對照組(P<0.01)。血清中和試驗結果錶明,免疫大鯢體內產生瞭抗 MCP蛋白的抗體,免疫後第28天抗體效價最高[1︰(370.01±31.55)]。真覈質粒pcDNA-MCP在免疫大鯢的肌肉、肝、脾和腎的組織錶達檢測結果顯示,免疫接種後第1、3、5、7、14、21、28天大鯢的肌肉、肝、脾和腎組織中均存在真覈質粒的分佈；RT-PCR結果顯示,免疫後第7天和28天,在大鯢的上述組織中均有目的基因的錶達。攻毒感染試驗結果顯示,免疫組大鯢相對免疫保護率可達73.3%。本研究為真覈錶達質粒 pcDNA-MCP 作為潛在候選疫苗應用于大鯢虹綵病毒病的預防和控製奠定瞭前期基礎。
근거대예홍채병독(Chinese giant salamander iridovirus, GSIV)주의각단백(major capsid protein, MCP)기인서렬설계특이성인물,경 PCR 확증득도 MCP 기인편마광1392 bp 전장서렬,장기정향극륭도진핵표체재체pcDNA3.1(+)중,구건중조질립병명명위pcDNA-MCP。장pcDNA-MCP질립전염대예기육세포계(GS-M),간접형광면역염색결과현시, MCP단백가재GS-M세포중표체,차전염후72 h적표체량현저고우전염후48 h；수집전염후72 h적GS-M세포,경Western blot검측,가검측도MCP단백적특이성표체。장진핵표체질립pcDNA-MCP이20μg/미적제량경배부기육주사면역건강대예(Andrias davidianus),분별재면역후제1、3、5、7、14、21、28、35천수궤종실험조여대조조중채양,미정맥채혈진행외주혈혈세포계수、백세포분류계수급측정혈청중화항체효개。결과표명,면역대예체내홍세포、중성립세포화단핵세포수량명현증가,홍세포수재제5천겁현저고우대조조(P<0.01)；중성립세포(neutrophil)분류백분비종제3천개시승고,제5천체도봉치(26.33±1.04)%,겁현저고우대조조(P<0.01)；단핵세포(monocyte)적변화추세화중성립세포상사,제7천체도봉치(15.83±0.76)%,겁현저고우대조조(P<0.01)。수후림파세포대량증식,제28천림파세포분류백분비체도봉치(68.33±1.53)%,겁현저고우대조조(P<0.01)。혈청중화시험결과표명,면역대예체내산생료항 MCP단백적항체,면역후제28천항체효개최고[1︰(370.01±31.55)]。진핵질립pcDNA-MCP재면역대예적기육、간、비화신적조직표체검측결과현시,면역접충후제1、3、5、7、14、21、28천대예적기육、간、비화신조직중균존재진핵질립적분포；RT-PCR결과현시,면역후제7천화28천,재대예적상술조직중균유목적기인적표체。공독감염시험결과현시,면역조대예상대면역보호솔가체73.3%。본연구위진핵표체질립 pcDNA-MCP 작위잠재후선역묘응용우대예홍채병독병적예방화공제전정료전기기출。
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.