恶性疟原虫自噬相关蛋白8基因原核重组表达及多克隆抗体制备
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摘要
目的通过pET-41 Ek/LIC载体在大肠杆菌工程菌中重组表达恶性疟原虫自噬相关蛋白8(PfAtg8)基因的重组蛋白GST-PfAtg8-6×His,并用蛋白免疫法制备小鼠抗自噬相关蛋白8(PfAtg8)的多克隆抗体,并验证其效价。方法体外培养恶性疟原虫3D7株,并提取DNA,PCR扩增PfAtg8序列全长,与pET-41 Ek/LIC载体通过基因重组直接连接,在大肠杆菌Rosetta~(TM)(DE3)PLysS工程菌中诱导表达重组蛋白GST-PfAtg8-6×His,蛋白免疫法制备鼠抗PfAtg8的多克隆抗体并用Western blots和酶联免疫吸附测定法(enzyme-linked immunosorbent assay, ELISA)进行效价验证。结果 PCR扩增PfAtg8基因并插入pET-41 Ek/LIC载体,转化入大肠杆菌。诱导表达并纯化重组蛋白,进而利用该蛋白免疫小鼠制备多克隆抗体,经验证该抗体可对重组蛋白进行特异性识别,且效价可达1∶100 000。结论成功表达、纯化了GST-PfAtg8-6×His重组蛋白,并免疫小鼠获得多克隆抗体血清。
Objective To express the GST-PfAtg8-6×His protein of Plasmodium falciparum autophagy-associated protein 8(PfAtg8) in Escherichia coli strain with pET-41 Ek/LIC vector, and to prepare mouse anti-PfAtg8 polyclonal antibodies by protein immunoassay.Methods Strain of 3D7 Plasmodium falciparum was cultured in vitro and total DNA was extracted. The full length of PfAtg8 was amplified by PCR and directly ligated with pET-41 Ek/LIC vector by nucleotide sequence recombination. The GST-PfAtg8-6×His recombinant protein was induced in Escherichia coli Rosetta~(TM)(DE3) PLysS,the mouse anti-PfAtg8 polyclonal antibodies was prepared by protein immunoassay and verified by Western blots and ELISA(enzyme-linked immunosorbent assay).Results PfAtg8 was amplified by PCR, directly ligated with pET-41 Ek/LIC vector and transformed into E. coli. The recombinant protein was induced and purified, and the polyclonal antibody was successfully prepared, which can specifically recognize the recombinant protein, and the serum titer was up to 1∶100000.Conclusion The GST-PfAtg8-6×His recombinant protein was expressed and purified, and the polyclonal antibody was successfully prepared by protein immunoassay.
Objective To express the GST-PfAtg8-6×His protein of Plasmodium falciparum autophagy-associated protein 8(PfAtg8) in Escherichia coli strain with pET-41 Ek/LIC vector, and to prepare mouse anti-PfAtg8 polyclonal antibodies by protein immunoassay.Methods Strain of 3D7 Plasmodium falciparum was cultured in vitro and total DNA was extracted. The full length of PfAtg8 was amplified by PCR and directly ligated with pET-41 Ek/LIC vector by nucleotide sequence recombination. The GST-PfAtg8-6×His recombinant protein was induced in Escherichia coli Rosetta~(TM)(DE3) PLysS,the mouse anti-PfAtg8 polyclonal antibodies was prepared by protein immunoassay and verified by Western blots and ELISA(enzyme-linked immunosorbent assay).Results PfAtg8 was amplified by PCR, directly ligated with pET-41 Ek/LIC vector and transformed into E. coli. The recombinant protein was induced and purified, and the polyclonal antibody was successfully prepared, which can specifically recognize the recombinant protein, and the serum titer was up to 1∶100000.Conclusion The GST-PfAtg8-6×His recombinant protein was expressed and purified, and the polyclonal antibody was successfully prepared by protein immunoassay.
引文
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[2]WHO.World malaria report[R].Geneva.2018.
[3]HOLZ L E,FERNANDEZ-RUIZ D,HEATH W R.Protective immunity to liver-stage malaria[J].Clin Transl Immunology,2016,5(10):e105.
