【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、HindⅢ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。更多还原

【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 BamHⅠ and 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.更多还原