活化蛋白激酶C受体1影响柔嫩艾美耳球虫子孢子入侵细胞的初步研究
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摘要
在前期实验中,本实验室利用i TRAQ技术研究了柔嫩艾美耳球虫子孢子感染宿主细胞后蛋白质组学的变化,获得了一批与子孢子入侵相关的宿主蛋白。本研究以鸡肌肉的c DNA为模板,对其中活化蛋白激酶C受体1(receptor for activated C kinase1,RACK1)进行克隆、表达及纯化,结果显示,成功获得RACK1-GST融合蛋白,并作为免疫原制备兔抗RACK1-GST多克隆抗体;利用RT-q PCR和Western blot检测子孢子感染DF-1细胞过程中RACK1的m RNA转录水平和蛋白表达水平。结果显示,子孢子感染细胞24~72 h时,细胞RACK1的转录水平和蛋白表达水平均明显提高;利用抗体抑制入侵试验检测RACK1多抗对子孢子入侵细胞的影响,结果显示,100μg/m L和200μg/m L RACK1-GST抗体作用细胞后对子孢子的入侵影响不大,而300μg/m L和400μg/m L作用细胞后能明显促进子孢子的入侵。以上结果表明,宿主RACK1对球虫子孢子入侵细胞起负调控的作用,但具体机制尚需深入研究。
At present, research on the mechanism of Eimeria invading host cells has mainly focused on the parasitic proteins and relevant host genes have been rarely studied. In our previous experiment, i TRAQ technology was used to study the proteomics changes in host cells after infection with Eimeria tenella sporozoites and a number of host proteins related to Eimeria invasion were obtained. In the present study, one of those host proteins, chicken receptor for activated C kinase 1(RACK1), was cloned by PCR with the c DNA from chicken muscles as template. The PCR products were subcloned to p GEX-6 P-1 vector and then prokaryotic recombinant expression plasmid p GEX-6 P-1-RACK1 was constructed. The recombinant protein RACK1-GST was expressed and purified for analysis in Western blot. The expressed RACK1-GST fusion protein was visualized as 61 k Da in molecular mass. The transcriptional and translational levels of RACK1 in DF-1 cells during Eimeria tenella sporozoite infection were detected by RT-q PCR and Western blot. The results showed that both transcriptional and protein expression levels of RACK1 in DF-1 cells significantly increased at 24~72 h post infection. The effect of RACK1 polyclonal antibodies on sporozoite invasion into cells was examined in antibody inhibition assay. Antibodies at 100 μg/m L and 200 μg/m L had little influence on the sporozoite invasion but antibody increase to 300 μg/m L and 400 μg/m L significantly enhanced this capability. These results showed that host RACK1 might play a negative role on Eimeria invasion. However, further studies are needed to illuminate the exact mechanism of how RACK1 negatively regulate Eimeria invasion.
At present, research on the mechanism of Eimeria invading host cells has mainly focused on the parasitic proteins and relevant host genes have been rarely studied. In our previous experiment, i TRAQ technology was used to study the proteomics changes in host cells after infection with Eimeria tenella sporozoites and a number of host proteins related to Eimeria invasion were obtained. In the present study, one of those host proteins, chicken receptor for activated C kinase 1(RACK1), was cloned by PCR with the c DNA from chicken muscles as template. The PCR products were subcloned to p GEX-6 P-1 vector and then prokaryotic recombinant expression plasmid p GEX-6 P-1-RACK1 was constructed. The recombinant protein RACK1-GST was expressed and purified for analysis in Western blot. The expressed RACK1-GST fusion protein was visualized as 61 k Da in molecular mass. The transcriptional and translational levels of RACK1 in DF-1 cells during Eimeria tenella sporozoite infection were detected by RT-q PCR and Western blot. The results showed that both transcriptional and protein expression levels of RACK1 in DF-1 cells significantly increased at 24~72 h post infection. The effect of RACK1 polyclonal antibodies on sporozoite invasion into cells was examined in antibody inhibition assay. Antibodies at 100 μg/m L and 200 μg/m L had little influence on the sporozoite invasion but antibody increase to 300 μg/m L and 400 μg/m L significantly enhanced this capability. These results showed that host RACK1 might play a negative role on Eimeria invasion. However, further studies are needed to illuminate the exact mechanism of how RACK1 negatively regulate Eimeria invasion.
引文
[1]孔繁瑶.家畜寄生虫学[M].2版.北京:中国农大出版社,2008.
[2]Shirley M W,Ivens A,Gruber A,et al.The Eimeriagenome projects:a sequence of events.[J].Trends Parasitol,2004,20(5):199-201.
[3]韩红玉,林矫矫,黄兵.鸡球虫病疫苗的研究进展[J].中国动物传染病学报,2007,15(6):40-46.
[4]Tomley F.Techniques for isolation and characterization of apical organelles from Eimeria tenella sporozoites[J].Methods,1997,13(2):171-176.
[5]宋翠萍,沈永林,汪志楷.柔嫩艾美球虫(Eimeria tenella)体外细胞培养[J].中国兽医学报,1998,18(2):151-155.