[4]李缜,李晶晶,邹洋,等.疟原虫表观遗传学研究进展[J].中国热带医学,2017,17(6):630-634.
[5]BESTEIRO S.Autophagy in apicomplexan parasites[J].Curr Opin Microbiol,2017,40:14-20.
[6]STOLZ A,ERNST A,DIKIC I.Cargo recognition and trafficking in selective autophagy[J].Nat.Cell Biol.,2014,16(6):495-501.
[7]VARBERGJ M,LAFAVERSK A,ARRIZABALAGA G,et al.Characterization of plasmodium atg3-atg8 interaction inhibitors identifies novel alternative mechanisms of action in toxoplasma gondii[J].Antimicrob Agents Chemother,2018,62(2):e01489-e01417.
[8]DATTA G,HOSSAINM E,ASAD M,et al.Plasmodium falciparumOTU-like cysteine protease(PfOTU)is essential for apicoplast homeostasis and associates with noncanonical role of Atg8[J].Cellular Microbiology,2017,19(9):e12748.
[9]ICHIMURA Y,KIRISAKO T,TAKAO T,et al.A ubiquitin-like system mediates protein lipidation[J].Nature,2000,408(6811):488-492.
[10]LEEY K,LEEJ A.Role of the mammalian ATG8/LC3family in autophagy:Differential and compensatory roles in the spatiotemporal regulation of autophagy[J].BMB Reports,2016,49(8):424-430.
[11]NGUYENH M,LIU S X,DAHER W,et al.Characterisation of two Toxoplasma PROPPINs homologous to Atg18/WIPI suggests they have evolved distinct specialised functions[J].PLoS One,2018,13(4):e0195921.
[12]TOMLINSA M,BEN-RACHED F,WILLIAMSR A,et al.Plasmodium falciparum ATG8 implicated in both autophagy and apicoplast formation[J].Autophagy,2013,9(10):1540-1552.
[13]WALCZAK M,GANESANS M,NILESJ C,et al.ATG8 is essential specifically for an autophagy-independent function in apicoplast biogenesis in blood-stage malaria parasites[J].MBio,2018,9(1):e02021-e02017.
[14]李缜,邹洋,贾永根,等.疟原虫和弓形虫的自噬作用研究进展[J].中国热带医学,2018,18(9):944-949.
[15]SLOBODKINM R,ELAZAR Z.The Atg8 family:Multifunctional ubiquitin-like key regulators of autophagy[J].Essays Biochem,2013,55:51-64.
[16]CERVANTES S,BUNNIKE M,SARAF A,et al.The multifunctional autophagy pathway in the human malaria parasite,Plasmodium falciparum[J].Autophagy,2014,10(1):80-92.
[17]NAVALE R,ATUL,ALLANKIA D,et al.Characterization of the autophagy marker protein Atg8 reveals atypical features of autophagy in Plasmodium falciparum[J].PLoS One,2014,9(11):e113220.
[18]KITAMURA K,KISHI-ITAKURA C,TSUBOI T,et al.Autophagyrelated atg8 localizes to the apicoplast of the human malaria parasite plasmodium falciparum[J].PLoS One,2012,7(8):e42977.
[19]TOMLINS A M,BEN-RACHED F,WILLIAMS R A,et al.Plasmodium falciparum ATG8 implicated in both autophagy and apicoplast formation[J].Autophagy,2013,9(10):1540-1552.
[20]EICKEL N,KAISER G,PRADO M,et al.Features of autophagic cell death in Plasmodium liver-stage parasites[J].Autophagy,2013,9(4):568-580.
[21]VOSS C,EHRENMAN K,MLAMBO G,et al.Overexpression of Plasmodium berghei ATG8 by Liver Forms Leads to Cumulative Defects in Organelle Dynamics and to Generation of Noninfectious Merozoites[J].MBio,2016,7(3):e00682-00686.
[22]COSTA G L,AMARAL L C,FONTES C J,et al.Assessment of copy number variation in genes related to drug resistance in Plasmodium vivax and Plasmodium falciparum isolates from the Brazilian Amazon and a systematic review of the literature[J].Malar J,2017,16(1):152.
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