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	Construction of single chain variable fragment (scfv) and biscfv-alkaline phosphatase fusion protein for detection of bacillus anthracis
	Wang, SH; Zhang, JB; Zhang, ZP; Zhou, YF; Yang, RF; Chen, J; Guo, YC; You, F; Zhang, XE
刊名	Analytical chemistry
	2006-02-15
卷号	78期号:4页码:997-1004
ISSN号	0003-2700
DOI	10.1021/ac0512352
通讯作者	Zhang, xe()
英文摘要	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 (e.v) labeled biscfv-6w10 (biscfv-6w10-eap) in e. coli cells. ne 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 paand 500-1000 bacterial cells in similar to 2 h, while the sensitivity of cy3-labeled protein chip reached 1 pg of pa and 50-100 cells within 2 h.
WOS关键词	NEURAMINIDASE ANTIBODY NC10 ; V-L DOMAIN ; ESCHERICHIA-COLI ; MULTIMERS ; DIABODIES ; PHAGE ; TRIABODIES ; IDENTIFICATION ; IMMUNOGLOBULIN ; SURFACE
WOS研究方向	Chemistry
WOS类目	Chemistry, Analytical
语种	英语
出版者	AMER CHEMICAL SOC
WOS记录号	WOS:000235574300004
内容类型	期刊论文
URI标识	http://www.corc.org.cn/handle/1471x/2375279
专题	武汉病毒研究所
通讯作者	Zhang, XE
作者单位	1.Chinese Acad Sci, Joint Res Grp Analyt Pathogen Microbiol, Wuhan Inst Virol, Wuhan 430071, Peoples R China 2.Chinese Acad Sci, Inst Biophys, Wuhan 430071, Peoples R China 3.State Key Lab Virol, Wuhan 430071, Peoples R China 4.State Key Lab Macromol, Wuhan 430071, Peoples R China 5.Huazhong Agr Univ, Coll Life Sci & Technol, State Key Lab Agr Microbiol, Wuhan 430079, Peoples R China 6.Fujian Agr & Forestry Univ, Coll Life Sci, Fujian 350002, Peoples R China 7.Acad Mil Med Sci, Inst Microbiol & Epidemiol, Beijing, Peoples R China
推荐引用方式 GB/T 7714	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[J]. Analytical chemistry,2006,78(4):997-1004.
APA	Wang, SH.,Zhang, JB.,Zhang, ZP.,Zhou, YF.,Yang, RF.,...&Zhang, XE.(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.
MLA	Wang, SH,et al."Construction of single chain variable fragment (scfv) and biscfv-alkaline phosphatase fusion protein for detection of bacillus anthracis".Analytical chemistry 78.4(2006):997-1004.

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