马尔堡病毒糖蛋白RBD基因的原核表达、纯化及多克隆抗体的制备  

Prokaryotic expression and purification of the receptor binding domain of GP protein of Marburg virus and the preparation of polyclonal antibody

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作  者:王琪[1,2] 盖微微[2,3] 闫飞虎[2] 冯娜[1,2] 吴芳芳[2] 赵梓淇[1,3] 曹增国[2] 李岭[2,3] 迟航[2] 金宏丽[2,3] 邱泊宁 崔健男[2,4] 赵永坤[2] 王铁成[2] 高玉伟[2,1] 王化磊[2,1] 杨松涛[2,1] 夏咸柱[2] WANG Qi GAI Wei-wei YAN Fei-hu FENG Na WU Fang-fang ZHAO Zi-qi CAO Zeng- guo LI Ling CHI Hang JIN Hong-li QIU Bo-ning CUI Jian-nan ZHAO Yong-kun WANG Tie-cheng GAO Yu-wei WANG Hua-lei YANG Song-tao XIA Xian-zhu(College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China Key Laboratory of Zoonosis Prevention and Control of Jilin Province/Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130122, China College of Veterinary Medicine , J ilin University ,Changchun 130062 ,China College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonosis, Yangzhou , Jiangsu 225009, China)

机构地区:[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]军事医学科学院军事兽医研究所/吉林省人兽共患病预防与控制重点实验室,吉林长春130122 [3]吉林大学动物医学学院,吉林长春130062 [4]东北农业大学动物医学学院

出  处:《中国兽医学报》2017年第7期1261-1267,共7页Chinese Journal of Veterinary Science

基  金:国家科技重大专项(重大新药创制)资助项目(2015ZX09102025)

摘  要:原核表达并纯化马尔堡病毒(Marburg virus,MARV)糖蛋白(glycoprotein,GP)受体结合域(receptor binding domain,RBD)蛋白,并以此为抗原免疫家兔制备抗MARV-GP-RBD多克隆抗体。参照GenBank提供的MARV GP全基因序列,找到主要抗原表位区域,设计特异性引物,采用PCR方法扩增RBD基因,扩增产物经双酶切(EcoRⅠ/XhoⅠ)后定向克隆至原核表达载体pET-30a(+),构建重组表达质粒pET-30a(+)-GP-RBD,转化BL21(DE3)感受态表达宿主菌,在不同条件下(时间、IPTG浓度、温度)诱导表达目的蛋白,并用His-Band N+柱进行亲和层析纯化;以纯化的重组pET-30a(+)-GP-RBD蛋白免疫家兔,制备多克隆抗体。通过SDS-PAGE、Western blot和IFA鉴定重组蛋白的反应原性及免疫原性。结果显示:PCR扩增到长度为453bp的RBD基因片段;构建的重组质粒pET-30a(+)-GP-RBD经双酶切后得到与目的片段长度相同的特异性条带,测序结果显示没有突变;转化产物在培养7h、终浓度为0.4mmol/L IPTG和37℃条件下能够充分诱导目的蛋白表达,得到相对分子质量为25 000的重组蛋白,主要以包涵体形式存在,BCA试剂盒产量测定,每升诱导的重组菌可纯化约20mg纯度较高的目的蛋白;Western blot检测证实重组pET-30a(+)-GP-RBD蛋白能同时被抗His标签的单抗和兔源多抗识别并发生特异性反应,证明重组蛋白有良好的反应原性;IFA鉴定证实所制备的兔源多抗能够特异性识别表达MARV GP蛋白的重组杆状病毒rBacmid-GP-VP40,证明重组蛋白具有良好的免疫原性。结果表明:成功表达、纯化了MARV GP RBD蛋白,并完成了兔源多抗的制备,为MARV亚单位疫苗的制备和抗原、抗体检测方法的建立奠定基础。Receptor binding domain(RBD) of GP protein of Marburg virus was expressed in pro karyotic cells and purified to produce the polyclonal antibody of rabbit against MARV-GP-RBD protein. According to the genes equences of MARV GP published in GenBank,a pair of printers was designed for amplifying the RBD of MARV GP by PCR method. Double digested PCR prod- ucts were cloned into the prokaryotic expression vector pET-30a(b) to generate the recombinant expression plasmid pET-30a(+)-GP-RBD,which was transformed into E. coli BL21 (DE3) for ex pressing MARV-GP-RBD proteins under variously inducing conditions(time,IPTG concentration, temperature). Recombinant protein was purified using His-Band Ni+ affinity chromatography and purified recombinant protein was used to immunize rabbit to produce polyclonal antibody. The im- munogenicity and reactivity of the recombinant protein was identified by SDS PAGE,Western blot and IFA. A fragment about 453 bp in length of RBD gene was successfully amplified,recombinant plasmid pET-30a(+) GP-RBD was constructed,correctly inserted into BI.21 (DE3) after PCR restriction analysis and sequencing. SDS-PAGE indentified a 25 000 protein which was fully ex- pressed as inclusion bodies with induction by 0.4 mmol/L IPTGat 37℃ for 7 h. After purification by His BandNi + ,affinity chromatography,20 mg target protein was produced from 1 L of shake flask culture which determined by BCA protein assay kit. Western blot confirmed that the recom binant protein could be recognized by monoclonal antibody against His and polyclonal antibody from rabbit,showing a good immunoreactivity;IFA test showed that polyclonal antibodies could i dentify recombinant baculovirus rBacmid-GP-VP40 expressing MARV GP, proving a good immunogenicity. RBD protein of GP of Maburg virus was successfully expressed and purified. In addition, polyclonal antibody was prepared,which laied the foundation for the research on subunit vac- cine and establishment of detection method of antigen and antibo

关 键 词:马尔堡病毒 糖蛋白受体结合域 原核表达与纯化 多克隆抗体 

分 类 号:S852.65[农业科学—基础兽医学] R535[农业科学—兽医学]

 

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