抗癌胚抗原(CEA)纳米抗体的筛选鉴定及双纳米抗体夹心ELISA检测CEA  

Screening and Identification of Anti-CEA Nanobody and Construction of a Double-nanobody ELISA for CEA Detection

作　　者：王新婷 胡倩倩 娄楚 杨天宁 李江伟[1] WANG Xinting;HU Qianqian;LOU Chu;YANG Tianning;LI Jiangwei(Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xinjiang University,Urumqi 830046,China)

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《中国生物工程杂志》2024年第2期48-58,共11页China Biotechnology

摘　　要：目的:筛选能与癌胚抗原(carcinoembryonic antigen,CEA)高亲和力结合的纳米抗体(nanobodies,Nbs),用于今后开发CEA的快速检测方法。方法:使用人CEA蛋白免疫双峰驼,以淋巴细胞cDNA为模板,采用巢式PCR扩增重链抗体可变区(VHH)基因,连接到pMECS载体,电转化TG1大肠杆菌,构建VHH cDNA文库。经辅助噬菌体M13K07感染后生成噬菌体展示文库,采用ELISA固相亲和淘洗方法,富集与CEA结合的VHH克隆,通过胞间质可溶性ELISA(PE-ELISA)筛选与CEA高结合的纳米抗体,并在WK6中进行IPTG诱导表达和亲和纯化。ELISA检测纳米抗体与CEA的结合特异性,竞争ELISA筛选配对纳米抗体构建夹心ELISA。结果:构建获得了库容为2.8×10^(8) cfu的VHH文库,菌液PCR表明VHH插入约为100%,随机挑取第2~3轮淘洗富集的90个克隆进行PE-ELISA检测,结果45个克隆为阳性,序列分析表明,它们编码7个纳米抗体序列。在WK6中,这些纳米抗体以可溶形式表达,经Ni亲和层析获得了纯度接近90%的重组纳米抗体。间接ELISA显示7株纳米抗体均特异结合CEA,其中2株纳米抗体3G2和3F4与CEA具有较高亲和力且呈浓度依赖,即使在高浓度(5μg/mL)下也不与无关蛋白溶菌酶结合,表现出较高特异性。竞争ELISA结果显示,3G2和3F4在结合CEA中没有竞争作用,表明3G2和3F4结合在CEA的不同表位。将3F4作为捕获抗体,3G2作为检测抗体,建立了双纳米抗体夹心ELISA方法(2Nbs-ELISA法),其对溶液中CEA的检测限(limit of detection,LOD)为1.8 ng/mL。结论:采用获得的两株CEA特异纳米抗体建立了夹心ELISA检测方法,对CEA的检测具有更高敏感性和特异性。Objective:To screen nanobodies that bind to carcino-embryonic antigen(CEA)with high affinity for the development of rapid detection methods for CEA in the future.Methods:Using human CEA protein to immunize Bactrian camels,lymphocyte cDNA was used as a template,and nested PCR was used to amplify the variable domain of heavy chain of heavy-chain antibody(VHH)gene,ligated to pMECS vector,and electroporated into TG1 E.coli to construct a VHH cDNA library.After infection with helper phage M13K07,a phage display library was generated,and VHH clones bound to CEA were enriched by biopanning in ELISA,nanobodies with high binding to CEA were screened by periplasmic soluble ELISA(PE-ELISA),and IPTG-induced expression and affinity purification were performed in WK6.The binding specificity of the nanobodies to CEA was by measured by ELISA,and the epitope overlap was detected by competitive ELISA to screen paired nanobodies for the construction of sandwich ELISAs.Results:A VHH library with a size of 2.8×10^(8) cfu was constructed.Colony PCR showed that VHH insertion was about 100%.A total of 90 clones enriched in the 2nd-3rd round were randomly selected for PE-ELISA detection,and 45 clones were positive.Sequence analysis showed that they encoded 7 nanobody sequences.In WK6,these nanobodies are expressed in a soluble form,and recombinant nanobodies with a purity close to 90% are obtained by Ni affinity chromatography.Indirect ELISA showed that all 7 Nbs specifically bound CEA,and 2 nanobodies 3G2 and 3F4 had high affinity and concentration-dependent binding to CEA,and did not bind to the unrelated protein lysozyme even at high concentrations(5μg/mL),showing high specificity.Competitive ELISA results showed that 3G2 and 3F4 did not compete in binding to CEA,indicating that 3G2 and 3F4 bind to different epitopes of CEA antigens.Using 3F4 as the capture antibody and 3G2 as the detection antibody,the double nanobody sandwich ELISA method(referred to as 2Nbs-ELISA)was established,and its limit of detection(LOD)for CEA in solution

关 键 词：CEA 纳米抗体 噬菌体展示 表位归类 双纳米抗体夹心ELISA 

分 类 号：Q816[生物学—生物工程]