杂合抗菌肽在毕赤酵母中的表达及其活性测定

Expression of Hybrid Antimicrobial Peptides in Pichia Yeast and Identification of Its Biological Activity

为获得溶血活性低、抗菌活性高的杂合抗菌肽,以家蝇抗菌肽Cec Md和中国林蛙抗菌肽Chensirin为母体肽,并结合毕赤酵母偏爱密码子的原则,设计出6条具有抗菌潜力的新型杂合抗菌肽,将其命名为CC22、CC28、CC29、CC30和CC34(1),CC34,利用SOE-PCR技术合成所需的目的基因,并将其克隆至毕赤酵母表达载体p GAPZαA,通过电击转化技术,将其转化至毕赤酵母SMD1168中,经含有Zeocin的抗性平板筛选阳性转化子,YPD液体培养72h后,经Tricine-SDSPAGE检测出目的蛋白,然后采用高效液相色谱法对其进行纯化。检测结果显示,表达产物CC29对大肠杆菌、鸡沙门氏菌的最小抑菌浓度(MIC)均为25μg/ml;CC34(1)对大肠杆菌表现相对较弱的抑制作用,最小抑菌浓度为100μg/ml;CC34对鸡沙门氏菌和金黄色葡萄球菌的最小抑菌浓度为50μg/ml;且杂合抗菌肽对有益菌均没有表现出抑制作用。6条杂合肽的溶血活性均呈现较低水平,其中表现出抗菌活性的3条抗菌肽中,以CC29的溶血活性最低,CC34(1)和CC34相对次之。结合抑菌活性,CC29和CC34的抑菌效果较为明显,从而确定溶血活性低且抗菌活性较高的CC29和CC34为新型杂合抗菌肽。

In order to obtain hybrid antimierobial peptides which have low haemolytie activity and strong antibacterial activity, six hybrid antimierobial peptides were designed depend on Musca domestica Cec Md and Rana Chensinensis as well as preference condon of Pichia yeast,named CC22, CC28, CC29, CC30, CC34（ 1 ） and CC34. The target genes of six hybrid antimicrobial peptides were synthesized successfully by SOE-PCR and, were cloned into the expression vector pGAPZc^A of Pichia pastoris. Which were transformed into Pichia pastoris SMDI168 by electrotransformation,and the positive transformants which containing hybrid antimicrobial peptides gene were selected using high concentrations of Zeocin. When the screened recombinant gene engineering bacteria were cultured 72 hours in YPD medium, the expression products were identified by Tricine-SDS-PAGE and purified by HPLC. The biological activity measurement results showed that the minimal inhibitory concentration（MIC） of CC29 to Escherichia coli and Salmonella paratyphus were 25 μg/ml; CC34（ 1 ） had lowerinhibitionon and the MIC to Escherichia coli was 100 μg/ml; the MIC of CC34 to Salmonella gallinarum and Staphylococcus aureus were 50μg/ml; six hybrid antimicrobial peptides had no antimicrobial effect on intestine bacteria. As for hemolytic activity, all of the antimicrobial peptides were low, CC29 was the lowest, CC34 （ 1 ） and CC34 was the higher in which showed the antibacterial activities of them. Considering the antibacterial activity, CC29 and CC34 were more effective on it, so two new hybrid antimicrobial peptides were obtained which had both more effective antibacterial activity and lower hemolytic activity.