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目的利用毕赤酵母诱导表达T4溶菌酶蛋白并测定其抗菌活性。方法T4溶菌酶(T4lysozyme)基因以N端融合的方式被准确插入到质粒pPIC9K的EcoR I-Not I位点内,得到分泌型重组表达载体 pPIC9K-T4L。该载体首先经限制性内切酶Sal I酶切,进而采用电击方法将线性化的重组质粒DNA导入到毕赤酵母中。经过梯度筛选得到多个单拷贝和多拷贝转化重组子。随机挑取部分重组子PCR扩增阳性克隆,经菌体培养和甲醇诱导后获得了分泌表达,表达产物存在于培养上清液中。结果表达蛋白经琼脂孔扩散抗菌实验显示抑菌圈明显;重组蛋白对金黄色葡萄球菌与肺炎链球菌均具有显著抑制作用;多拷贝与单拷贝重组表达子没有抗菌活性差异与蛋白表达量的差异;加热煮沸对于T4溶菌酶蛋白的抗菌活性无明显影响。结论T4溶菌酶在毕赤酵母中得以成功诱导与表达;表达产物不受拷贝数影响并具热稳定性。
Objective To express T4 lysozyme protein by Pichia pastoris and determine its antibacterial activity. Methods The T4 lysozyme gene was inserted into EcoR I-Not I site of plasmid pPIC9K by N-terminal fusion to obtain the secreted recombinant expression vector pPIC9K-T4L. The vector was first digested with restriction enzyme Sal I, and then the linearized recombinant plasmid DNA was electroporated into Pichia pastoris. After gradient screening, multiple single copy and multiple copy transformation recombinants are obtained. The positive clones were amplified by polymerase chain reaction (PCR), and the secreted proteins were obtained after being induced by methanol and methanol. The expressed products existed in the culture supernatant. Results The expression of the expressed protein by agar diffusion experiments showed that the inhibition zone significantly; recombinant protein of Staphylococcus aureus and Streptococcus pneumoniae were significantly inhibited; multi-copy and single copy of the recombinant expression of non-antibacterial activity and protein expression differences ; Heating boiled T4 lysozyme protein antibacterial activity had no significant effect. Conclusion T4 lysozyme was successfully induced and expressed in Pichia pastoris. The expression product was not affected by the copy number and was thermostable.