山羊痘病毒毒力因子KLP2的表达及多克隆抗体的制备
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摘要
为了研究羊痘病毒(capripox virus),制备其毒力因子KLP2的多克隆抗体,试验采用基因同源重组的方法分别构建了羊痘病毒两个结构域(KLP2-1和KLP2-2)的原核表达载体并进行测序鉴定。提取质粒后转化大肠杆菌BL21感受态细胞并用IPTG诱导表达,目的蛋白采用SDS-PAGE和Western blotting检测,并用KCl染色切胶纯化,纯化蛋白加弗氏佐剂免疫家兔,制备的抗体用Western blotting和ELISA检测评价。结果表明,试验成功构建了表达载体p ET42b-KLP2-1和p ET42b-KLP2-2,测序结果显示KLP2-1和KLP2-2基因大小分别为657和984 bp,分别表达了约27和38 ku的蛋白,与理论值相符。切胶纯化的蛋白浓度在1~2 mg/m L之间,制备的抗体用Western blotting检测发现,蛋白有明显的特异条带,ELISA检测抗体的效价均为1∶512。说明试验成功表达了KLP2-1、KLP2-2蛋白,制备了相应的多克隆抗体。
In order to study capripox virus,and prepare polyclonal antibody of its virulence factor KLP2,the prokaryotic expression vector of two structure domains( KLP2-1 and KLP2-2) were built with the method of homologous recombination genes and identified by sequencing. The plasmid were transformed into E. coli BL21 and inducing expressed by IPTG,objective protein were tested by SDS-PAGE and Western blotting,and purified by KCl dyeing rubber cutting.The rabbits were immuned with freund's adjuvant,the prepared antibody were evaluated by Western blotting and ELISA test. The results showed that the expression vector of p ET42b-KLP2-1 and p ET42b-KLP2-2 were built successfully,and KLP2-1 and KLP2-2 genes were 657 and 984 bp,and expressed about 27 and 38 ku protein,respectively,it was consistent with theories value. The concentrations of rubber cutting purified protein was 1 to 2 mg/m L. The antibody showed obvious specific bands detecting by Western blotting,and its titer were 1 ∶ 512 detecting by ELISA. This result indicated that KLP2-1 and KLP2-2 proteins were expressed successfully,and the corresponding polyclonal antibody were prepared.
In order to study capripox virus,and prepare polyclonal antibody of its virulence factor KLP2,the prokaryotic expression vector of two structure domains( KLP2-1 and KLP2-2) were built with the method of homologous recombination genes and identified by sequencing. The plasmid were transformed into E. coli BL21 and inducing expressed by IPTG,objective protein were tested by SDS-PAGE and Western blotting,and purified by KCl dyeing rubber cutting.The rabbits were immuned with freund's adjuvant,the prepared antibody were evaluated by Western blotting and ELISA test. The results showed that the expression vector of p ET42b-KLP2-1 and p ET42b-KLP2-2 were built successfully,and KLP2-1 and KLP2-2 genes were 657 and 984 bp,and expressed about 27 and 38 ku protein,respectively,it was consistent with theories value. The concentrations of rubber cutting purified protein was 1 to 2 mg/m L. The antibody showed obvious specific bands detecting by Western blotting,and its titer were 1 ∶ 512 detecting by ELISA. This result indicated that KLP2-1 and KLP2-2 proteins were expressed successfully,and the corresponding polyclonal antibody were prepared.
引文
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[21]冯小黎.重组包涵体蛋白质的折叠复性[J].生物化学与生物物理进展,2001,28(4):482-485.
[22]黄庶识,卢明倩,李冰.重组大肠杆菌表达可溶性蛋白和包涵体过程的拉曼光谱实时分析[J].中国激光,2014,41(12):1-9.
[2]赵志荀,吴国华,颜新敏,等.羊痘病毒及其疫苗研究进展[J].动物医学进展,2010,31(4):77-81.
[3]Tulman E R,Afonso C L,Lu Z,et al.The genomes of sheeppox and goatpox viruses[J].Journal of Virology,2002,76(12):6054-6061.
[4]Okamoto T,Campbell S,Mehat N,et al.Sheeppox virus SPPV14 encodes a Bcl-2-like cell death inhibitor that countersa distinct set of mammalian proapoptotic proteins[J].J Virol,2012,86(21):11501-11511.
[5]Aromolaran K A,Benzow K A,Koob M D,et al.The Kelch-like protein 1 modulates P/Q-type calcium current density[J].Neuroscience,2007,145(3):841-850.
[6]陈轶霞.羊痘病毒基因组及其功能研究概况[J].中国兽医学报,2015,35(2):340-344.
[7]Shchelkunov S,Totmenin A,Kolosova I,et al.Speciesspecific differences in organization of orthopoxvirus Kelch-like proteins[J].Virus Genes,2002,24(2):157-162.
[8]Adams J C,Kelso R,Cooley L,et al.The Kelch repeat super family of proteins:Propellers of cell function[J].Trends Cell Biol,2000,10:17-24.
[9]Stogios P J,Downs G S,Jauhal J J S,et al.Sequence and structural analysis of BTB domain proteins[J].Genome Biology,2005,6(10):R82.
[10]Pires de Miranda M,Reading P C,Smith G L.The vaccinia virus Kelch-like protein C2L affects calcium-independent adhesion to the extracellular matrix and inflammation in a murine intradermal model[J].Gen Virol,2003,84:2459-2471.
[11]Zhao C,Sun Y,Zhao Y,et al.Immunogenicity of amulti-epitope DNA vaccine against hangta virus[J].Hum Vaccin IM-Muonther,2012,8(2):208-215.
[12]江楠,文明,许乐仁,等.羊痘病毒致宿主细胞病变研究进展[J].动物医学进展,2012,33:75-80.
[13]鲍长磊,何亚鹏,张琪,等,山羊痘病毒疫苗株O基因的克隆、序列分析及原核表达[J].西北农业学报,2016,25(4):502-507.
[14]贾慧娜,罗海玲.多克隆抗体制备方法的研究进展[J].中国草食动物科学,2012,S1:66-69.
[15]吴锦艳,田宏,陈妍,等.基于山羊痘病毒P32蛋白ELISA检测方法的建立[J].中国兽医学报,2014,34(12):1906-1911.
[16]颜新敏,吴国华,李健,等.羊痘在中国的流行现状分析[J].中国农学通报,2010,26(24):6-9.
[17]Li Y W,Hu J J.Identification of four goatpox virus outbreaks in Xinjiang of China in 2010[J].IEEE,2011,2:1003-1006.
[18]芦晓立.羊痘病毒P32基因与羊CD58基因共表达质粒的构建及其表达产物免疫活性分析[D].兰州:甘肃农业大学,2010.
[19]陈轶霞,刘俊林,王明明,等.羊痘病毒多表位核酸疫苗的构建及表达[J].中国兽医学报,2013,33(8):1196-1200.
[20]冯延叶.变性蛋白复性装置研制及包涵体蛋白变性和复性技术研究[D].上海:华东理工大学,2013.
[21]冯小黎.重组包涵体蛋白质的折叠复性[J].生物化学与生物物理进展,2001,28(4):482-485.
[22]黄庶识,卢明倩,李冰.重组大肠杆菌表达可溶性蛋白和包涵体过程的拉曼光谱实时分析[J].中国激光,2014,41(12):1-9.