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Construction of single chain variable fragment (ScFv) and BiscFv-alkaline phosphatase fusion protein for detection of Bacillus anthracis.
Anal Chem. 2006 Feb 15; 78(4):997-1004.AC

Abstract

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.

Authors+Show Affiliations

Joint Research Group of Analytical Pathogen Microbiology, Wuhan Institute of Virology and Institute of Biophysics, Chinese Academy of Sciences, Wuhan 430071, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16478089

Citation

Wang, Shi-Hua, et al. "Construction of Single Chain Variable Fragment (ScFv) and BiscFv-alkaline Phosphatase Fusion Protein for Detection of Bacillus Anthracis." Analytical Chemistry, vol. 78, no. 4, 2006, pp. 997-1004.
Wang SH, Zhang JB, Zhang ZP, et al. Construction of single chain variable fragment (ScFv) and BiscFv-alkaline phosphatase fusion protein for detection of Bacillus anthracis. Anal Chem. 2006;78(4):997-1004.
Wang, S. H., Zhang, J. B., Zhang, Z. P., Zhou, Y. F., Yang, R. F., Chen, J., Guo, Y. C., You, F., & Zhang, X. E. (2006). Construction of single chain variable fragment (ScFv) and BiscFv-alkaline phosphatase fusion protein for detection of Bacillus anthracis. Analytical Chemistry, 78(4), 997-1004.
Wang SH, et al. Construction of Single Chain Variable Fragment (ScFv) and BiscFv-alkaline Phosphatase Fusion Protein for Detection of Bacillus Anthracis. Anal Chem. 2006 Feb 15;78(4):997-1004. PubMed PMID: 16478089.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Construction of single chain variable fragment (ScFv) and BiscFv-alkaline phosphatase fusion protein for detection of Bacillus anthracis. AU - Wang,Shi-Hua, AU - Zhang,Ji-Bin, AU - Zhang,Zhi-Ping, AU - Zhou,Ya-Feng, AU - Yang,Rui-Fu, AU - Chen,Jia, AU - Guo,Yong-Chao, AU - You,Fan, AU - Zhang,Xian-En, PY - 2006/2/16/pubmed PY - 2007/3/30/medline PY - 2006/2/16/entrez SP - 997 EP - 1004 JF - Analytical chemistry JO - Anal. Chem. VL - 78 IS - 4 N2 - 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. SN - 0003-2700 UR - https://www.unboundmedicine.com/medline/citation/16478089/Construction_of_single_chain_variable_fragment__ScFv__and_BiscFv_alkaline_phosphatase_fusion_protein_for_detection_of_Bacillus_anthracis_ L2 - https://dx.doi.org/10.1021/ac0512352 DB - PRIME DP - Unbound Medicine ER -