大容量天然鼠源核糖体展示单链抗体文库的构建与鉴定
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摘要
为了构建大容量、天然鼠源核糖体展示(ribosome display,RD)单链抗体(single-chain fragment variant,scFv)文库。通过提取SPF级小鼠脾脏总RNA,反转录为cDNA,利用引物分别扩增鼠抗体的VH、VL和Cκ基因,采用重叠延伸PCR(SOE-PCR)方法,通过Linker((Gly4Ser)3)相互拼接,构建单链抗体scFv基因文库和核糖体展示基因文库;经连接转化,对所构建基因文库进行菌落PCR鉴定和序列测定分析。所构建核糖体展示基因文库库容量约为5.6×1013,基因片段全长大小约为1 100bp,基因文库序列具有较好的完整性和多样性。本试验成功构建了一个大容量、天然鼠源核糖体展示单链抗体文库,为进一步筛选单链抗体奠定基础。
To construction of high-capacity,naive mouse ribosome display single chain Fv library.Total RNA was extracted from the spleen cell of the specific pathogen free mouse followed RTPCR using random primer.The heavy chain variable region gene(VH),light chain variable region gene(VL)and the kappa chain constant region gene(Cκ)of the immunoglobulin were amplified respectively by PCR.They were linked by a linker encoding(Gly4Ser)3splicing and overlap extension PCR(SOE-PCR)to construct single chain Fv library and ribosome display library.The positive clones were identified by PCR,sequenced after linked and transformed to competent cells.The constructed ribosome display library showed a good integrity and diversity,with a capacity of5.6×1013 and a gene length of about 1 100 bp.This study has constructed a high-capacity,naive mouse ribosome display single chain Fv library successfully,which would play roles in screening scFv.
To construction of high-capacity,naive mouse ribosome display single chain Fv library.Total RNA was extracted from the spleen cell of the specific pathogen free mouse followed RTPCR using random primer.The heavy chain variable region gene(VH),light chain variable region gene(VL)and the kappa chain constant region gene(Cκ)of the immunoglobulin were amplified respectively by PCR.They were linked by a linker encoding(Gly4Ser)3splicing and overlap extension PCR(SOE-PCR)to construct single chain Fv library and ribosome display library.The positive clones were identified by PCR,sequenced after linked and transformed to competent cells.The constructed ribosome display library showed a good integrity and diversity,with a capacity of5.6×1013 and a gene length of about 1 100 bp.This study has constructed a high-capacity,naive mouse ribosome display single chain Fv library successfully,which would play roles in screening scFv.
引文
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[6] Chen S S,Yang Y M,Barankiewicz T J.Selection of IgE-binding aptameric green fluorescent protein(ApGFP)by the ribosome display(RD)platform[J].Biochem Biophys Res Commun,2008,374(3):409-414.
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[8] Sun Y,Ning B,Gao Z,et al.Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library[J].PLoS One,2012,7(3):e33186.
[9] Lee M S,Kwon M H,Kim K H,et al.Selection of scFvs specific for HBV DNA polymerase using ribosome Display[J].J Immunol Methods,2004,284,147-157.
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[12] Mollova S,Retter I,Müller W.Visualising the immune repertoire[J].BMC Systems Biol,2007,1(1):30.
[13] Lamla T,Erdmann V A.Searching sequence space for high-affinity binding peptides using ribosome display[J].J Mol Biol,2003,329(2):381-388.
[14] He M,Taussig M J.Ribosome display:cell-free protein display technology[J].Brief Funct Genomic Proteomic,2002,1:204-212.
[15] Li F,Su P,Lin C,et al.Ribosome display and selection of human anti-placental growth factor scFv derived from ovarian cancer patients[J].Protein Pept Lett,2010,17:585-590.
[2] Nina E,Christopher J.Applications of single-chain variable fragment antibodies in therapeutics and diagnostics[J].Biotechnol Adv,2009,27:502-520.
[3] Hanes J,PlückthunA.In vitro selection and evolution of function proteins by using ribosome display[J].Proc Natl Acad Sci U S A,1997,94(10):4937-4942.
[4] Mattheakis L C,Bhatt R R,Dower W J.An in vitro polysome display system for identifying ligands from very large peptide libraries[J].Proc Natl Acad Sci U S A,1994,91(19):9022-9026.
[5] Zhao X L,Chen W Q,Yang Z H,et al.Selection and affinity maturation of human antibodies against rabies virus from a scFv gene library using ribosome display[J].J Biotechnol,2009,144(4):253-258.
[6] Chen S S,Yang Y M,Barankiewicz T J.Selection of IgE-binding aptameric green fluorescent protein(ApGFP)by the ribosome display(RD)platform[J].Biochem Biophys Res Commun,2008,374(3):409-414.
[7] Li F,Su P P,Lin C,et al.Ribosome display and selection of human anti-placental growth factor scFv derived from ovarian cancer patients[J].Protein Pept Lett,2010,17(5):585-590.
[8] Sun Y,Ning B,Gao Z,et al.Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library[J].PLoS One,2012,7(3):e33186.
[9] Lee M S,Kwon M H,Kim K H,et al.Selection of scFvs specific for HBV DNA polymerase using ribosome Display[J].J Immunol Methods,2004,284,147-157.
[10] He M Y,Taussig M J.Antibody-ribosome-mRNA(ARM)complexes as efficient selection particles for in vitro display and evolution of antibody combining sites[J].Nucleic Acids Res,1997,25,5132-5134.
[11] Levinson H J S,Mudgett-Hunter M.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 U S A,1988,85:5879-5883.
[12] Mollova S,Retter I,Müller W.Visualising the immune repertoire[J].BMC Systems Biol,2007,1(1):30.
[13] Lamla T,Erdmann V A.Searching sequence space for high-affinity binding peptides using ribosome display[J].J Mol Biol,2003,329(2):381-388.
[14] He M,Taussig M J.Ribosome display:cell-free protein display technology[J].Brief Funct Genomic Proteomic,2002,1:204-212.
[15] Li F,Su P,Lin C,et al.Ribosome display and selection of human anti-placental growth factor scFv derived from ovarian cancer patients[J].Protein Pept Lett,2010,17:585-590.