黄沙鳖β-防御素Hs-BD1的原核表达及抗菌活性检测

Prokaryotic expression and antibacterial activity test of Hs-BD1, β-defensin of Huangsha soft-shelled turtle

【目的】构建黄沙鳖β-防御素1(Hs-BD1)原核表达并分析其抗菌活性,为深入分析Hs-BD1的生理功能及开发新型黄沙鳖抗菌药物提供理论依据。【方法】构建Hs-BD1成熟肽序列的重组表达载体pCold-TF-Hs-BD1,采用异丙基硫代半乳糖苷(IPTG)诱导重组蛋白rHs-BD1表达,以十二硫酸酯钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western blotting)检测重组蛋白rHs-BD1的表达情况。采用Ni柱亲和层析法对重组蛋白rHs-BD1进行纯化,分析其对几种革兰氏阳性和阴性菌的抑菌活性、溶血性和抗氧化能力。【结果】SDS-PAGE检测结果显示,重组蛋白rHs-BD1在电泳凝胶约58.1 kDa处出现特异性条带,其分子量大小与预测值相符。经Western blotting检测,重组蛋白rHs-BD1可与Anti-6×His Tag抗体发生特异性反应,表明Hs-BD1基因在原核表达系统成功表达,经纯化的重组蛋白rHs-BD1含量为400.0μg/mL。对已纯化的重组蛋白rHs-BD1进行体外抗菌活性检测,结果显示其对无乳链球菌(Streptococcus agalactiae) HPG1和金黄色葡萄球菌(Staphylococcus aureus)HPN1的最小抑菌浓度(MIC)为40.00μg/mL,对大肠杆菌(Escherichia coli)YLG1、类志贺邻单胞菌(Plesiomons shigelloides)DAL1和迟钝爱德华氏菌(Edwardsiella tarda)FCH1的MIC为20.00μg/mL,表明重组蛋白rHs-BD1对一些革兰氏阳性和阴性菌具有较强的抗菌活性。20.0、40.0、60.0和80.0μg/mL重组蛋白rHs-BD1对新鲜黄沙鳖血细胞的溶血率分别为1.5%、2.7%、3.3%和4.2%,20.0、40.0、80.0和120.0μg/mL重组蛋白rHs-BD1对1,1-二苯基-2-苦肼基(DPPH)自由基的清除率分别为2.6%、5.2%、11.6%和17.0%。【结论】经原核表达获得的Hs-BD1重组蛋白rHs-BD1具有一定的广谱抗菌活性和抗氧化能力,且溶血作用较弱,对黄沙鳖机体毒副作用较低,可用于新型黄沙鳖抗菌药物开发。

【Objective】The prokaryotic expression of β-defensin 1 of Huangsha soft-shelled turtle(Hs-BD1) were constructed and its antibacterial activity were analyzed, so as to give a theoretical evidence for the depth analysis of its physiological function and the development of new antibiotics.【Method】The recombinant expression vector pCold-TF-Hs-BD1 of the mature peptide sequence of Hs-BD1 was constructed. The recombinant protein rHs-BD1 expression was acquired by the induction of isopropyl thiogalactoside(IPTG). The expression of the recombinant protein rHs-BD1 was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblotting(Western blotting). The recombinant protein rHs-BD1 was purified using Ni column affinity chromatography method and the effect of the purified recombinant protein rHs-BD1 on the antibacterial activity against several gram-positive and gram-negative bacteria, the hemolysis and antioxidant activity of were analyzed.【Result】The test result of SDS-PAGE showed that a specific band was displayed at about 58.1 kDa of electrophoresis gel, which was consistent with the predicted molecular weight value. Western blotting test showed that the recombinant protein Hs-BD1 could react specifically with Anti-6×His Tag antibody, indicating the successful expression of Hs-BD1 gene in prokaryotic expression system. The concentration of purified recombinant protein rHs-BD1 was 400.0 μg/mL. In vitro antibacterial activity test of purified recombinant protein rHs-BD1 showed that minimal inhibitory concentrations(MICs) of recombinant protein rHs-BD1 were 40.0 μg/mL against Streptococcus agalactiae HPG1 and Staphylococcus aureus HPN1,and 20.0 μg/mL against Escherichia coli YLG1,Plesiomons shigelloides DAL1 and Edwardsiella tarda FCH1,indicating that the recombinant protein rHs-BD1 had a strong antibacterial activity against some gram-positive and gram-negative bacteria. The hemolysis rates of the recombinant protein rHs-BD1 with the concentration of 20.0,40.0,60.0 and 80.0 μg/mL were 1.5%,2.7%,3.3% and 4.2% to fresh Huangsha soft-shelled turtle’s blood cells. And the scavenging rates against DPPH(1,1-Diphenyl-2-picrylhydrazyl) free radical of the recombinant protein rHs-BD1 with the concentration of 20.0,40.0,80.0 and 120.0 μg/mL were 2.6%,5.2%,11.6% and 17.0%,respectively.【Conclusion】The recombinant protein rHs-BD1 of Hs-BD1 obtained by prokaryotic expression may have a certain broad spectrum antibacterial activity and antioxidant activity, and the hemolysis activity is fairly weak. It had low toxicity and weak side effect on the body of Huangsha soft-shelled turtle and could be used for the development of new antibiotic for Huangsha soft-shelled turtle.