镰形扇头蜱防御素的原核表达及其抑菌活性的分析  

Prokaryotic expression and antimicrobial activity of defensin in Rhipicephalus haemaphysaloides

作　　者：崔濮凡 张永英 宋登阔 石玉祥 钟翠红 权陶芬 李书德 王方方 CUI Pu-fan;ZHANG Yong-ying;SONG Deng-kuo;SHI Yu-xiang;ZHONG Cui-hong;QUAN Tao-fen;LI Shu-de;WANG Fang-fang(College of Life Science and Food Engineering,Hebei University of Engineering,Handan 056000,China)

机构地区：[1]河北工程大学生命科学与食品工程学院,河北邯郸056000

出　　处：《中国预防兽医学报》2024年第10期1086-1091,1106,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：河北省自然科学基金(C2020402007)。

摘　　要：为分析镰形扇头蜱防御素RhD的生物学功能和抑菌活性,本研究利用在线网站预测RhD的分子生物学特性,采用MegAlign软件分析GenBank中不同物种防御素氨基酸序列之间的相似性,利用MEGA 7构建不同物种防御素氨基酸序列的系统进化树。结果显示,RhD基因开放阅读框为225 bp,编码75个氨基酸,具有Knot1结构域、信号肽,为疏水蛋白。RhD的二级及三级结构均为α-螺旋、β-折叠和无规则卷曲等结构,且与变异革蜱防御素(Q86QI5.1.A)三级结构相似性最高。不同物种防御素氨基酸序列的相似性结果显示,RhD与血红扇头蜱防御素的相似性最高为93.2%,变异度最低为7.1%;与长角血蜱防御素的相似性为82.4%,变异度为20.1%,与其余物种防御素相似性较低,且变异度较高;不同物种防御素氨基酸序列的系统进化树结果显示,RhD与血红扇头蜱防御素、长角血蜱防御素位于同一进化分支,与黑腹果蝇、埃及伊蚊、家蚕等防御素同属一大分支。采用PCR从镰形扇头蜱唾液腺中扩增已切除信号肽编码基因的RhD基因,克隆至表达载体pET-30a中构建重组质粒pET30a-RhD,并经PCR和测序鉴定正确后,转化E.coli BL21(DE3)感受态细胞,经IPTG诱导后利用镍离子亲和层析柱纯化重组RhD蛋白(rRhD),采用SDS-PAGE检测r RhD的表达及纯化效果,使用BCA蛋白定量试剂盒测定纯化后rRhD浓度,采用western blot鉴定rRhD的反应原性。SDS-PAGE结果显示,在15 ku处出现目的蛋白条带,且其主要以可溶性形式表达,纯化后rRhD浓度约为110μmol/L。Western blot显示在15 ku处出现特异性条带,与His标签单克隆抗体(MAb)的反应原性较强。分别利用牛津杯法和微量二倍稀释法检测RhD(110μmol/L~1.72μmol/L)对常见3种细菌的抑菌活性。结果显示,rRhD对大肠杆菌和沙门菌的平均抑菌圈直径基本一致,但均极显著低于阳性对照组(硫酸卡那霉素)(P<0.01),对金黄色葡萄球菌的平均抑菌圈直径显In order to analyze the biological function and antibacterial activity of Rhipicephalus haemaphysaloides defensin(RhD),the molecular biological characteristics of RhD were predicted using online websites,the similarities between the amino acid sequences of defensins from different species in GenBank were analyzed using MegAlign software,and the phylogenetic tree of the amino acid sequences of defensins from different species was constructed using MEGA 7.The results showed that the open reading frame of the RhD gene was 225bp,encoding 75 amino acids,with a Knot1 domain,a signal peptide,and was a hydrophobic protein.The secondary and tertiary structures of RhD were all alpha-helix,beta-pleated sheet,and random coil structures,and the tertiary structure was most similar to the Dermacentor variabilis defensing protein(Q86QI5.1.A).The similarity of amino acid sequences of defensions from different species showed that the similarity of RhD with the tick defensin protein of Rhipicephalus sanguineus was the highest at 93.2%,with the lowest variation of 7.1%;the similarity with the tick defensin protein of Haemaphysalis longicornis was 82.4%,with a variation of 20.1%;the similarity with the defensin proteins of other species was lower,and the variation was higher;the phylogenetic tree of amino acid sequences of defensin proteins from different species showed that RhD was on the same evolutionary branch as the tick defensin proteins of Rhipicephalus sanguineus and Haemaphysalis longicornis,and belonged to the same branch as the defensin proteins of Drosophila melanogaster,Aedes aegypti,Bombyx mori,etc.The RhD gene with the signal peptide coding gene removed was amplified from the salivary gland of Rhipicephalus haemaphysaloides by PCR and cloned it into the expression vector pET-30a to construct the recombinant plasmid pET30a-RhD.After being confirmed by PCR and sequencing,the recombinant plasmid was transformed into E.coli BL21(DE3)competent cells.After IPTG induction,the recombinant RhD protein(rRhD)was purified using a

关 键 词：镰形扇头蜱 防御素 抑菌活性 

分 类 号：S855.9[农业科学—临床兽医学]