猪β防御素2成熟肽在酵母中的表达

Expressing of porcine β-defensin-2 mature peptide in the yeast

摘要:

摘要：【目的】将猪β防御素2成熟肽基因片段正确整合到酵母基因组染色体上，从而得到稳定的猪β防御素2成熟肽的毕赤酵母表达株。实现猪β防御素2成熟肽的表达。【方法】首先参考酵母偏爱密码子，设计3段引物序列，利用PCR技术扩增得到β防御2成熟肽基因，构建了重组质粒pPIC9k-GST-pBD-2和pPIC9k-pBD-2。将线性化的重组质粒电转化到毕赤酵母KM71细胞中。最后筛选得到酵母阳性克隆，通过不断调节表达条件，实现猪β防御素2成熟肽的表达。【结果】将GST-pBD-2基因序列和pBD-2基因序列分别成功整合到酵母KM71基因组中，重组毕赤酵母工程菌构建成功；重组酵母蛋白GST-pBD-2和PBD-2都成功获得了表达；PBD-2成熟肽表达上清对猪霍乱沙门氏菌弱毒株C500有一定的抑制作用。【结论】获得表达pBD-2成熟肽的酵母菌株，本实验是用真核细胞表达pBD-2成熟肽的一次探索，为后续大量表达pBD-2成熟肽方法的研究打下了基础。

Abstract:

Abstract: [Objective] Porcine β-defensin-2 mature peptide gene fragments were correctly integrated into genome chromosome of the yeast KM71, which was stable Pichia pastoris strains expressing porcine β-defensin-2 mature peptide. Porcine β-defensin-2 mature peptide was expressed successfully. [Method] Considering the biased codon usage of Pichia pastoris, we designed three primers and used PCR technology to get the gene fragment of pBD-2 mature peptide. We obtained the recombinant vectors of pPIC9K-pBD-2 and pPIC9k-GST-pBD-2. The recombinant plasmids linearized were transformed into yeast KM71 by electroporation. By changing the parameters of expression environment, screening yeast clones induced by methanol expressed the Porcine β-defensin-2 mature peptide finally. [Result] GST-Porcine β-defensin-2 and Porcine β-defensin-2 gene fragments were successfully integrated into genome chromosome of the yeast KM71, We finally constructed the recombinant yeast; Protein GST-Porcine β-defensin-2 and Porcine β-defensin-2 were expressed successfully. The peptide of Porcine β-defensin-2 can restrain the Salmonella choleraesuis (C500-). [Conclusion] We got the recombinant yeast which can express porcine β-defensin-2 mature peptide. This study is an exploration to express Porcine β-defensin-2 in eukaryotic cells, It established foundation for the subsequent study of large-scale expression of Porcine β-defensin-2 mature peptide.