| 摘要: |
| 【目的】 克隆猪圆环病毒 2 型陕西分离株（PCV-2 SX株）ORF2 基因，并进行序列分析及原核表达。【方法】 根据 GenBank 公布的 PCV-2 ORF2 基因的核苷酸序列，设计并合成1对特异性引物，应用 PCR 方法扩增 PCV-2 SX株 ORF2 全长基因，将其克隆入 pGEM-T 载体中，进行测序及序列分析。然后，将ORF2 基因亚克隆入原核表达载体 pET-32a 中，在大肠杆菌 BL21中进行 IPTG 诱导表达，对表达产物进行 SDS-PAGE 和 Western-blotting 鉴定。【结果】 扩增到了 702 bp 的 PCV-2 ORF2 全长基因。序列分析结果表明，PCV-2 SX株 ORF2 基因与广西分离株（EF675237，China GX）和巴西分离株（DQ861802, am21）的核苷酸序列同源性均达98.0%，氨基酸序列同源性均达98.3%，与其他毒株的核苷酸序列和氨基酸序列同源性分别在89.9%~97.9%和 88.5%~98.0%。SDS-PAGE 可检测到分子质量约为 48 ku 的融合蛋白，主要以可溶性蛋白形式存在。Western blotting 分析表明，重组蛋白可被 PCV-2 阳性血清所识别。【结论】 成功克隆了 PCV-2 SX株ORF2 基因,并进行了原核表达。 |
| 关键词: 猪圆环病毒2型 陕西分离株 ORF2基因 序列分析 原核表达 |
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| 基金项目:陕西省科技攻关项目（2009K02-01）；教育部新世纪优秀人才支持计划（NCET-07-0701）［基金项目］(西北农林科技大学 动物医学院，陕西 杨凌 712100) |
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| Cloning,sequence analysis and prokaryotic expression of ORF2 gene of PCV-2 Shaanxi strain |
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| Abstract: |
| 【Objective】 PCV-2 SX strain ORF2 gene was cloned, analyzed and expressed in E.coli.【Method】 According to the published complete nucleotide sequence of PCV-2 in GenBank，a pair of primers specific to the full length of ORF2 gene were designed.The ORF2 gene fragment amplified by PCR was cloned into pGEM T vector.The recombinant plasmid was sequenced and ORF2 gene was compared with other Genebank’s PCV-2 strains.ORF2 gene segment was amplified from recombinant plasmid by PCR,then the ORF2 gene was cloned into vector pET-32a for prokaryotic expression in E.coli.【Result】 The ORF2 gene of Shaanxi strain was 702 bp in full-length.It shared higher nucleotide and amino acid sequences homology with different strains of our country and foreign countries.The recombinant fusion proteins were highly expressed in E.coli BL21 in the form of soluble protein.The molecular weight of the expressed fusion protein was approximately 28 ku.Western blot showed that the recombinant protein can be recognized by PCV-2 positive serum.【Conclusion】 PCV-2 Shaanxi strain ORF2 gene was cloned and expressed in E.coli successfully. |
| Key words: PCV-2 Shaanxi strain ORF2 gene sequence analysis prokaryotic expression |
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