猪源多肽类抗生素PR-39真核表达及体外活性研究

Eukaryotic Expression of Porcine PR-39 and Its Antimicrobial Activity In Vitro

PR-39通过非穿孔形式抑制DNA和蛋白质的合成,从而对革兰氏阴性菌和阳性菌具有抗菌活性。此外,在伤口修复、炎症、缺血再灌注损伤以及诱导血管生成等方面也发挥着重要的生物学功能,因此开发PR-39具有重大的意义。用人工合成的PR-39基因为模板,通过PCR扩增获得PR-39片段,将其克隆到载体pHIL-S1构建重组质粒pHIL-S1-PR-39,并电转化至毕赤酵母(Pichia pastoris)GS115中,用MD和MM平板筛选重组子,经甲醇诱导其表达后,Tricine-SDS-PAGE分析表达蛋白,通过琼脂扩散试验检测PR-39的抑菌活性。通过MD和MM平板筛选出Mut+的转化子,甲醇诱导表达产物经Tricine-SDS-PAGE得到大约4kD的重组蛋白,并能够抑制大肠杆菌、沙门氏菌和金黄色葡萄球菌的生长。本研究获得了能高效稳定表达具有生物学活性的重组PR-39的基因工程菌,为抗菌肽工业化生产奠定了实验基础。

PR-39 has bactericidal properties that are unique in that it kills by a non-pore-forming mechanism that inhibits DNA and protein synthesis in gram-positive and gram-negative bacteria. Besides, other effects of PR-39 include in-creased syndecan expression, a proteoglycan involved in wound repair, prevent neutrophil recruitment in the develop-ment of inflammation and myocardial ischemia-reperfusion injury, induced of angiogenesis. So it is necessary to develop recombinant PR-39. According to the codon usage preference of pichia pastoris, a PR-39 gene was synthesized and the PCR was performed to obtain PR-39 fragment using the synthetic PR-39 gene as template. Then PR-39 fragment was cloned into plasmid pHIL-S1. The resulting plasmid pHIL-S1-PR-39 was linearized and transformed into pichia pastoris by electroporation, Mut＋ transformants were selected by MD and MM, then induced recombinant expression with methanol, analysed recombinant protein by Tricine-SDS-PAGE, doing agar diffusion to detect the antibacterial activi-ties of PR-39. To select Mut^＋ transformants by MD and MM, and induce the expression of recombinant yeast by methanol, a molecular mass of expressed PR-39 was about 4 kD by Tricine-SDS-PAGE analysis, and the recombinant protein showed certain antimicrobial activity against Escherichia coli, Salmonella, Staphylococcus aureus. These re-sults indicated that a recombinant strain which can effectively and stably produce recombinant porcine PR-39 with high bioactivity was obtained.