大片形吸虫丝氨酸/苏氨酸蛋白磷酸酶2A的原核表达及不同时期表达水平分析

doi:10.16431/j.cnki.1671-7236.2023.03.025

摘要/Abstract

摘要： 【目的】克隆大片形吸虫丝氨酸/苏氨酸蛋白磷酸酶2A(Ser/Thr protein phosphatases 2A,PP2A)基因并构建原核表达载体,获得原核表达蛋白,为探究PP2A基因功能及其在大片形吸虫的发育中的作用奠定基础。【方法】提取大片形吸虫成虫虫体组织总RNA,应用RT-PCR方法扩增PP2A基因的完整编码区序列,并以双酶切的方式连接pET-28a(＋)载体;经酶切、测序鉴定后获得阳性质粒pET-28a-PP2A,将pET-28a-PP2A重组质粒转化大肠杆菌BL21(DE3)感受态细胞进行IPTG诱导表达,并对重组蛋白的诱导条件进行优化;通过SDS-PAGE及Western blotting检测大片形吸虫PP2A基因的表达效果;应用实时荧光定量PCR检测PP2A基因在大片形吸虫虫卵、囊蚴、6周龄童虫和成虫阶段中的表达情况。【结果】克隆的大片形吸虫PP2A基因编码区序列长1161 bp,编码386个氨基酸,分子式为C₁₉₈₀H₃₁₀₅N₅₃₅O₅₆₆S₂₄,分子质量为44.23 ku。生物信息学分析结果显示,大片形吸虫PP2A基因编码的蛋白为PP2Ac,分子功能为信号转导(GO:0004871),生物学过程为蛋白去磷酸化(GO:0006470),细胞组分为细胞质(GO:0005829)。PP2A蛋白无信号肽,不存在跨膜结构域,为亲水性蛋白,共含有37个磷酸位点。SDS-PAGE和Western blotting结果显示,大片形吸虫PP2A在30 ℃、0.8 mmol/L IPTG诱导6 h时表达量最大,表达产物主要以包涵体的形式存在,且Western blotting检测结果显示,重组蛋白可被抗His标签识别;实时荧光定量PCR结果显示,PP2A基因在大片形吸虫6周龄童虫阶段表达量最高,极显著高于其他阶段(P＜0.01);在囊蚴阶段的表达量最低,与虫卵阶段差异不显著(P＞0.05)。【结论】本研究成功构建了大片形吸虫PP2A原核表达载体并获得相应蛋白,为研究PP2A基因在大片形吸虫生长发育中的功能奠定了基础。

关键词: 大片形吸虫; 丝氨酸/苏氨酸蛋白磷酸酶2A(PP2A); 原核表达

Abstract: 【Objective】 The aim of this study was to clone the serine/threonine protein phosphatase 2A (Ser/Thr protein phosphatases 2A,PP2A) gene of Fasciola gigantica and construct a prokaryotic expression vector to obtain the prokaryotic expression protein,so as to lay a foundation for exploring the function of PP2A gene and its role in the development of Fasciola gigantica.【Method】 Total RNA was extracted from adult trematodes,the complete coding region of PP2A gene was amplified by RT-PCR and ligated to pET-28a(＋) vector by double restriction endonuclease digestion and sequencing,the positive plasmid was named as recombinant plasmid pET-28a-PP2A.The recombinant plasmid pET-28a-PP2A was transformed into E.coli expression strain BL21 (DE3) competent cell for IPTG induction,and the induction conditions of the recombinant protein were optimized.The expression effect of PP2A gene of Fasciola gigantica was detected by SDS-PAGE and Western blotting.Real-time PCR was used to detect the expression of PP2A gene in eggs,metacercaria,6-week-old juvenile and adults of Fasciola gigantica.【Result】 The coding region of PP2A gene of Fasciola gigantica was 1 161 bp,encoding 386 amino acids.The molecular formula was C₁₉₈₀H₃₁₀₅N₅₃₅O₅₆₆S₂₄ and the molecular weight was 44.23 ku.The results of bioinformatics analysis showed that the protein edited by PP2A gene was PP2Ac,the molecular function was signal transduction (GO:0004871),the biological process was protein dephosphorylation (GO:0006470),and the cell component was cytoplasm (GO:0005829).PP2A protein had no signal peptide and no transmembrane domain.It was a hydrophilic protein with 37 phosphate sites.The results of SDS-PAGE and Western blotting showed that the expression of PP2A in Fasciola gigantica was the highest at 30 ℃ and 0.8 mmol/L IPTG inducing for 6 h.The expressed product was mainly in the form of inclusion body,and the recombinant protein could be recognized by anti-His tag by Western blotting detection.The results of Real-time PCR showed that the expression of PP2A gene was the highest in the 6-week-old juvenile stage,which was extremely significant higher than that in other stages (P＜0.01),and the lowest in the metacercaria stage,and had no significant difference with the egg stage(P＞0.05).【Conclusion】 In this study,the prokaryotic expression vector of Fasciola gigantica PP2A was successfully constructed and the corresponding protein was obtained,which laid a foundation for studying the function of PP2A gene in the growth and development of Fasciola gigantica.

