[1]马巍娜,刘雪林,宋宏彬,等.盐酸克伦特罗生物素化单链抗体在大肠埃希氏菌中的表达[J].现代检验医学杂志,2016,31(04):44-46,50.[doi:10.3969/j.issn.16717-414.2016.04.011]
　MA Wei-na,LIU Xue-lin,SONG Hong-bin,et al.Expression of Clenbuterol Biotinylated ScFv in Escherichia Coli[J].Journal of Modern Laboratory Medicine,2016,31(04):44-46,50.[doi:10.3969/j.issn.16717-414.2016.04.011]
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盐酸克伦特罗生物素化单链抗体在大肠埃希氏菌中的表达()

《现代检验医学杂志》[ISSN:/CN:]

卷:
第31卷
期数:
2016年04期
页码:
44-46,50
栏目:
论著
出版日期:
2016-08-10

文章信息/Info

Title:
Expression of Clenbuterol Biotinylated ScFv in Escherichia Coli
文章编号:
1671-7414(2016)04-044-04
作者:
马巍娜1,刘雪林2,宋宏彬2,沈建良1,黄友章1,刘 毅1,向 丹1
1.中国人民解放军海军总医院血液科,北京 100037;
2.中国人民解放军军事医学科学院十所,北京 100039
Author(s):
MA Wei-na1,LIU Xue-lin2,SONG Hong-bin2,SHENJian-liang1,HUANG You-zhang1, LIU-Yi1,XIANG Dan1
1.Department of Hematology,General Hospital of PLA Navy, Beijing 100037,China;
2.PLA Disease Control and Prevention,Beijing 100039,China
关键词:
噬菌体表面展示技术;  筛选;  盐酸克伦特罗;  生物素;  表达
分类号:
R378.21; R392.11
DOI:
10.3969/j.issn.16717-414.2016.04.011
文献标志码:
A
摘要:
目的 提取盐酸克伦特罗(CLB)生物素化抗体片段蛋白进行表达,为下一步纯化及农兽药品快速检测奠定基础。方法 确定一株生物素化CLB噬菌体抗体 “pHEN1-BHNS-CLB1”对其进行蛋白表达,即从噬菌粒“PHEN1-BHNS-CLB1”切下scFv基因片段,亚克隆至能引导可溶性表达的载体pCANTAB5E上,完成了重组质粒的构建,将所构质粒转化大肠埃希氏菌感受态细胞BL21。命名“5E-BHNS-CLB1”,并对“5E-BHNS-CLB1”进行PCR及酶切鉴定后,测序分析。结果 ELISA结果证明其具有较好的亲和性,竞争抑制ELISA、与gp120蛋白及三聚氰胺的交叉反应ELISA结果均证明其具有很好的特异性,片段大小与预想目的片段大小一致,SDS-PAGE及Western blotting的结果显示,其分子量大小与目的一致,并能与抗E-tag标签抗体结合显色。结论 说明表达的蛋白能与表达载体5E后带有E-Tag标签的短肽作用,含有E-Tag标签,是我们要表达的目的蛋白,为下一步纯化蛋白创造了条件。
Abstract:
Objective to extract clenbuterol hydrochloride(CLB)biotinylated antibody fragments of protein expression,purification andlay the foundation for the rapid detection of pesticide and veterinary drug next.Methods Determined biotinylated phage antibodies to CLB“pHEN1 BHNS CLB1” for protein expression.The scFv gene was cut from the phagemid “PHEN1-BHNS-CLB1”,cloned into the vector pCANTAB5E can guide the soluble expression,completed the construction of recombinant plasmid,the plasmid was transformed into E.coli competent cells BL21.Named “5E-BHNS-CLB1”,and “5E-BHNS-CLB1” by PCR and enzyme digestion,sequencing.Results The ELISA results proved that it had better affinity,competitiveinhibition of ELISA,ELISA and gp120 cross reaction results indicated that the protein and melamine had good specificity,the scFv gene was cut from the phagemid “PHEN1-BHNS-CLB1”,cloned into the vector pCANTAB5E could guide the solubleexpression,completed the construction of recombinant plasmid,the plasmidwas transformed into E.coli competent cells BL21.Named “5E-BHNS-CLB1”,and“5E-BHNS-CLB1” by PCR and enzyme digestion,sequencing,fragment size and expected fragment size consistent.SDS-PAGE and Western blotting results showedthat consistency in their molecular size and purpose,and with the label of antie-tag antibody combined with color.Conclusion Proteinexpression and expression vector 5E with e-tag label short peptide,containingthe E-tag label is we want to table of the target protein,as a purified protein to create the conditions.

