抑制金黄色葡萄球菌的乳酸菌抗菌肽基因筛选及表达鉴定

Screening and expression identification of Staphylococcus aureus-inhibiting peptide genes from lactic acid bacteria

以具有抑菌效果的L2、L16、L19等7株乳酸菌为模板,通过PCR扩增验证部分菌株中含有PlnF、PlnE、PlnN、PlnJ、PlnK等抗菌肽基因,以pET28a为载体并在抗菌肽基因两端引入Nco I和Xho I酶切位点,经双酶切和T4连接酶反应构建pET28a-抗菌肽重组质粒,转化E.coli BL21(DE3),培养至OD_(600)=0.6时,加入0.5mmol/L的IPTG诱导6h,菌体在400 W、超声4s、间歇5s条件下破碎后以8mol/L尿素进行变性处理和Ni柱纯化透析复性后的蛋白与相对应未纯化的蛋白相比,仅PlnF纯化蛋白对金黄色葡萄球菌具有抑菌效果[抑菌圈直径为(13.43±0.21)mm],而其他蛋白几乎无抑菌活性。进一步通过Tricine-SDS-PAGE电泳和nanoLC-ESI-MS/MS验证,目的蛋白与PlnF抗菌肽具有97.6%的同源性。

The 7 strains with antimicrobial activity were screened as samples and verification by PCR amplification has antimicrobial peptide genes of PlnF,PlnE,PlnN,PlnJ,and PlnK.Then designed aprimer to introduce Nco I and Xho I sites into both ends of the antibacterial peptide genes with the treated of pET28 aplasmid to obtain recombinant plasmid using T4 ligase,and the pET28 a-peptide was transformed into E.coli BL21（DE3）.The experiment results showed that the inhibition zone of the pET28 a-peptide was the PlnF,after 0.5 mmol/L IPTG inducted for 6 hafter the OD_（600）=0.6 of the E.coli LB.The cell in the 400 W,ultrasonic for 4 s,with intermittent 5 s for crushing,and then with 8 mol/L urea denaturation and renaturation with Ni purified,the PlnF purified protein has the antibacteria effects to the Staphylococcus aureus with（13.43±0.21）mm compare with the control group.Further by Tricine-SDS-PAGE electrophoresis and nanoLC ESI/MS/MS verification,the homology of the target protein and PlnF antimicrobial peptide with 97.6%.