CAJ | 학술논문

对扩增的Tomlinson I 噬菌体抗体库进行3轮特异性富集,挑取单菌落,采用 ELISA、PCR 及测序比对分析鉴定阳性噬菌体单链抗体( scFv)；以宿主替换的方式将阳性噬菌体 scFv 基因导入 Escherichia coli HB2151中进行可溶性表达,scFv 过柱纯化后,建立针对Cry1F毒素的IC-ELISA 检测方法。以Cry1B、Cry1C、Cry1 Ab、Cry1 Ac 作为竞争抑制物,分析scFv的特异性,以Cry1 F毒素在玉米中的添加回收试验评价检测方法的实用性。结果显示,从第3轮富集库中筛选到了11株具有 Cry1F 毒素结合活性的噬菌体 scFv菌株,经PCR 鉴定都含有目的条带,对其中2株( H1、G9)进行测序,证实为人源化的 scFv。选取 G9号阳性噬菌体scFv进行可溶性表达,纯化后收集到的 scFv 浓度为256μg/ml。建立的 IC-ELISA 检测方法检测灵敏度( IC10)为5.99 ng/ml,抑制中浓度( IC50)为0.410μg/ml,线性检测范围( IC20~ IC80)为0.107~0.713μg/ml。 scFv(G9)对Cry1B、Cry1C具有一定结合活性,交叉反应率分别达到了20.92%和15.59%,但不识别Cry1Ab 和Cry1Ac,交叉反应率均低于0.1%。添加回收试验结果表明,基于scFv(G9)建立的IC-ELISA 检测方法稳定性和重复性都比较好。
대확증적Tomlinson I 서균체항체고진행3륜특이성부집,도취단균락,채용 ELISA、PCR 급측서비대분석감정양성서균체단련항체( scFv)；이숙주체환적방식장양성서균체 scFv 기인도입 Escherichia coli HB2151중진행가용성표체,scFv 과주순화후,건립침대Cry1F독소적IC-ELISA 검측방법。이Cry1B、Cry1C、Cry1 Ab、Cry1 Ac 작위경쟁억제물,분석scFv적특이성,이Cry1 F독소재옥미중적첨가회수시험평개검측방법적실용성。결과현시,종제3륜부집고중사선도료11주구유 Cry1F 독소결합활성적서균체 scFv균주,경PCR 감정도함유목적조대,대기중2주( H1、G9)진행측서,증실위인원화적 scFv。선취 G9호양성서균체scFv진행가용성표체,순화후수집도적 scFv 농도위256μg/ml。건립적 IC-ELISA 검측방법검측령민도( IC10)위5.99 ng/ml,억제중농도( IC50)위0.410μg/ml,선성검측범위( IC20~ IC80)위0.107~0.713μg/ml。 scFv(G9)대Cry1B、Cry1C구유일정결합활성,교차반응솔분별체도료20.92%화15.59%,단불식별Cry1Ab 화Cry1Ac,교차반응솔균저우0.1%。첨가회수시험결과표명,기우scFv(G9)건립적IC-ELISA 검측방법은정성화중복성도비교호。
A large phage antibody library ( Tomlinson I) was employed to generate single-chain fragment variable (scFv) antibody against Cry1F toxin by affinity panning, and single colonies were randomly selected from amplified Tomlinson I after 3 rounds of panning. The positive clones were confirmed by ELISA, PCR, and sequencing. The positive phage scFv gene was introduced into Escherichia coli HB2151 by replacement of host for soluble expression. After elution and purification, protein scFv was achieved. An indirect competitive ELISA ( IC-ELISA ) was then de-veloped for the determination of Cry1F toxin using scFv as antibody. Totally eleven positive clones with distinct nucleotide sequences and intact scFv gene were confirmed to be specific for the Cry1F toxin, among which, H1 and G9 positive clones were the humanized scFv, which were confirmed by DNA sequencing. G9 positive phage was chosen for soluble expression and the concentration of purified scFv reached 256μg/ml. The concentration of Cry1F toxin spiked in corn sample (IC20 ~IC80 ) detected by IC-ELISA ranged from 0. 107 to 0. 713μg/ml, and the median inhibitory concentration ( IC50 ) and detec-tion limit ( IC10 ) were 0. 410 μg/ml and 5. 99 ng/ml, respectively. The cross-reactivities for Cry1B and Cry1C were 20. 92% and 15. 59%, and were less than 0. 1% for Cry1Ab、Cry1Ac. The results suggested that the IC-ELISA developed in this study had a good repeatability and stability.