| 摘要: |
| [目的]探讨猪圆环病毒2型(PCV-2)中ORF5所编码非结构蛋白(NS)的功能.[方法]根据GenBank已发表PCV-2基因组序列设计特异引物,分别以HZ0201株(AY188355)、美国98-15237株病毒DNA为模板,采用PCR法扩增PCV-2 ORF5基因,并构建了pGEX-4T-1-ORF5原核表达载体和pEGFP-C2-ORF5真核表达载体,对ORF5重组蛋白进行SDS-PAGE电泳、Western blot分析, 用真核表达载体pEGFP-C2-ORF5转染PCV-2易感细胞PK-15.[结果]PCV-2 ORF5基因长162 bp,编码54个氨基酸;其所编码蛋白在E.coli中以包涵体形式存在,分子质量大小约为32 ku.[结论]PCV-2 ORF5编码重组蛋白在真核PK-15细胞中成功表达. |
| 关键词: 猪圆环病毒2型 ORF5基因 原核表达 真核表达 纯化 |
| DOI： |
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| 基金项目:世界银行贷款高校发展项目，国家自然科学基金 |
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| Expression of ORF5-encoding NS protein for type 2 of porcine circovirus |
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| Abstract: |
| 【Objective】 The study was to explore the relationship between structure and function of non structural protein(NS protein),a putative protein encoded by open reading frame 5(ORF5) gene of PCV-2.【Method】 The specific primers were designed and synthesized according to the published sequences of ORF5 gene of PCV-2 by GenBank.The complete DNA fragment of ORF5 gene was obtained by PCR from viral DNA of PCV-2 USA strain(98-15237) and HangZHou strain(HZ0201) respectively,and inserted into prokaryotic expression vector pGEX-4T-1-ORF5 and eukaryotic expression vector pEGFP-C2-ORF5.The ORF5 reconbinant protein was analyzed by SDS-PAGE electrophoresis and Western blot,and eukaryotic expression vector pEGFP-C2-ORF5 was transfected to PK-15 cells.【Result】 The results showed that the complete open reading frame(ORF) of ORF5 gene encoding 54 amino acids was 162 bp in length;PCV-2 ORF5 recombinant protein expressed in E.coli mainly exsisted as inclusion body;The molecular weight of ORF5 encoding product with GST was 32 ku. 【Conclusion】 PCV-2-ORF5 encoding product was expressed in cellular nucleus and cytoplasma of PK-15 cells. |
| Key words: Porcine circovirus type 2(PCV-2) ORF5 gene prokaryotic expression eukaryotic expression purification |