参考文献/References:

[1] 颜春荣,刘贤进,余向阳,等.氟虫腈单克隆抗体制备及其性能分析[J].江苏农业学报,2003,19(3):166-169. Yan CR,Liu XJ,Yu XY,et al.Monoclonal antibody preparation and its property analysis for fipronil[J].Jiangsu Journal of Agricultural Sciences,2003,19(3):166-169.
[2] 赵肃清,孙远明,张春艳.甲胺磷单克隆抗体制备及鉴定[J].免疫学杂志,2003,19(2):142-145. Zhao SQ,Sun YM,Zhang CY.Production and identification of anti-methamidophos monoclonal antibodies[J].Immunology Journal,2003,19(2):142-145.
[3] 何继红,沈建忠.磺胺二甲嘧啶单克隆抗体的研制[J].中国兽医杂志,2003,39(1):8-11. He JH,Shen JZ.Development of monoclonal antibodies againt sulfamethazine[J].Chinese Journal of Veterinary Medicine,2003,39(1):8-11.
[4] 何方洋,邱阳生,杨根海,等.克伦特罗单克隆抗体的制备与鉴定[J].中国兽医科技,2001,31(6):30-32. He FY, Qiu YS, Yang GH,et al.Preparation and characterization ofmonoclonal antibodies clenbuterol[J].Chinese Journal of Veterinary Science and Technology,2001,31(6):30-32.
[5] Shelver WL, Smith DJ, Berry ES.Production and characterization of a monoclonal antibody against the beta-a drenergic agonist ractopamine[J].Journal of Agricaltural and Food Chemistry,2000,48(9):4020-4026.
[6] Griffiths AD,Malmqvist M,Marks JD,et al.Human anti-self antibodies with high specificity from phage display libraries[J].EMBO J,1993,12(2):725-734.
[7] Mcelhiney J,Lawton LA,Porter AJR.Detection and quantification of microcystins(cyanobacterial hepatotoxins)with recombinant abtibody fragments isolated from a native human phage display liberary[J].FEMS Microbiology Letter,2000,193(1):83-88.
[8] Nissim A,Hoogenboom HR,Tomlinson IM,et al.Antibody fragments from a‘single pot'phage display library as immunochemical reagents[J].EMBO J,1994,13(3):692-694.
[9] Tomlinson IM,Walter G,Marker JD,et al.The re-pertoire of human germline VH sequences reveals about fifty groups of VH segments with different hyervariable loops[J].J Mol Biol,1992,227(3):776-798.
[10] Sun Y,Li WJ,Ma JS,et al.Soluble expression,purification andcharacterization of single-chain Fv catalytic antibody(sFv -2F3)[J].Chemical Research in Chinese Universities,2004,20(3):317-322.
[11] Tordsson JM,Ohlsson LG,Abrahmsen LB,et al.Phage-selected primate antibodies fused to superantigenes for immunotherapy of malignant melanoma[J].Cancer Immunol Immunother,2000,48(12):691-702.
[12] Walewski JL,Gutierrez JA,Branch-Elliman W,et al.Mutation master:profiles of substitutions in hepatitis C virus RNA of the core:alternate readingframe,and NS2 coding regions[J].RNA,2002,8(5):557-571.
[13] 乔媛媛,王 琰,陈晓穗,等.大容量噬菌体抗体库的构建及鉴定[J].中华微生物学与免疫学杂志,2004,24(3):194-197. Qiao YY,Wang Y,Chen XS,et al.Construction of a large singlechain phage antibody library[J].Chinese Journal of Microbiology and Immunology,2004,24(3):194-197.
[14] 张建琼,谢 维,张雪萍,等.人源抗丙型肝炎病毒噬菌体抗体库的构建、筛选及表达[J].上海免疫学杂志,2000,20(5):304-307. Zhang JQ,Xie W,Zhang XP,et al.Construction,screening and expression of hepatitis C virus specific phage antibody combinatorial library[J].ShanghaiJournal of Immunology,2000,20(5):304-307.
[15] 傅 蓉,刘 刚,戴红梅,等.利用HER2/neu胞外配体结合区2从噬菌体抗体库中筛选抗体及其初步鉴定[J].生物技术通讯,2006,17(4):574-576. Fu R,Liu G,Dai HM,et al.Screening and identifying of anti-RLDS antibodies from phage antibody libraries[J].Letters in Biotechnology,2006,17(4):574-576.
[16] 葛晓冬,刘友生,王晓东,等.人源噬菌体抗体库的构建及抗人NH-LBP抗体的筛选与鉴定[J].细胞与分子免疫学杂志,2005,21(2):180-184. Ge XD,Liu YS,Wang XD,et al.Construction of human phage antibody librayand screening and characterization of phage antibodies against N terminal fragment of human lipopolysaccharide binding protein[J].Chinese Journal of Cellular and Molecular Immunology,2005,21(2):180-184.
[17] Meulemans EV,Slobbe R,Wasterval P,et al.Selection of phage-displayed antibodies specific for a cytoskeletal antigen by competitive elution with a monoclonal antibody[J].J Mol Biol,1994,244(4):353-360.
[18] 马巍娜,刘雪林,宋宏彬,等.抗甲硝唑人源scFv抗体筛选与鉴定[J].现代检验医学杂志,2010,25(4):7-10. Ma WN,Liu XL,Song HB,et al.Screening and cha-racterization of human phage antibody to metronidazole[J].Journal of Modern Laboratory Medicine,2010,25(4):7-10.
[19] 马巍娜,刘雪林,宋宏彬,等.三聚氰胺人源scFv抗体筛选与鉴定[J].现代检验医学杂志,2012,27(3):10-13. Ma WN,Liu XL,Song HB,et al.Screening and cha-racterization of human phage antibody to melamine[J].Journal of Modern Laboratory Medicine,2012,27(3):10-13.

备注/Memo

备注/Memo:
基金项目:国家863计划科学基金资助项目(项目编号:2006BAK02A09)。
作者简介:马巍娜(1983-),女,硕士研究生,微生物控制技术,Tel:010-66958485,E-mail:mv4477@sina.com。
更新日期/Last Update: 2016-08-10