太平洋牡蛎防御素在毕赤酵母中的重组表达及其抑菌活性

Recombinant expression of Crassostrea gigas defensin in Pichia pastoris and its antibacterial activity

摘要:

防御素是一类富含精氨酸和半胱氨酸的内源性阳离子抗菌肽，是软体动物抵御各种病原微生物侵染的重要免疫因子。太平洋牡蛎防御素 (Crassostrea gigas defensin，CgD) 近羧基端的43个氨基酸残基构成了其成熟肽区域，决定了CgD的生物学活性。首先通过逆转录PCR和设计特异性引物从太平洋牡蛎外套膜中分离并扩增到3?端添加和不添加6×His标签的两种目的基因CgDH+和CgDH–；与pPICZαA连接后构建的重组表达载体(pPICZαA-CgDH+和pPICZαA-CgDH–) 电转至毕赤酵母Pichia pastoris X-33中，使用1.0%甲醇诱导表达目的蛋白CgDH+和CgDH–，最适培养条件为29 ℃、250 r/min、72 h；通过固化金属离子亲和层析 (IMAC) 获得分子量为5.78 kDa的纯化的重组蛋白CgDH+，根据其蛋白质浓度推算表达量为2.32 mg/L。经MALDI-TOF-TOF质谱分析证明纯化产物即为预期的目的蛋白。抑菌试验结果显示分别含重组蛋白CgDH+和重组蛋白CgDH–的培养液上清对金黄色葡萄球菌Staphylococcus aureus和铜绿假单孢菌Pseudomonas aeruginosa都具有抑菌活性，表明重组蛋白中6×His标签的存在与否并不影响其生物学活性。

Abstract:

Defensins are endogenous cationic antimicrobial peptides rich in arginine and cysteine residues. They are important immune factors resisting pathogenic bacteria infection for mollusks. The 43 amino acid residues near the carboxyl terminal for Crassostrea gigas defensin (CgD) form its mature peptide region, responsible for the biological activity of CgD. First, two target genes, CgDH+ (with 6×His-tag at 3? end) and CgDH- (without 6×His-tag at 3? end) were separated and amplified by RT-PCR with specific primers from Crassostrea gigas mantle. These two target genes were ligated to the expression vector pPICZαA to construct recombinant expression vectors, pPICZαA-CgDH+ and pPICZαA-CgDH-, which were transformed into competent Pichia pastoris X-33 cells by electroporation respectively. The recombinant target proteins, CgDH+ and CgDH-, were induced for 72 h with 1% methanol at 29 °C and 250 r/min. The recombinant CgDH+ (5.78 kDa) was purified by immobilized metal affinity chromatography (IMAC), and identified by MALDI-TOF-TOF analysis, demonstrating that it was the expected target protein. Based on the concentration of the purified product, the estimated yield of recombinant CgDH+ was 2.32 mg/L. Antimicrobial assay showed that the culture medium supernatant containing recombinant CgDH+ and recombinant CgDH-, respectively, had activities against Staphylococcus aureus and Pseudomonas aeruginosa, indicating that the existence of 6×His tag in the recombinant proteins do not affect their biological activities.