Key words: Fasciola gigantica; serine/threonine protein phosphatase 2A (PP2A); prokaryotic expression

中图分类号:

S852.73

章志涛, 李祥龙, 孔令丽, 罗雨昕, 王冬英. 大片形吸虫丝氨酸/苏氨酸蛋白磷酸酶2A的原核表达及不同时期表达水平分析[J]. 中国畜牧兽医, 2023, 50(3): 1107-1117.

ZHANG Zhitao, LI Xianglong, KONG Lingli, LUO Yuxin, WANG Dongying. Prokaryotic Expression of Serine/Threonine Protein Phosphatase 2A in Fasciola gigantica and Expression Analysis at Different Stages[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1107-1117.

参考文献

[1] CALVANI N,SLAPETA J.Fasciola species introgression:Just a fluke or something more?[J].Trends in Parasitology,2021,37(1):25-34.
[2] PIEDRAFITA D,SPITHILL T W,SMITH R E,et al.Improving animal and human health through understanding liver fluke immunology[J].Parasite Immunology,2010,32(8):572-581.
[3] MAS-COMA S,BARGUES M D,VALERO M A.Human fascioliasis infection sources,their diversity,incidence factors,analytical methods and prevention measures[J].Parasitology,2018,145(13):1665-1699.
[4] SAH R,KHADKA S,LAKHEY P J,et al.Human case of Fasciola gigantica-like infection,review of human fascioliasis reports in Nepal,and epidemiological analysis within the South Central Asia[J].Acta Parasitologica,2018,63(3):435-443.
[5] FAIRWEATHER I,BRENNAN G P,HANNA R,et al.Drug resistance in liver flukes[J].International Journal for Parasitology:Drugs and Drug Resistance,2020,12:39-59.
[6] WEBB C M,CABADA M M.Recent developments in the epidemiology,diagnosis,and treatment of Fasciola infection[J].Current Opinion in Infectious Diseases,2018,31(5):409-414.
[7] ARINO J,VELAZQUEZ D,CASAMAYOR A.Ser/Thr protein phosphatases in fungi:Structure,regulation and function[J].Microbial Cell Factories,2019,6(5):217-256.
[8] KAMADA R,KUDOH F,ITO S,et al.Metal-dependent Ser/Thr protein phosphatase PPM family:Evolution,structures,diseases and inhibitors[J].Pharmacology & Therapeutics,2020,215:107622.
[9] CASAMAYOR A,ARINO J.Controlling Ser/Thr protein phosphatase PP1 activity and function through interaction with regulatory subunits[J].Advances in Protein Chemistry and Advances in Structural Biology,2020,122:231-288.
[10] SHI Y.Serine/threonine phosphatases:Mechanism through structure[J].Cell,2009,139(3):468-484.
[11] HOFFMAN A,TALESKI G,SONTAG E.The protein serine/threonine phosphatases PP2A,PP1 and calcineurin:A triple threat in the regulation of the neuronal cytoskeleton[J].Molecular and Cellular Neuroscience,2017,84:119-131.
[12] KHALIFE J,FREVILLE A,GNANGNON B,et al.The multifaceted role of protein phosphatase 1 in Plasmodium[J].Trends in Parasitology,2021,37(2):154-164.
[13] ZHU X,SUN L,HE Y,et al.Plasmodium berghei serine/threonine protein phosphatase PP5 plays a critical role in male gamete fertility[J].International Journal for Parasitology,2019,49(9):685-695.
