昆虫死亡素thanatin与斑点叉尾鮰β-防御素在毕赤酵母中的表达及其抑菌效果评价

Expression of insect thanatin and Ictalurus punetaus β-defensin and antibacterial effect in Pichia pastoris

旨在利用毕赤酵母表达系统表达出具有良好抑菌效果的重组抗菌肽。选定昆虫死亡素thanatin基因和斑点叉尾鮰β-防御素基因(命名为defbl),按毕赤酵母偏好性密码子优化后,将用T2A剪切肽连接的共表达基因(命名为TD)及各单基因分别构建至pMD19-T载体上,经EcoRⅠ、SalⅠ双酶切后亚克隆至表达载体pPICZɑA上,经SacⅠ线性化后电转化至毕赤酵母GS115感受态细胞中,通过Zeocin抗性及PCR筛选出阳性菌株,利用1%甲醇进行诱导表达,72 h后收集培养基上清液,冻干浓缩并纯化后,进行Western blot检测及体外抑菌试验、溶血性试验。结果显示,成功构建重组毕赤酵母工程菌株GS115-pPICZαA-thanatin、GS115-pPICZαA-defbl和GS115-pPICZαA-TD,通过甲醇诱导成功获得重组蛋白,纯化后Western blot检测结果显示,thanatin组和defbl组分别在2.3 kDa和7.8 kDa处出现特异性条带,TD组同时出现上述2条带,蛋白大小均与预期相符;3个重组蛋白浓度分别为600、300和700μg/mL;体外抑菌试验及溶血性试验结果显示,各重组蛋白均具有良好的抑菌作用,TD重组蛋白抑菌效果最佳,且所获蛋白均具有较低溶血性。研究表明,利用酵母表达系统成功获得具有良好抑菌效果且溶血性低的重组抗菌肽,相较之下,TD组抗菌肽抑菌效果最佳。本研究结果为后续进一步探索上述抗菌肽的功能及临床应用奠定基础。

This study was to express recombinant antimicrobial peptides with high antibacterial activity in a Pichia pastoris expression system.The insect thanatin gene and the Ictalurus punetaus β-defensin gene(named defbl)were optimizated according to P.pastoris codon usage frequency.The co-expression genes(named TD)which were linked with the T2A shear peptide and the two single genes were constructed into the pMD19-T vector,respectively.They were then subcloned into the expression vector pPICZɑA.After double digestion with EcoRⅠand SalⅠ,they were linearized by SacⅠand transferred into competent P.pastoris GS115 cells.Positive transformants were selected by Zeocin and PCR,and then 1% methanol was used to induce the expression of the target genes.72 hours later,the culture medium supernatant was collected,lyophilized and purified for Western blot detection,in vitro antibacterial test and hemolytic test.The results showed that the recombinant Pichia yeast engineered strains GS115-pPICZaA-thanatin,GS115-pPICZaA-defbl and GS115-pPICZaA-TD were obtained successfully.By methanol induction,the recombinant proteins were obtained and purified.The Western blot results showed that there were specific bands appearing at 2.3 kDa and 7.8 kDa in the thanatin and defbl groups,respectively,and the two bands both appeared in the TD group.The protein sizes were all consistent as expected.The protein concentrations were 600μg/mL,300μg/mL,and 700μg/mL,respectively.The results of in vitro antibacterial test and hemolytic test of recombinant antimicrobial peptides showed that all the proteins had good antibacterial effect and lower hemolysis.TD produced the best antibacterial effect.To sum up,in this study,recombinant antimicrobial peptides with good antibacterial effect and low hemolysis were obtained by the yeast expression system,with the peptides of the TD group exhibiting the best antibacterial effect.The present results laid a foundation for further exploration of the function and clinical application of the above antimicrobial peptides.