Wang Shi-Hua, Zhang Ji-Bin, Zhang Zhi-Ping, Zhou Ya-Feng, Yang Rui-Fu, Chen Jia, Guo Yong-Chao, You Fan, Zhang Xian-En
Joint Research Group of Analytical Pathogen Microbiology, Wuhan Institute of Virology and Institute of Biophysics, Chinese Academy of Sciences, Wuhan 430071, China.
Anal Chem. 2006 Feb 15;78(4):997-1004. doi: 10.1021/ac0512352.
This paper describes an attempt for convenient and sensitive detection of Bacillus anthracis with single chain variable fragment (scFv)-based protein chip. Phage display technology was employed to generate scFv by using the protective antigen (PA) of B. anthracis for immunization. V(H) and V(L) genes of the scFv were amplified separately by reverse transcriptase-PCR from mRNA of immunized mice and then assembled into scFv gene with a linker DNA sequence. The scFv gene was inserted into a phagemid vector pCANTAB-5E and then transformed into Escherichia coli TG1 to yield recombinant phages after infection with helper phage M13KO7. After six rounds of panning with PA, the phage clones displaying scFv fragments of the antibody were selected by ELISA. One phage clone scFv-6w10 showing the strongest positive signal in ELISA was selected. To enhance the affinity of the scFv-6w10, a recombinant bivalent single-chain Fv antibody (biscFv-6w10) directed against PA was constructed and tested in functional assays. The affinity of the biscFv-6w10 was much higher than that of scFv-6w10 and reached 6.5 x 10(9) M(-1). An expression system was constructed for the production of E. coli alkaline phosphatase (EAP) labeled biscFv-6w10 (biscFv-6w10-EAP) in E. coli cells. The expressed fusion protein retained both antigen-specific binding and enzymatic activity and thus directly served as an enzyme-labeled antibody. Detections of PA and bacterial cells of B. anthracis using biscFv-6w10-EAP and Cy3-labeled biscFv-6w10 were performed on a protein chip. The fusion protein (biscFv-6w10-EAP) chip could detect 10 pg of PA and 500-1000 bacterial cells in approximately 2 h, while the sensitivity of Cy3-labeled protein chip reached 1 pg of PA and 50-100 cells within 2 h.
本文描述了一种利用基于单链可变片段（scFv）的蛋白质芯片方便、灵敏地检测炭疽芽孢杆菌的尝试。采用噬菌体展示技术，用炭疽芽孢杆菌的保护性抗原（PA）免疫来产生scFv。通过逆转录聚合酶链反应（RT-PCR）从免疫小鼠的mRNA中分别扩增scFv的重链可变区（V(H)）和轻链可变区（V(L)）基因，然后用连接子DNA序列组装成scFv基因。将scFv基因插入噬菌粒载体pCANTAB-5E，再用辅助噬菌体M13KO7感染后转化到大肠杆菌TG1中以产生重组噬菌体。用PA进行六轮淘选后通过酶联免疫吸附测定（ELISA）选择展示抗体scFv片段的噬菌体克隆。选择了在ELISA中显示最强阳性信号的一个噬菌体克隆scFv-6w10。为提高scFv-6w10的亲和力，构建了针对PA的重组二价单链Fv抗体（biscFv-6w10）并进行功能测定。biscFv-6w10的亲和力远高于scFv-6w10，达到6.5×10⁹ M⁻¹。构建了一个表达系统用于在大肠杆菌细胞中生产大肠杆菌碱性磷酸酶（EAP）标记的biscFv-6w10（biscFv-6w10-EAP）。表达的融合蛋白保留了抗原特异性结合和酶活性，因此直接用作酶标记抗体。利用biscFv-6w10-EAP和Cy3标记的biscFv-6w10在蛋白质芯片上对PA和炭疽芽孢杆菌的细菌细胞进行检测。融合蛋白（biscFv-6w10-EAP）芯片在约2小时内可检测到10 pg的PA和500 - 1000个细菌细胞，而Cy3标记的蛋白质芯片在2小时内的灵敏度达到1 pg的PA和50 - 100个细胞。
