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表达猪繁殖与呼吸综合征病毒N蛋白的重组腺病毒的构建及其免疫原性分析
引用本文:刘庆庆,吴鹏,李培东,张江伟,陈创夫,肖陈诚.表达猪繁殖与呼吸综合征病毒N蛋白的重组腺病毒的构建及其免疫原性分析[J].中国畜牧兽医,2022,49(3):1024-1031.
作者姓名:刘庆庆  吴鹏  李培东  张江伟  陈创夫  肖陈诚
作者单位:1. 石河子大学动物科技学院, 石河子 832000;2. 人兽共患传染性疾病防治协同创新中心, 石河子 832000;3. 石河子大学生命科学学院, 石河子 832000
基金项目:西部地区高发人兽共患传染性疾病协同创新专项(2013-179)
摘    要:【目的】 利用腺病毒AdMax系统表达载体表达猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)核衣壳蛋白(nucleocapsid protein),并研究其免疫原性。【方法】 参考GenBank中已公布的PRRSV N基因序列(登录号:KT945017.1),人工合成PRRSV N基因,并将其连接至腺病毒穿梭载体pDC316-mCMV-EGFP,转化大肠杆菌Top10感受态细胞,构建重组穿梭质粒pDC316-N。将重组穿梭质粒pDC316-N与AdMax腺病毒系统的骨架质粒PBHGLOX (delta) E1,3Cre共同转染293A细胞,获得重组腺病毒rAd-N,对获得的重组腺病毒液进行PCR和测序鉴定,用鉴定正确的rAd-N病毒液感染293A细胞,对该重组腺病毒进行扩大培养,检测病毒的TCID50,并用RT-PCR和Western blotting检测重组腺病毒的表达和反应原性。用重组腺病毒免疫小鼠,收集血清用PRRSV抗体检测试剂盒检测其抗体水平,初步评价其对小鼠的免疫效果。【结果】 PCR扩增出1条大小为400 bp的PRRSV N基因条带,测序结果正确,表明重组腺病毒构建成功,浓缩后测得其半数组织培养感染剂量(TCID50)为10-10.239。RT-PCR和Western blotting检测结果证实目的基因在基因和蛋白水平上均可得到正确表达,蛋白分子质量约为14 ku。小鼠特异性抗体检测表明,重组腺病毒rAd-N免疫小鼠后可使小鼠快速产生PRRSV特异性抗体,与对照组差异显著(P<0.05),其中重组腺病毒与Gel佐剂配合使用时效果最好,最高可达7.84 U/L。【结论】 本研究成功构建表达PRRSV N蛋白的重组腺病毒,其具有良好的免疫原性,为建立针对PRRSV抗体的间接ELISA检测方法和进一步研发PRRSV抗体检测试剂盒奠定基础。

关 键 词:猪繁殖与呼吸综合征病毒(PRRSV)  核衣壳蛋白  重组腺病毒  免疫原性  
收稿时间:2021-08-05

Construction of Recombinant Adenovirus Expressing Porcine Reproductive and Respiratory Syndrome Virus N Protein and Its Immunogenicity Analysis
LIU Qingqing,WU Peng,LI Peidong,ZHANG Jiangwei,CHEN Chuangfu,XIAO Chencheng.Construction of Recombinant Adenovirus Expressing Porcine Reproductive and Respiratory Syndrome Virus N Protein and Its Immunogenicity Analysis[J].China Animal Husbandry & Veterinary Medicine,2022,49(3):1024-1031.
Authors:LIU Qingqing  WU Peng  LI Peidong  ZHANG Jiangwei  CHEN Chuangfu  XIAO Chencheng
Institution:1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;2. Collaborative Innovation Center for Prevention and Control of Zoonotic Infectious Diseases, Shihezi 832000, China;3. College of Life Sciences, Shihezi University, Shihezi 832000, China
Abstract:【Objective】 The aim of this study was to express the nucleocapsid protein of Porcine reproductive and respiratory syndrome virus (PRRSV) by Adenovirus AdMax system expression vector, and to study its immunogenicity.【Method】 Referring to the PRRSV N gene sequence published in GenBank (accession No.:KT945017.1), the PRRSV N gene was synthesized and connected to the Adenovirus shuttle vector pDC316-mCMV-EGFP, transformed into E.coli Top10 competent cells, and the recombinant shuttle plasmid pDC316-N was constructed.The recombinant shuttle plasmid pDC316-N and the framework plasmid PBHGLOX(delta)E1, 3Cre of AdMax Adenovirus system were co-transfected into 293A cells to obtain the recombinant Adenovirus rAd-N.The obtained recombinant Adenovirus solution was identified by PCR and sequencing.The 293A cells were infected with the identified correct rAd-N virus solution.The recombinant Adenovirus was expanded for culture and the TCID50of the virus was detected, RT-PCR and Western blotting were used to detect the expression and reactogenicity of recombinant Adenovirus.Mice were immunized with recombinant Adenovirus, the serum was collected, the antibody level was detected with PRRSV antibody detection kit, and the immune effect on mice was preliminarily evaluated.【Result】 A 400 bp PRRSV N gene band was amplified by PCR.The sequencing results were correct, indicating that the recombinant Adenovirus was successfully constructed.After concentration, it was determined that its TCID50 was 10-10.239.The results of RT-PCR and Western blotting confirmed that the target gene was correctly expressed at the gene and protein levels, and the molecular weight of the protein was about 14 ku.The detection of mouse specific antibody showed that mice immunized with recombinant Adenovirus rAd-N could quickly produce specific antibody against PRRSV, which was significantly different from the control group (P<0.05).The effect of recombinant Adenovirus combined with Gel adjuvant was the best, which was up to 7.84 U/L.【Conclusion】 The recombinant Adenovirus expressing PRRSV N protein was successfully constructed, which had good immunogenicity, and laid a foundation for the establishment of indirect ELISA for PRRSV antibody and the further development of PRRSV antibody detection kit.
Keywords:Porcine reproductive and respiratory syndrome virus (PRRSV)  nucleocapsid protein  recombinant Adenovirus  immunogenicity  
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