刚地弓形虫TgPRF的原核表达、多克隆抗体制备与应用
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摘要
目的原核表达弓形虫TgPRF蛋白,制备多克隆抗体并评价其作为内参抗体的可行性。方法以刚地弓形虫RH株速殖子cDNA为模板,PCR扩增TgPRF编码基因,克隆至pGEX-4T-1载体,转化至大肠埃希氏菌(Escherichia coli)BL21。经IPTG诱导表达后,SDS-PAGE分析重组蛋白表达情况。利用GST标签亲和层析法纯化重组蛋白。以纯化后蛋白为抗原免疫新西兰大白兔,制备抗TgPRF多克隆抗体。收集弓形虫RH株速殖子,以制备的多克隆抗体为一抗,Western blotting检测抗体特异性及效价。收集ATc调控下不同时间点的弓形虫iKO-Ty-TgAPH虫株速殖子进行Western blotting,以抗Ty和抗TgPRF抗体为一抗,检测TgAPH和TgPRF表达量的变化。结果 SDS-PAGE显示:TgPRF在诱导4 h后表达量趋于饱和,相对分子量为52 ku,且呈可溶性表达。弓形虫RH株速殖子总蛋白进行的Western blotting显示:抗TgPRF多克隆抗体可识别内源性的TgPRF,且该多克隆抗体稀释至1∶10 000时仍可见明显条带;iKO-TyTgAPH虫株速殖子总蛋白进行的Western blotting显示:TgAPH的表达量随ATc作用时间的增加呈递减趋势,而TgPRF表达量基本维持恒定。结论本实验制备的抗TgPRF多克隆抗体特异性和效价良好,可作为衡量弓形虫特定蛋白表达量的内参抗体,也可直接检测内源性TgPRF的表达。
Objective To generate rabbit polyclonal antibodies against the recombinant protein GST-TgPRF to be used as a loading control antibody. Methods Using the tachyzoite cDNA of Toxoplasma gondii RH strain as template, the TgPRF coding gene was amplified by PCR, cloned into pGEX-4 T-1 vector, and transformed into Escherichia coli BL21. After IPTG induced expression, SDS-PAGE was used to analyze the expression of recombinant protein. The recombinant protein was purified by GST label affinity chromatography. Anti-TgPRF polyclonal antibody was prepared by immunizing New Zealand white rabbits with purified protein antigen. The tachyzoites of T. gondii RH strain were collected, and the polyclonal antibodies prepared were used as primary antibodies, and the specificity and titer of the antibodies were detected by Western blotting.The tachyzoites of Toxoplasma iKO-Ty-TgAPH strains at different time points regulated by ATc were collected for Western blotting, and Ty and anti-TgPRF were used as primary antibodies to detect the changes in the expression levels of TgAPH and TgPRF. Results SDS-PAGE showed a 52 ku band of expressed proteins post IPTG-induction 4 h. Western blotting of the total protein of tachyzoites of Toxoplasma RH strain showed that anti-TgPRF polyclonal antibody could recognize endogenous TgPRF, and obvious bands could be seen when the polyclonal antibody was diluted to 1∶10 000;Western blotting of the total protein of tachyzoites of iKO-Ty-TgAPH showed that the expression of TgAPH decreased with the increase of ATc, while the expression of TgPRF remained basically constant. Conclusion The rabbit anti-TgPRF polyclonal antibody generated in this experiment showed a good specificity and sensitivity. It can be used as an internal control in western blot analyses and may be used as well as in other immunological applications.
Objective To generate rabbit polyclonal antibodies against the recombinant protein GST-TgPRF to be used as a loading control antibody. Methods Using the tachyzoite cDNA of Toxoplasma gondii RH strain as template, the TgPRF coding gene was amplified by PCR, cloned into pGEX-4 T-1 vector, and transformed into Escherichia coli BL21. After IPTG induced expression, SDS-PAGE was used to analyze the expression of recombinant protein. The recombinant protein was purified by GST label affinity chromatography. Anti-TgPRF polyclonal antibody was prepared by immunizing New Zealand white rabbits with purified protein antigen. The tachyzoites of T. gondii RH strain were collected, and the polyclonal antibodies prepared were used as primary antibodies, and the specificity and titer of the antibodies were detected by Western blotting.The tachyzoites of Toxoplasma iKO-Ty-TgAPH strains at different time points regulated by ATc were collected for Western blotting, and Ty and anti-TgPRF were used as primary antibodies to detect the changes in the expression levels of TgAPH and TgPRF. Results SDS-PAGE showed a 52 ku band of expressed proteins post IPTG-induction 4 h. Western blotting of the total protein of tachyzoites of Toxoplasma RH strain showed that anti-TgPRF polyclonal antibody could recognize endogenous TgPRF, and obvious bands could be seen when the polyclonal antibody was diluted to 1∶10 000;Western blotting of the total protein of tachyzoites of iKO-Ty-TgAPH showed that the expression of TgAPH decreased with the increase of ATc, while the expression of TgPRF remained basically constant. Conclusion The rabbit anti-TgPRF polyclonal antibody generated in this experiment showed a good specificity and sensitivity. It can be used as an internal control in western blot analyses and may be used as well as in other immunological applications.
