鸡源性抗FB_1噬菌体单链抗体库的构建与鉴定
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摘要
目的构建免疫鸡源性噬菌体单链抗体基因文库,筛选鉴定抗伏马菌素B1(fumonisin B1,FB1)的单链抗体。方法制备抗原免疫来亨母鸡,提取脾脏RNA反转录后通过PCR扩增获得单链抗体编码基因,构建单链抗体基因文库,噬菌体展示技术筛选抗FB1单链抗体,并利用表达ELISA检测抗体的亲和力。结果构建的单链抗体库的质量较高,通过噬菌体展示淘选和ELISA鉴定获得5个抗FB1的单链抗体。结论成功构建鸡源性抗FB1噬菌体单链抗体库,并鉴定获得抗FB1的单链抗体,表明鸡源性噬菌体单链抗体库可用于分离FB1等小分子半抗原的特异单链抗体。
To construct a phage-displayed single-chain variable fragment(sc Fv) antibody library and screen chicken-derived sc Fv antibodies against fumonisin B1(FB1), Leghorn chickens were immunized with prepared antigen. Total RNA was extracted from spleen and sc Fv coding genes were reverse-transcripted and amplified.Then, FB1-specific antibodies were isolated by phage display technology from the library. The selected sc Fv genes were induced in Escherichia coli to express proteins, and the protein binding capacities were determined by enzyme-linked immunosorbent assay(ELISA). The results showed that the constructed sc Fv antibody library had a good positive ratio, and five anti-FB1 sc Fv antibodies were obtained by phage-display panning and ELISA identification. In conclusion, a phage-displayed sc Fv antibody library is successfully constructed and chicken sc Fv antibodies against FB1 are selected, indicating that chicken-derived phage-displayed sc Fv antibody library could be utilized to isolate specific sc Fv antibodies against small molecules(haptens).
To construct a phage-displayed single-chain variable fragment(sc Fv) antibody library and screen chicken-derived sc Fv antibodies against fumonisin B1(FB1), Leghorn chickens were immunized with prepared antigen. Total RNA was extracted from spleen and sc Fv coding genes were reverse-transcripted and amplified.Then, FB1-specific antibodies were isolated by phage display technology from the library. The selected sc Fv genes were induced in Escherichia coli to express proteins, and the protein binding capacities were determined by enzyme-linked immunosorbent assay(ELISA). The results showed that the constructed sc Fv antibody library had a good positive ratio, and five anti-FB1 sc Fv antibodies were obtained by phage-display panning and ELISA identification. In conclusion, a phage-displayed sc Fv antibody library is successfully constructed and chicken sc Fv antibodies against FB1 are selected, indicating that chicken-derived phage-displayed sc Fv antibody library could be utilized to isolate specific sc Fv antibodies against small molecules(haptens).
引文
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[2]Yazar S,Omurtag GZ.Fumonisins,trichothecenes and zearalenone in cereals[J].Int J Mol Sci,2008,9(11):2062-2090.
[3]Wang JH,Zhang JB,Li HP,et al.Pathogenicity of Fusarium temperatum isolated from maize in China[J].JPhytopath,2014,162(3):147-157.
[4]Wang Y,Liu S,Zheng H,et al.T-2 toxin,zearalenone and fumonisin B1in feedstuffs from China[J].Food Addit Contam Part B Surveill,2013,6(2):116-122.
[5]Han Z,Ren Y,Liu X,et al.A reliable isotope dilution method for simultaneous determination of fumonisins B1,B2and B3in traditional Chinese medicines by ultra-highperformance liquid chromatography-tandem mass spectrometry[J].J Sep Sci,2010,33(17/18):2723-2733.
[6]Binder E.Managing the risk of mycotoxins in modern feed production[J].Ani Feed Sci Technol,2007,133(1/2):149-166.
[7]Turner NW,Subrahmanyam S,Piletsky SA.Analytical methods for determination of mycotoxins:a review[J].Anal Chim Acta,2009,632(2):168-180.
[8]Liu JL,Hu ZQ,Xing S,et al.Attainment of 15-fold higher affinity of a Fusarium-specific single-chain antibody by directed molecular evolution coupled to phage display[J].Mol Biotechnol,2012,52(2):111-122.
[9]Hu ZQ,Li HP,Zhang JB,et al.A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains[J].Anal Chim Acta,2013,764:84-92.
[10]Xue S,Li HP,Zhang JB,et al.Chicken single-chain antibody fused to alkaline phosphatase detects Aspergillus pathogens and their presence in natural samples by direct sandwich enzyme-linked immunosorbent assay[J].Anal Chem,2013,85(22):10992-10999.
[11]Azcona Olivera JI,Abouzied MM,Plattner RD,et al.Generation of antibodies reactive with fumonisins B1,B2,and B3by using cholera toxin as the carrier-adjuvant[J].Appl Environ Microbiol,1992,58(1):169-173.
[12]Zou L,Xu Y,Li Y,et al.Development of a single-chain variable fragment antibody-based enzyme-linked immunosorbent assay for determination of fumonisin B1in corn samples[J].J Sci Food Agric,2014,94(9):1865-1871.
[13]Hu ZQ,Li HP,Wu P,et al.An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay[J].Anal Chim Acta,2015,867:74-82.
[14]Reynaud CA,Anquez V,Grimal H,et al.Ahyperconversion mechanism generates the chicken light chain preimmune repertoire[J].Cell,1987,48(3):379-388.
[15]Reynaud CA,Dahan A,Anquez V,et al.Somatic hyperconversion diversifies the single VHgene of the chicken with a high incidence in the D region[J].Cell,1989,59(1):171-183.
[16]Mc Cormack WT,Tjoelker LW,Thompson CB.Immunoglobulin gene diversification by gene conversion[J].Prog Nucleic Acid Res Mol Biol,1993,45:27-45.
[17]Hu ZQ,Liu JL,Li HP,et al.Generation of a highly reactive chicken-derived single-chain variable fragment against Fusarium verticillioides by phage display[J].Int J Mol Sci,2012,13(6):7038-7056.
[18]Huston JS,Levinson D,Mudgett Hunter M,et al.Protein engineering of antibody binding sites:recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli[J].Proc Natl Acad Sci USA,1988,85(16):5879-5883.
[19]Smith GP.Filamentous fusion phage:novel expression vectors that display cloned antigens on the virion surface[J].Science,1985,228(4705):1315-1317.
[20]Mc Cafferty J,Griffiths AD,Winter G,et al.Phage antibodies:filamentous phage displaying antibody variable domains[J].Nature,1990,348(6301):552-554.
[21]Schoonbroodt S,Steukers M,Viswanathan M,et al.Engineering antibody heavy chain CDR3 to create a phage display Fab library rich in antibodies that bind charged carbohydrates[J].J Immunol,2008,181(9):6213-6221.
[22]Fukuda I,Kojoh K,Tabata N,et al.In vitro evolution of single-chain antibodies using m RNA display[J].Nucleic Acids Res,2006,34(19):e127.