| 摘要: |
| 【目的】 构建猪圆环病毒2型(PCV2)ORF2原核表达质粒载体，高效重组表达PCV2壳蛋白Cap，为进一步建立PCV2 ELISA的检测方法及Cap蛋白单克隆抗体的制备研究奠定基础。【方法】 根据猪圆环病毒2型ORF2基因序列设计带有BamHⅠ、Hind Ⅲ酶切位点的引物，进行ORF2的PCR体外扩增。用扩增的ORF2基因构建pMD18-T-ORF2质粒，经测序检测正确后，与pET-32a（+）连接获得原核表达重组质粒pET-32a-ORF2。重组质粒pET-32a-ORF2转化BL21-ΔE3后用IPTG诱导表达，对获得的纯化表达产物进行了SDS-PAGE电泳、Western blot检测。【结果】 PCR扩增得到了预期580 bp的目的基因，连接载体后经测序完全正确；pET-32a-ORF2在BL21-ΔE3得到高效表达，获得以包涵体形式存在的体外重组原核表达PCV2 Cap蛋白；Western blot检测结果表明，所得到的蛋白能够识别PCV2多克隆抗体。【结论】 重组质粒pET-32a-ORF2在BL21-ΔE3高效表达重组PCV2 Cap蛋白，且具有免疫学生物活性。 |
| 关键词: 猪圆环病毒2型 ORF2 原核表达 |
| DOI： |
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| 基金项目:陕西省科技攻关项目（2008K02-05-2） |
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| Prokaryotic expression of ORF2 gene of porcine circovirus type 2 in E.coli system |
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| Abstract: |
| 【Objective】 In order to study monoclonal antibody of porcine circovirus type 2(PCV2) Cap protein and establish the ELISA detection method for PCV2,a procaryotic expression vector of PCV2-ORF2 was reconstructed,which can express Cap protein highly efficiently.【Method】 To amplify the PCV2-ORF2 in vitro,a pair of primers with BamHⅠand Hind Ⅲ endonuclease sites were designed by primer premier 5.The amplified ORF2 was cloned into pET-32a(+) when plasmid pMD18-T-ORF2 was reconstructed after the amplified ORF2 was contirmed right by sequencing.The reconstructed pET-32a-ORF2 was transformed into BL21-ΔE3 and the fusion proteins were expressed when it was induced by 1 mmol/L IPTG.The expression products of reconstructed pET-32a-ORF2-SDS-PAGE undernent electrophoresis and Western blot detection.【Result】 A 580 bp fragment as expected was acquired by PCR amplification and confirned right by sequeding.The expression products of reconstructed pET-32a-ORF2 is a fusiion protein and can bind with polyclonal antibody of ORF2 by SDS-PAGE electrophoresis and Western blot detection.【Conclusion】 The high expression proteins of the reconstructed pET-32a-ORF2 in BL21-ΔE3 have a good immunoreactiviby. |
| Key words: porcine circovirus type 2 ORF2 procaryotic expression |