[14] CIESLA J,FRACZYK T,RODE W.Phosphorylation of basic amino acid residues in proteins:Important but easily missed[J].Acta Biochimica Polonica,2011,58(2):137-148.
[15] KERK D,MATTICE J F,VALDES-TRESANCO M E,et al.The origin and radiation of the phosphoprotein phosphatase (PPP) enzymes of eukaryotes[J].Scientific Reports, 2021,11(1):13681.
[16] OLSEN J V,BLAGOEV B,GNAD F,et al.Global,in vivo,and site-specific phosphorylation dynamics in signaling networks[J].Cell,2006,127(3):635-648.
[17] YANG C,ARRIZABALAGA G.The serine/threonine phosphatases of apicomplexan parasites[J].Molecular Microbiology,2017,106(1):1-21.
[18] BAESHEN M N,AL-HEJIN A M,BORA R S,et al.Production of biopharmaceuticals in E.coli:Current scenario and future perspectives[J].Journal of Microbiology and Biotechnology,2015,25(7):953-962.
[19] GUPTA S K,SHUKLA P.Advanced technologies for improved expression of recombinant proteins in bacteria:Perspectives and applications[J].Critical Reviews in Biotechnology,2016,36(6):1089-1098.
[20] LIU M,WANG B,WANG F,et al.Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner[J].Applied Microbiology and Biotechnology,2019,103(15):6071-6079.
[21] MARKOVA S V,LARIONOVA M D,GORBUNOVA D A,et al.The disulfide-rich metridia luciferase refolded from E.coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells[J].Journal of Photochemistry and Photobiology B:Biology,2017,175:51-57.
[22] GUPTA S K,SHUKLA P.Sophisticated cloning,fermentation,and purification technologies for an enhanced therapeutic protein production:A review[J].Frontiers in Pharmacology,2017,8:419.
[23] MALEKIAN R,SIMA S,JAHANIAN-NAJAFABADI A,et al.Improvement of soluble expression of GM-CSF in the cytoplasm of Escherichia coli using chemical and molecular chaperones[J].Protein Expression and Purification, 2019,160:66-72.
[24] AULD S K,TINSLEY M C.The evolutionary ecology of complex lifecycle parasites:Linking phenomena with mechanisms[J].Heredity (Edinb),2015,114(2):125-132.
[25] CHAUDHURI M.Cloning and characterization of a novel serine/threonine protein phosphatase type 5 from Trypanosoma brucei[J].Gene,2001,266(1-2):1-13.
[26] WIMMER M,SCHMID B,TAG C,et al.Ascaris suum:Protein phosphotyrosine phosphatases in oocytes and developing stages[J].Experimental Parasitology,1998,88(2):139-145.
[27] KHALIFE J,FREVILLE A,GNANGNON B,et al.The multifaceted role of protein phosphatase 1 in Plasmodium[J].Trends in Parasitology,2021,37(2):154-164.
[28] DOBSON S,BRACCHI V,CHAKRABARTI D,et al.Characterization of a novel serine/threonine protein phosphatase (PfPPJ) from the malaria parasite,Plasmodium falciparum[J].Molecular and Biochemical Parasitology,2001,115(1):29-39.