猪β防御素2与猪γ干扰素在毕赤酵母中的融合表达及生物学活性比较

Fusion expression and bioactivity comparison of porcine β-defensin-2 and porcine interferon-gamma in Pichia pastoris

摘要:

为了研究猪β防御素2(PBD-2) 与猪γ干扰素 (PoIFNγ)，采用重叠延伸PCR法合成嵌合编码序列PBD-2-PoIFNγ，在此序列的基础上扩增PoIFNγ基因，并分别克隆入毕赤酵母表达载体pPICZaA，构建重组表达质粒pPICZaA-PBD-2-PoIFNγ与pPICZaA-PoIFNγ。经Sac I线性化，电击转化巴斯德毕赤酵母X33，筛选阳性重组子，在含0.5％甲醇的BMMY培养基中诱导72 h。SDS-PAGE及Western blotting结果表明，所获得的重组子能够分别分泌表达PBD-2-PoIFNγ融合蛋白与PoIFNγ。琼脂扩散法和细胞病变抑制法未检测到融合蛋白的抑菌和抗病毒活性，而PoIFNγ具有明显的抗病毒活性。圆二色谱分析显示PoIFNγ与PBD-2-PoIFNγ的螺旋和无规则卷曲含量差别较大，推测是融合蛋白未能正确折叠影响了PBD-2-PoIFNγ的活性。

Abstract:

In order to study PBD-2 and PoIFNγ, the chimeric gene PBD-2-PoIFNγ was synthesized by overlap extension PCR, and amplified PoIFNγ on the basis of this sequence, then cloned into yeast expression vector pPICZaA separately to get the recombinant plasmid pPICZaA-PBD-2-PoINFγ and pPICZaA-PoINFγ. The recombinant plasmid was digested by Sac I and introduced into Pichia pastoris X33 cells by electroporation. Positive clones were screened and cultivated in BMMY medium containing 0.5% methanol for 72 h. SDS-PAGE and Western blotting analysis showed that the screened recombinant could secrete PBD-2-PoINFγ and PoINFγ separately. The activity of fusion protein was not detected by cytopathic effect inhibition assay and agar diffusion assay, but detected obvious antiviral activity of PoINFγ. The helix and random coil contents was showed vary greatly between PoIFNγ and PBD-2-PoINFγ by circular dichroism analysis. It was speculated that the fusion protein was not correctly folded and may affect the activity of PBD-2-PoINFγ.