淋巴细胞功能相关的抗基因-1(LFA-1)是淋巴细胞和其他白细胞上发现的一种描述良好的整合素,已知在白血病和淋巴瘤中过度表达。这种受体在T细胞活化等免疫反应中起着重要作用。白细胞-细胞相互作用,以及白细胞种群的贩运。随后,LFA-1的结合剂成为癌症和自身免疫疗法的潜在候选者.这项研究使用噬菌体展示技术来构建和表征针对LFA-1的高亲和力单链片段可变(scFv)抗体。在表达式之后,净化,透析,和重组LFA-1蛋白的浓度,四只雌性BALB/c小鼠免疫,提取脾细胞的mRNA,并合成了cDNA。通过使用SOEingPCR通过72-bp接头连接扩增的VH/Vκ片段来构建scFv文库。接下来,将scFv基因片段克隆到pComb-3XSS噬菌粒载体中;因此,开发了噬菌体文库。选择过程涉及三轮噬菌体生物淘选,多克隆,和单克隆噬菌体ELISA。通过SDS-PAGE在阳性克隆中选择并表征AF17,西方印迹,间接ELISA,和计算机内分析。研究结果表明成功构建了针对LFA-1的高亲和力scFv文库。通过SDS-PAGE证实了AF17生产的准确性及其与LFA-1的结合能力,蛋白质印迹,和ELISA。这项研究强调了针对LFA-1的高亲和力AF17用于靶向T淋巴细胞用于治疗目的的潜在应用。
Lymphocyte function-associated antigene-1 (LFA-1) is a well-described integrin found on lymphocytes and other leukocytes, which is known to be overexpressed in leukemias and lymphomas. This receptor plays a significant role in immune responses such as T-cell activation, leukocyte cell-cell interactions, and trafficking of leukocyte populations. Subsequently, binders of LFA-1 emerge as potential candidates for cancer and autoimmune therapy. This study used the phage display technique to construct and characterize a high-affinity single-chain fragment variable (scFv) antibody against LFA-1. After expression, purification, dialysis, and concentration of the recombinant LFA-1 protein, four female BALB/c mice were immunized, splenocyte\'s mRNA was extracted, and cDNA was synthesized. A scFv library was constructed by linking the amplified VH/Vκ fragments through a 72-bp linker using SOEing PCR. Next, the scFv gene fragments were cloned into the pComb-3XSS phagemid vector; thus, the phage library was developed. The selection process involved three rounds of phage-bio-panning, polyclonal, and monoclonal phage ELISA. AF17 was chosen and characterized among the positive clones through SDS-PAGE, Western blotting, indirect ELISA, and in-silico analyses. The results of the study showed the successful construction of a high-affinity scFv library against LFA-1. The accuracy of the AF17 production and its ability to bind to the LFA-1 were confirmed through SDS-PAGE, Western blot, and ELISA. This study highlights the potential application of the high-affinity AF17 against LFA-1 for targeting T lymphocytes for therapeutic purposes.