伤寒沙门菌Mig-14蛋白部分密码子优化后的表达及多克隆抗体制备
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摘要
通过原核表达Mig-14蛋白,制备相应多克隆抗体,用于后续Mig-14分子功能研究,为探寻Mig-14拮抗PB分子机制奠定基础.经密码子偏爱性分析显示mig-14基因含有8%的稀有密码子,综合考虑密码子偏好性、GC含量、限制性酶切位点等影响因素对密码子进行优化,重新合成mig-14基因,PCR获得周质空间部分,克隆至表达载体pET22b(+),并转入大肠埃希菌JM109中表达,KCl染色后切胶纯化的蛋白作为抗原免疫家兔,制备多克隆抗体.Mig-14蛋白多克隆抗体的制备为后续免疫共沉淀研究Mig-14的作用蛋白奠定了基础.
In order to explore the molecular function of Mig-14,we try to express Mig-14and prepare the anti-Mig-14polyclonal antibody.Codon preference analysis showed mig-14gene contains 8%of rare codons.In this study,mig-14was synthesized by considering the codon bias,GC cotent,restriction site et al.mig-14in the periplasm which acquired by PCR using synthesized mig-14as template was cloned into pET22b(+)and expressed in E.coli JM109.The recombinant protein Mig-14-p(Mig-14in the periplasm)was purified by cutting the gel slices with KCl stain and was used as antigen to prepare polyclonal antibody.This anti-Mig-14-p polyclonal antibody can be used to research the role of mig-14in Salmonella enterica serovar Typhi.
In order to explore the molecular function of Mig-14,we try to express Mig-14and prepare the anti-Mig-14polyclonal antibody.Codon preference analysis showed mig-14gene contains 8%of rare codons.In this study,mig-14was synthesized by considering the codon bias,GC cotent,restriction site et al.mig-14in the periplasm which acquired by PCR using synthesized mig-14as template was cloned into pET22b(+)and expressed in E.coli JM109.The recombinant protein Mig-14-p(Mig-14in the periplasm)was purified by cutting the gel slices with KCl stain and was used as antigen to prepare polyclonal antibody.This anti-Mig-14-p polyclonal antibody can be used to research the role of mig-14in Salmonella enterica serovar Typhi.
引文
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[12]姜静,孙其飞,陈勇,等.切胶免疫制备腮腺液高丰度蛋白多克隆抗体[J].北京口腔医学,2007,15(5):254-256.
[2]Valdivia R H,Cirillo D M,Lee A K,et a1.mig-14is a Horizontally acquired,host-induced gene required for salmonella enterica lethal infection in the murine model of typhoid fever infect immun[J].Infection and Immunity,2000,68(12):7126-7131.
[3]Valdivia R H,Falkow S.Fluorescence-based isolation of bacterial genes expressed within host cells[J].Science,1997,277(5334):2007-2011.
[4]Gallegos M T,Schleif R,Bairoch A,et a1.Arac/XylS family of transcriptional regulators[J].Microbiol Mol Biol Rev,1997,61(4):393-410.
[5]Jayaraj S,Reid R,Santi D V.GeMS:an advanced software package for designing synthetic genes[J].Nucleic Acids Res,2005,33(9):3011-3016.
[6]鞠爱萍,吴亮,沈进,等.弓形虫棒状体蛋白18的原核表达及鉴定[J].江苏大学学报:医学版,2013,23(3):207-211.
[7]Deleage G,Combet C,Blanchet C,et al.ANTHEPROT:an integrated protein sequence analysis software with client/server capabilities[J].Comput Biol Med,2001,31:259-267.
[8]Parker J M,Guo D,Hodges R S.New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data:correlation of predicted surface residues with antigenicity and X-ray-derived accessible sites[J].Biochemistry,1986,25:5425-5432.
[9]Lithwick G,Margalit H.Hierarchy of sequence-dependent features associated with prokaryotic translation[J],Genome Res,2003,13(12):2665-2673.
[10]Angov E.Codon usage:Nature's roadmap to expression and folding of proteins[J],Biotechnol J,2011,6(6):650-659.
[11]Tuller T,Waldman Y Y,Kupiec M,et al.Translation efficiency is determined by both codon bias and folding energy[J],Proc Natl Acad Sci U S A,2010,107(8):3645-3650.
[12]姜静,孙其飞,陈勇,等.切胶免疫制备腮腺液高丰度蛋白多克隆抗体[J].北京口腔医学,2007,15(5):254-256.