| 猪繁殖与呼吸综合征病毒GP5蛋白单克隆抗体的制备及鉴定 |
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| 引用本文： | 李志杰,提金凤,李舫,沈美艳,袁东方,苏成文.猪繁殖与呼吸综合征病毒GP5蛋白单克隆抗体的制备及鉴定[J].动物医学进展,2016(11):36-42. |
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| 作者姓名： | 李志杰 提金凤 李舫 沈美艳 袁东方 苏成文 |
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| 作者单位： | 山东畜牧兽医职业学院,山东潍坊,261061 |
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| 基金项目： | 潍坊市科技发展计划项目(20122050)；山东省农业重大应用技术创新课题(规模化畜禽场清洁生产关键技术研究与示范)；国家星火计划项目(2013GA740071) |
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| 摘 要： | 为制备猪繁殖与呼吸综合征病毒(PRRSV)GP5蛋白单克隆抗体,首先构建猪PRRSV GP5基因的真核重组表达载体pCDNA-GP5,将其作为免疫原,对Balb/c小鼠进行免疫。取免疫4次后的小鼠脾细胞与骨髓瘤细胞SP2/0进行融合。构建PRRSV GP5基因的原核重组表达载体pGEX-6P-GP5,将其转化至大肠埃希菌Rosetta(DE3)并诱导表达。以表达的原核蛋白GP5作为单克隆抗体的筛选抗原,通过间接ELISA进行杂交瘤细胞株的检测和筛选。本研究成功构建了GP5基因的真核重组表达载体pCDNA-GP5,表达并纯化了GP5蛋白。经2~3次亚克隆后获得3株针对GP5蛋白的杂交瘤细胞株,IFA和Western blot对3株杂交瘤细胞株分泌的单克隆抗体进行了鉴定。GP5蛋白单克隆抗体的成功研制,为进一步建立特异性PRRSV检测方法奠定了基础。
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| 关 键 词： | 猪繁殖与呼吸综合征病毒 GP5 蛋白 真核表达 单克隆抗体 |
Preparation and Identification of Monoclonal Antibodies Against GP5 Protein of Porcine Reproductive and Respiratory Syndrome Virus |
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| Abstract: | The eukaryotic recombinant expression plasmid of GP5 gene of porcine reproductive and respira-tory syndrome virus (PRRSV)GP5 gene was constructed and the recombinant plasmid was named pcDNA-GP5.The recombinant plasmid was used to inoculate Balb/c mice as the immunogen.After 4 times of im-munization,spleen cells from mice were fused with SP2/0 cells.The prokaryotic recombinant expression plasmid of GP5 gene of PRRSV was constructed and the recombinant plasmid was named pGEX-6P-GP5. The plasmid pGEX-6P-GP5 was transformed to E.coli Rosetta(DE3)and induced to express protein.The expressed protein was used to screen the monoclonal antibodies against GP5 protein by indirect ELISA. Three hybridoma cell lines were abtained after 2-3 subclones,and monoclonal antibodies against GP5 pro-tein were identified by IFA and Western blot. |
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| Keywords: | Porcine reproductive and respiratory syndrome virus eukaryotic expression monoclonal anti-body |
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