引文
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[8]JIA Y G,MARQ J B,BISIO H,et al.Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma Gondii[J].EMBO J,2017,36(21):3250-3267.
[9]LLINáS M,DEITSCH K W,VOSS T S.Plasmodium gene regulation:Far more to factor in[J].Trends Parasitol,2008,24(12):551-556.
[10]NEAL L M,KNOLL L J.Toxoplasma gondii profilin promotes recruitment of Ly6Chi CCR2+inflammatory monocytes that can confer resistance to bacterial infection[J].PLoS Pathog,2014,10(6):e1004203.
[11]KOBLANSKY A A,JANKOVIC D,OH H,et al.Recognition of profilin by Toll-like receptor 12 is critical for host resistance to Toxoplasma gondii[J].Immunity,2013,38(1):119-130.
[12]SKILLMAN K M,DAHER W,MA C I,et al.Toxoplasma gondii profilin acts primarily to sequester G-actin while formins efficiently nucleate actin filament formation in vitro[J].Biochemistry,2012,51(12):2486-2495.
[13]XU Y,WANG X J,LIU J,et al.Toxoplasma gondii rhoptry protein38(TgROP38)affects parasite invasion,egress,and induces IL-18 secretion during early infection[J].Acta Biochim Biophys Sin(Shanghai),2018,50(8):766-775.
[14]ALAM A,BHATNAGAR R K,RELAN U,et al.Proteolytic activity of Plasmodium falciparum subtilisin-like protease 3 on parasite profilin,a multifunctional protein[J].Mol Biochem Parasitol,2013,191(2):58-62.
[15]RAETZ M,KIBARDIN A,STURGE C R,et al.Cooperation of TLR12 and TLR11 in the IRF8-dependent IL-12 response to Toxoplasma gondii profilin[J].J Immunol,2013,191(9):4818-4827.2019-04-29
[2]熊乙丁,杨瑞,岳续鹏,等.Profilin在弓形虫侵入宿主细胞及诱导免疫应答中作用的研究进展[J].中国人兽共患病学报,2017,33(12):1110-1114.
[3]UBOLDI A D,WILDE M L,MCRAE E A,et al.Protein kinase Anegatively regulates Ca2+signalling in Toxoplasma gondii[J].PLoSBiol,2018,16(9):e2005642.
[4]DARVILL N,DUBOIS D J,ROUSE S L,et al.Structural basis of phosphatidic acid sensing by APH in apicomplexan parasites[J].Structure,2018,26(8):1059-1071.e6.
[5]贾永根,闫爱霞,黄敏君,等.刚地弓形虫TgPH1蛋白的重组表达及其与磷脂酰肌醇结合性的鉴定[J].中国热带医学,2019,19(3):201-204.
[6]PLATTNER F,YAROVINSKY F,ROMERO S,et al.Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response[J].Cell Host Microbe,2008,3(2):77-87.
[7]PINO P,SEBASTIAN S,KIM E A,et al.A tetracycline-repressible transactivator system to study essential genes in malaria parasites[J].Cell Host Microbe,2012,12(6):824-834.
[8]JIA Y G,MARQ J B,BISIO H,et al.Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma Gondii[J].EMBO J,2017,36(21):3250-3267.
[9]LLINáS M,DEITSCH K W,VOSS T S.Plasmodium gene regulation:Far more to factor in[J].Trends Parasitol,2008,24(12):551-556.
[10]NEAL L M,KNOLL L J.Toxoplasma gondii profilin promotes recruitment of Ly6Chi CCR2+inflammatory monocytes that can confer resistance to bacterial infection[J].PLoS Pathog,2014,10(6):e1004203.
[11]KOBLANSKY A A,JANKOVIC D,OH H,et al.Recognition of profilin by Toll-like receptor 12 is critical for host resistance to Toxoplasma gondii[J].Immunity,2013,38(1):119-130.
[12]SKILLMAN K M,DAHER W,MA C I,et al.Toxoplasma gondii profilin acts primarily to sequester G-actin while formins efficiently nucleate actin filament formation in vitro[J].Biochemistry,2012,51(12):2486-2495.
[13]XU Y,WANG X J,LIU J,et al.Toxoplasma gondii rhoptry protein38(TgROP38)affects parasite invasion,egress,and induces IL-18 secretion during early infection[J].Acta Biochim Biophys Sin(Shanghai),2018,50(8):766-775.
[14]ALAM A,BHATNAGAR R K,RELAN U,et al.Proteolytic activity of Plasmodium falciparum subtilisin-like protease 3 on parasite profilin,a multifunctional protein[J].Mol Biochem Parasitol,2013,191(2):58-62.
[15]RAETZ M,KIBARDIN A,STURGE C R,et al.Cooperation of TLR12 and TLR11 in the IRF8-dependent IL-12 response to Toxoplasma gondii profilin[J].J Immunol,2013,191(9):4818-4827.2019-04-29