[6]王自文.柔嫩艾美耳球虫钙依赖蛋白激酶4功能特性的初步研究[D].北京:中国农业科学院,2016.
[7]Jahn D,Matros A,Bakulina A Y,et al.Model structure of the immunodominant surface antigen of Eimeria tenella identified as a target for sporozoite-neutralizing monoclonal antibody[J].Parasitol Res,2009,105(3):655-668.
[8]Mochly-Rosen D,Khaner H,Lopez J.Identification of intracellular receptor proteins for activated protein kinase C[J],Proc Natl Acad Sci USA,1991,88(9):3997-4000.
[9]Del Vecchio I,Zuccotti A,Pisano F,et al.Functional mapping of the promoter region of the GNB2L1 human gene coding for RACK1 scaffold protein[J].Gene,2009,430(1-2):17-29.
[10]Bird R J,Baillie G S,Yarwood S J.Interaction with receptor for activated C-kinase 1(RACK1)sensitizes the phosphodiesterase PDE4D5 towards hydrolysis of c AMP and activation by protein kinase C[J].Biochem J,2010,432(1):207-216.
[11]Kiely P A,Baillie G S,Lynch M J,et al.Tyrosine 302in RACK1 is essential for insulin-like growth factor-Imediated competitive binding of PP2A and beta1 integrin and for tumor cell proliferation and migration[J].J Biol Chem,2008,283(34):22952-22961
[12]Mourton T,Hellberg C B,Burden-Gulley S M,et al.The PTPmu protein-tyrosine phosphatase binds and recruits the scaffolding protein RACK1 to cell-cell contacts[J].J Biol Chem,2001,276(18):14896-14901.
[13]Scholtyseck E,Mehlhorn H,Hammond D M.Electron microscope studies of microgametogenesis in Coccidia and related groups[J].Z Parasitenkd,1972,38(2):95-131.
[14]李有文.狂犬病病毒P蛋白与宿主RPL9的相互作用及其调控病毒复制的研究[D].武汉:华中农业大学,2015.
[15]张世伟.新城疫病毒调控宿主YB-1蛋白的初步研究[D].合肥:安徽农业大学,2016.
[2]Shirley M W,Ivens A,Gruber A,et al.The Eimeriagenome projects:a sequence of events.[J].Trends Parasitol,2004,20(5):199-201.
[3]韩红玉,林矫矫,黄兵.鸡球虫病疫苗的研究进展[J].中国动物传染病学报,2007,15(6):40-46.
[4]Tomley F.Techniques for isolation and characterization of apical organelles from Eimeria tenella sporozoites[J].Methods,1997,13(2):171-176.
[5]宋翠萍,沈永林,汪志楷.柔嫩艾美球虫(Eimeria tenella)体外细胞培养[J].中国兽医学报,1998,18(2):151-155.
[6]王自文.柔嫩艾美耳球虫钙依赖蛋白激酶4功能特性的初步研究[D].北京:中国农业科学院,2016.
[7]Jahn D,Matros A,Bakulina A Y,et al.Model structure of the immunodominant surface antigen of Eimeria tenella identified as a target for sporozoite-neutralizing monoclonal antibody[J].Parasitol Res,2009,105(3):655-668.
[8]Mochly-Rosen D,Khaner H,Lopez J.Identification of intracellular receptor proteins for activated protein kinase C[J],Proc Natl Acad Sci USA,1991,88(9):3997-4000.
[9]Del Vecchio I,Zuccotti A,Pisano F,et al.Functional mapping of the promoter region of the GNB2L1 human gene coding for RACK1 scaffold protein[J].Gene,2009,430(1-2):17-29.
[10]Bird R J,Baillie G S,Yarwood S J.Interaction with receptor for activated C-kinase 1(RACK1)sensitizes the phosphodiesterase PDE4D5 towards hydrolysis of c AMP and activation by protein kinase C[J].Biochem J,2010,432(1):207-216.
[11]Kiely P A,Baillie G S,Lynch M J,et al.Tyrosine 302in RACK1 is essential for insulin-like growth factor-Imediated competitive binding of PP2A and beta1 integrin and for tumor cell proliferation and migration[J].J Biol Chem,2008,283(34):22952-22961
[12]Mourton T,Hellberg C B,Burden-Gulley S M,et al.The PTPmu protein-tyrosine phosphatase binds and recruits the scaffolding protein RACK1 to cell-cell contacts[J].J Biol Chem,2001,276(18):14896-14901.
[13]Scholtyseck E,Mehlhorn H,Hammond D M.Electron microscope studies of microgametogenesis in Coccidia and related groups[J].Z Parasitenkd,1972,38(2):95-131.
[14]李有文.狂犬病病毒P蛋白与宿主RPL9的相互作用及其调控病毒复制的研究[D].武汉:华中农业大学,2015.
[15]张世伟.新城疫病毒调控宿主YB-1蛋白的初步研究[D].合肥:安徽农业大学,2016.