基于磁珠和双抗夹心免疫分析的Bt Cry1Ac毒素单链抗体筛选与鉴定

Selection and Identification of Anti-Cry1Ac scFv from a Phage Display Antibody Library by Magnetic Beads and Sandwich ELISA Immunoassay

【目的】通过设计并优化生物素-亲和素标记磁珠筛选策略,建立针对Cry1Ac毒素的新型双抗夹心ELISA检测方法,为研究灵敏、快速的Bt毒素的检测方法建立一种新的检测模式。【方法】采用磁珠液相正负筛选方法,经过4轮筛选后,对每轮筛选后抗Cry1Ac毒素特异性结合噬菌体抗体的富集效果进行鉴定,通过单克隆ELISA方法从第4轮筛选得到的次级库中获得特异性结合Cry1Ac的阳性克隆,并对其进行PCR鉴定和DNA测序,确定有完整插入的scFv片段后进行后期相关试验。将相对结合活性最好的阳性克隆scFv-A12替换宿主菌HB2151中进行可溶性诱导表达并纯化。利用纯化后的scFv-A12作为"捕获抗体",Anti-Cry1Ac兔多克隆抗体作为"检测抗体",建立针对Cry1Ac的双抗夹心ELISA检测方法。【结果】以生物素化的Cry1Ac蛋白为包被原,利用噬菌体抗体库对其进行了4轮液相筛选。结果显示,相对产出率随着筛选轮数的增加而提高,第4轮较第1轮提高了42倍;单克隆噬菌体ELISA鉴定抗Cry1Ac噬菌体抗体,以试验孔OD450/阴性孔OD450大于2.1为阳性标准,从第4轮筛选后的培养皿中随机挑选了300个菌落,经单克隆ELISA鉴定出6个阳性克隆,分别命名为hsCry1Ac-C5、hsCry1Ac-D8、hsCry1Ac-C12、hsCry1Ac-A12、hsCry1Ac-F9和hsCry1Ac-F4。其中,hsCry1Ac-A12具有相对较高的亲和力。对上述6个阳性克隆进行菌液PCR检测发现,在935 bp处均有明显的目的条带,通过对其进行测序和氨基酸序列比对,最终得到4株不同氨基酸序列的阳性克隆。hsCry1Ac-A12在成功替换宿主菌HB2151后,经1 mmol·L-1 IPTG诱导表达,于30℃条件下培养过夜。其表达产物经His Trap HP镍亲和柱纯化后SDS-PAGE检测,hsCry1Ac-A12蛋白分子量约为30 kD。通过方阵滴定对抗体浓度进行优化后,利用单链抗体为"捕获抗体",多抗为"检测抗体",建立了Cry1Ac双抗夹心ELISA检测方法。在1.25—2.43μg·mL-1线性检测范围内,线性回归方程为Y=0.2522X+1.2832(R2=0.9564),检测限为1.08 ng·mL-1。【结论】利用磁珠液相筛选方法,建立了针对Cry1Ac毒素的双抗夹心ELISA检测方法,为快速、准确检测转基因食品中Cry1Ac提供了一种新的检测模式。

[Objective] By using the biotin-avidin labeled magnetic bead screening strategy, this paper aims at establishing a new type of double-antibody sandwich ELISA detection method for CrylAc toxin, so as to build a new detection mode to analyze the sensitive and rapid Bt toxin detection method. [Method] The enrichment ofCrylAc phage-scFvs was investigated after each round of panning （total four rounds） by using magnetic beads with subtractive panning. Positive clones as specific binding to CrylAc were selected from the secondary library, which was gained from the fourth round of panning by the method of monoclonal ELISA, and after the PCR identification and DNA sequencing of these positive clones, post-relevant tests were conducted when confirming the complete insertion of the scFvs. Meanwhile, the positive clone, scFv-A12, with relatively best binding activity, was chosen to replace the host strain HB2151 for the purpose of induced-soluble expression and purification. And the purified scFv-A12 was regarded as ＂capture antibody＂, the anti-CrylAc rabbit polyclonal antibody as ＂detection antibody＂, thus to establish the detection method of double sandwich ELISA for CrylAc. [Result] With the coating antigen of biotinylated CrylAc protein, four rounds of panning were conducted by using the antibody phage library. The results show that, along with the increase of panning rotmds, the relative productivity was enhanced, and the data of the fourth round was increased by 42 times compared with that of the first round. The positive standard for monoclonal phage ELISA to identify anti-CrylAc phage antibody was decided by the figure gained from dividing test hole OD450 by negative hole OD450, which should be over 2.1. Three hundred bacterial clones were selected randomly from the Petri dish after the fourth panning, in which six positive clones were identified by monoclonal ELISA, being respectively named as hsCrylAc-C5, hsCrylAc-D8, hsCrylAc-C12, hsCrylAc-A12, hsCrylAc-F9 and hsCrylAc-F4. Among them, hsCrylAc-A12 had a relatively high affinity. After the bacteria PCR detection upon above six positive clones, the results show that the target band was obvious in the place of 935 bp, and with the sequencing and amino acid sequence alignment on the target band, four positive clones with different amino acid sequences were gained. After successfully replaced the host strain HB2151, hsCrylAc-A12 was cultured at 30℃ with 1 mmol·L-1 IPTG for inducing expression. Through the His Trap HP nickel affinity column purification and the SDS-PAGE identification of its expression product, the molecular weight of hsCrylAc-A12 protein was approximately 30 kD. After optimizing the antibody concentration by titration test and taking advantage of the single chain antibody as ＂capture antibody＂, polyvalent antibody as ＂detection antibody＂, the detection method of double antibody sandwich ELISA was established. Within the linear range of 1.25-2.43 gg-mL-1, the linear regression equation was Y=0.2522X＋1.2832（R2=0.9564）, with the limit of detection 1.08 ng.mL-1. [Conclusion] This paper adopted the method of magnetic beads with subtractive panning, the detection method of double-antibody sandwich ELISA for CrylAc toxin was established after the expression and purification of the selected clone A12 with best binding activity which provide a new detection mode for detecting the CrylAc contained in GMF rapidly and accurately.