林氏扇头蜱Kunitz型蛋白酶抑制剂Boophilin-H2原核表达及抗凝活性  

Prokaryotic expression and anticoagulant activity of Boophilin-H2,a protease inhibitor of Rhipicephalus Linnaei Kunitz type

作　　者：赵培真 李尧 余玲玲 谢子芳 陈杰[1] 彭维祺 黎明成 赵建国[1] 管庆丰[1] ZHAO Peizhen;LI Yao;YU Lingling;XIE Zifang;CHEN Jie;PENG Weiqi;LI Mingcheng;ZHAO Jianguo;GUAN Qingfeng(School of Life and Health Sciences,Hainan International One Health Institute,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]海南大学生命健康学院,海南全健康国际研究院,海南海口570228

出　　处：《中国热带医学》2024年第9期1106-1111,共6页China Tropical Medicine

基　　金：海南省重大科技计划项目(No.ZDKJ2021035);海南省自然科学基金项目(No.324RC446)。

摘　　要：目的克隆林氏扇头蜱Boophilin-H2基因,构建重组表达载体,在大肠埃希菌中表达Boophilin-H2重组蛋白,并检测其抗凝活性。方法通过设计特异性引物,以林氏扇头蜱饱血成蜱RNA反转为cDNA为模板,PCR扩增Bo⁃ophilin-H2基因片段,克隆、连接到质粒pSmart-I,构建重组表达载体pSmart-I/Boophilin-H2,BamHⅠ和XhoⅠ双酶切验证重组表达载体,将表达载体转入大肠埃希菌BL21(DE3)感受态中,经异丙基-β-D-硫代吡喃半乳糖苷低温诱导表达,Ni-NTA Resin纯化重组蛋白,12.5%SDS-PAGE检测重组蛋白表达及纯化情况,使用3.8%枸橼酸钠采血管收集新西兰大白兔股静脉血,离心分离吸取上层血浆,利用体外凝血四项检测盘检测重组蛋白抗凝活性。结果扩增并克隆出林氏扇头蜱Boophilin-H2基因片段387 bp,构建出原核表达载体pSmart-I/Boophilin-H2,双酶切验证载体构建成功,转入大肠埃希菌中经0.8 mmol/L IPTG诱导表达16 h,SDS-PAGE检测表明重组蛋白表达在上清液中,主要以可溶形式存在,编码的Boophilin-H2重组蛋白大小在35000左右,纯化Boophilin-H2重组蛋白进行抗凝活性检测表明,重组蛋白能显著延长活化部分凝血活酶时间(activated partial thromboplastin time,APTT),并具有浓度依赖性,重组蛋白对凝血酶时间、凝血酶原时间、纤维蛋白原凝血时长的影响差异无统计学意义。结论本研究构建Boophilin-H2表达载体,其表达产物具有很强的APTT抗凝活性,揭示了重组蛋白发挥作用在内源凝血途径,可能作用于内源性凝血因子Ⅷ、Ⅺ、Ⅻ从而阻止血液凝固,为进一步蜱类防控疫苗和抗凝血药物的研发提供了参考依据和理论基础。Objective To clone Rhipicephalus linnaei Boophilin-H2 gene,construct the recombinant expression vector,express the Boophilin-H2 recombinant protein in Escherichia coli,and assess its anticoagulant activity.Methods Specific primers were designed to amplify the Boophilin-H2 gene fragment using cDNA,synthesized from engorged Rhipicephalus linnaei tick RNA through reverse transcription,as a template.The gene fragment was cloned and connected to plasmid pSmartI,and the recombinant expression vector pSmart-I/Boophilin-H2 was constructed.The recombinant expression vector was verified by double restriction enzyme digestion with BamHⅠand XhoⅠ,transferred into the competent state of Escherichia coli BL21(DE3),and expressed under low-temperature induction with IPTG.The recombinant protein was purified by Ni-NTA Resin,and its expression and purification were detected by 12.5%SDS-PAGE.The femoral venous blood of New Zealand white rabbits was collected by 3.8%sodium citrate blood collection tube,and the upper plasma was centrifugally separated to measure the anticoagulant activity of the recombinant protein using four test plates of in vitro coagulation.Results A 387 bp gene fragment of Boophilin-H2 of Rhipicephalus linnaei was successfully amplified and cloned;the prokaryotic expression vector pSmart-I/Boophilin-H2 was constructed and verified by double enzyme digestion.Following induction with 0.8 mmol/L IPTG for 16 hours in Escherichia coli,SDS-PAGE showed that the recombinant protein was expressed in the supernatant primarily in a soluble form,with the Boophilin-H2 recombinant protein approximately 35000 in size.The anticoagulant activity assays of the purified recombinant protein Boophilin-H2 showed that the recombinant protein significantly prolongs the activated partial thromboplastin time(APTT)in a concentration-dependent manner,while its effects on thrombin time(TT),prothrombin time(PT),and fibrinogen(FIB)levels were not significant.Conclusions The expression vector for BoophilinH2 was successfully constructed,and

关 键 词：林氏扇头蜱 Kunitz型蛋白酶抑制剂 原核表达 抗凝活性 

分 类 号：R384.4[医药卫生—医学寄生虫学]