三疣梭子蟹C型溶菌酶在毕赤酵母中的表达及其抑菌活性

Expression of Swimming Crab C-type Lysozyme in Pichia pastoris and Its Bacteriostatic Activity

C型溶菌酶是存在于各种生物组织中的重要的先天性免疫蛋白,是开发天然抑菌剂的良好候选者。以甲壳类动物三疣梭子蟹(Portunus trituberculatus)C型溶菌酶成熟肽为研究对象,首先通过RT-PCR克隆其编码基因"ptlyC";以pPICZαA为表达载体,构建重组表达载体pPICZαA-ptlyC,电转至毕赤酵母(Pichia pastoris)X-33后,通过高浓度的博来霉素筛选到高拷贝酵母转化子;在28℃、250 r/min和pH6.0的条件下,经1%的甲醇诱导表达72 h,得到含重组ptlyC的培养液上清;经固化金属离子亲和层析(IMAC)纯化,得到分子量约为25.3 kD的纯化产物。通过菌落计数法证明含重组ptlyC的培养液上清具有抑制革兰氏阳性的枯草芽孢杆菌(Bacillus subtilis)的活性;通过琼脂糖凝胶扩散法证明纯化的重组ptlyC具有抑制革兰氏阴性的铜绿假单胞菌(Pseudomonas aeruginosa)、副溶血性弧菌(Vibrio parahaemolyticus)和沙门氏菌(Salmonella lignieres)的活性。

C-type lysozyme is an innate immune protein existing in different tissues of all organisms,thus it is a favorable candidate for developing natural bacteriostatic agent.In this study,mature peptide of C-type lysozyme from crustacean swimming crab(Portunus trituberculatus)was the object of recombinant DNA expression.Firstly,cDNA fragment encoding the C-type lysozyme mature peptide was cloned by RT-PCR,and was named as“ptlyC”.The ptlyC was ligated to pPICZαA vector to construct recombinant expression vector pPICZαA-ptlyC;then the pPICZαA-ptlyC was transformed into competent Pichia pastoris X-33 cells.Yeast transformants containing multi-copy gene insertion were screened by high concentration of zeocin.Recombinant ptlyC was induced with 1.0%(V/V)methanol at 28℃,250 r/min and pH6.0 for 72 h.The culture medium supernatant containing recombinant ptlyC was subjected to immobilized metal affinity chromatography(IMAC)for purification,and the molecular mass of the purified ptlyC was about 25.3 kD.Micro liquid-bacterial colony notation demonstrated that the culture medium supernatant containing recombinant ptlyC had a bacteriostatic effect on gram-positive Bacillus subtilis.Further,agar well diffusion assays confirmed that the purified recombinant ptlyC inhibited gram-negative Pseudomonas aeruginosa,Vibrio parahaemolyticus and Salmonella lignieres.