CAJ | 학술논문

目的筛选氯菊酯人源单链可变区(scFv)抗体,为研究快速检测试剂盒奠定基础.方法采用噬菌体表面展示技术,以氯菊酯作为抗原,固相包被于Nunc板,应用半合成的人源单链可变区抗体文库技术,从噬菌体单链可变区抗体库中经过3轮"吸附-洗脱-扩增"筛选过程,随机挑选抗氯菊酯的100个克隆,利用酶联免疫吸附法(ELISA)、交叉反应及竞争抑制实验,对其进行免疫学检测和鉴定,获得与氯菊酯结合活性较强的scFv阳性克隆,从噬菌体抗体阳性克隆中提取质粒,经酶切鉴定Sfi Ⅰ/Not Ⅰ后,亚克隆到pCANTAB5E载体;转化大肠杆菌XL1-Blue,提取质粒进行酶切鉴定分析.结果经过筛选100个克隆中有18株克隆ELISA的吸光度(A值)-490 nm波长(A490nm)值较高,将这些噬菌体上清与牛血清白蛋白(BSA)进行交叉反应,确定有5株交叉反应较弱,结合3次ELISA重复实验的A值及竞争抑制实验结果,最后确定1株阳性克隆.亚克隆到pCANTAB5E载体;转化感受态细胞XL1-Blue.结论提取质粒酶切片段与目的相符;为下一步其特异性亲和力的研究创造了条件.
목적 사선록국지인원단련가변구(scFv)항체,위연구쾌속검측시제합전정기출.방법 채용서균체표면전시기술,이록국지작위항원,고상포피우Nunc판,응용반합성적인원단련가변구항체문고기술,종서균체단련가변구항체고중경과3륜"흡부-세탈-확증"사선과정,수궤도선항록국지적100개극륭,이용매련면역흡부법(ELISA)、교차반응급경쟁억제실험,대기진행면역학검측화감정,획득여록국지결합활성교강적scFv양성극륭,종서균체항체양성극륭중제취질립,경매절감정Sfi Ⅰ/Not Ⅰ후,아극륭도pCANTAB5E재체;전화대장간균XL1-Blue,제취질립진행매절감정분석.결과 경과사선100개극륭중유18주극륭ELISA적흡광도(A치)-490 nm파장(A490nm)치교고,장저사서균체상청여우혈청백단백(BSA)진행교차반응,학정유5주교차반응교약,결합3차ELISA중복실험적A치급경쟁억제실험결과,최후학정1주양성극륭.아극륭도pCANTAB5E재체;전화감수태세포XL1-Blue.결론 제취질립매절편단여목적상부;위하일보기특이성친화력적연구창조료조건.
Objective To screen permethrin human single-chain variable region (scFv) antibody for aims of developing rapid detection kit. Methods Phage display technology was used in this study. Permethrin was solid phase coated on Nunc plate as antigen. Semi-synthetic single-chain variable region of human antibody library technology was applied, and single chain variable region was screened from phage antibody library after 3 rounds "adsorption - elution - amplification" of the selection process. 100 clones were random selected as resistance to permethrin clones , enzyme-linked immunosorbent assay (ELISA), crossreactivity and competitive inhibition experiments were used to validate permethrin binding activity with strong scFv clones from the selected phage antibody clones plasmid. The plasmid was digested with restriction enzyme Sfi Ⅰ / Not Ⅰ and subcloned into pCANTAB5E vector. After transformed into E. coli XL1BIue, the plasmid was identified by restriction enzyme analysis. Results After screening in 100 clones, 18 clones had high ELISA absorbance values ( A value) at 490nm wavelength ( A490nm), then bovine serum albumin (BSA) cross-reactions identified five weak cross-reaction. Combined with the triplicate ELISA and competitive inhibition experiment results, one positive clone was acquired at last. And this clone was subcloned into pCANTAB5E vector and transformed into competent cells XL1-Blue. Conclusion Plasmid fragment was consistent with the purpose, which provided the foundation for further study of its specific affinity.