猪圆环病毒2型Cap蛋白部分基因序列与大肠杆菌LTB成熟肽基因的融合表达及免疫原性研究

Fusion expression of PCV-2 Cap partial gene and E.coli heat-labile toxin B subunit gene and immunonicity of recombinant protein

为了获得猪圆环病毒2型(Porcine circovirus type 2,PCV-2)衣壳蛋白(Cap)基因3’端396 bp的片段与大肠杆菌不耐热肠毒素B亚单位(LTB)成熟肽编码区融合基因的高效表达,并检验重组蛋白的免疫保护效果。以含PCV-2全基因组的pMDT PCV-2质粒为模板,用PCR的方法扩增PCV-2 Cap基因,并将其克隆到pET28a(+)中,构建得到pET-Cap质粒,利用EcoRⅠ和HindⅢ双酶切切下Cap基因3’末端396 bp的片段,插入到pET-LTB原核表达质粒LTB基因的3’末端,从而构建pET-LTB△Cap原核表达质粒,将其转化大肠杆菌BL21(DE3)pLysS后,用IPTG诱导重组菌,用SDS-PAGE电泳和Western blot检测诱导物。表达产物经初步纯化后用昆明系小白鼠做免疫保护实验。结果表明:pET-LTB△Cap重组融合表达质粒在大肠杆菌中实现了高效表达,融合蛋白分子量约为29Ku,表达量约占菌体蛋白的36.67%,融合蛋白能被PCV-2阳性血清识别。攻毒后,所有小鼠临床表现正常。用PCR检测有1/8的免疫小鼠感染了PCV-2,而对照组8只小鼠都感染了PCV-21。PCV-2 Cap基因3’端396 bp的片段与LTB基因融合能实现高效表达,表达产物免疫的小白鼠可抵抗PCV-2的攻击此研究为PCV-2基因工程疫苗的研究奠定了基础。

To obtain high level expression of fusion gene of partial PCV-2 Cap subunit （LTB）, the Cap gene of PCV-2 was amplified by PCR from pMDTPCV-2 terminus 396 bp fragment （△cap） was released by double digestion with EcoR Ⅰ and gene and Escherichia coli heat-labile toxin B and cloned into pET28a （＋） vector. The 3＇ Hind Ⅲ and cloned into pET-LTB plasmids.The recombinant plasmid pET-LTB△cap was transformed into BL21 （DE3） LysS and high-level expression of the fusion protein （up to 36.7 % of total protein） was obtained under IPTG induction. Analysis by SDS-PAGE and Western blot showed that the fusion protein had a molecular weight around 29 Ku and could be specifically recognized by PCV-2 antibodies. Mice immunized with the recombinant LTB△cap protein were protected against PCV-2 challenge, indicating that the fusion protein had good immunogenicity.