毕赤酵母糖基化对布鲁菌P39抗原诱导巨噬细胞吞噬的影响
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摘要
P39蛋白是通过蛋白组学从布鲁菌中鉴定出的具有较高免疫原性的优势抗原蛋白之一。本研究对布鲁菌的优势抗原蛋白P39基因在毕赤酵母中进行表达,并对其进行纯化及Western blot鉴定,利用核磁共振接合PNGaseF糖苷酶进行蛋白糖基化鉴定,并研究其与巨噬细胞相互作用的速度。结果表明,P39蛋白在毕赤酵母GS115中呈分泌表达,表达量可达5.05 g/L。核磁共振结果表明,重组P39蛋白被糖基化修饰。修饰后的蛋白被巨噬细胞捕获效应明显强于原核蛋白,说明糖基化修饰可能影响目标蛋白与细胞相互作用的效率,为进一步研究糖基化对P39蛋白的影响及布鲁菌糖基化疫苗候选抗原的研究提供参考。
P39 protein is one of the most predominant antigenic proteins identified from Brucella by proteomics. First,the dominant antigen protein gene P39 was expressed in Pichia pastoris,and the expressed protein was purified and identified by Western blot. Then,theglycosylation of the protein was identified by NMR(nuclear magnetic resonance)conjugated PNGaseF glycosylase,and the interaction speedbetween the protein and macrophage cell was studied. The results showed that P39 protein was secreted in P. pastoris GS115 and the expressionlevel was up to 5.05 g/L. NMR results demonstrated that the recombinant P39 protein was modified by glycosylation. The macrophage captureeffect of the modified protein was significantly stronger than prokaryotic protein,indicating that the glycosylation modification may affect theefficiency of the interaction between the target protein and the cell,which provides a reference for further exploring the effect of glycosylation on P39 protein and candidate antigen of Brucella glycosylated vaccine.
P39 protein is one of the most predominant antigenic proteins identified from Brucella by proteomics. First,the dominant antigen protein gene P39 was expressed in Pichia pastoris,and the expressed protein was purified and identified by Western blot. Then,theglycosylation of the protein was identified by NMR(nuclear magnetic resonance)conjugated PNGaseF glycosylase,and the interaction speedbetween the protein and macrophage cell was studied. The results showed that P39 protein was secreted in P. pastoris GS115 and the expressionlevel was up to 5.05 g/L. NMR results demonstrated that the recombinant P39 protein was modified by glycosylation. The macrophage captureeffect of the modified protein was significantly stronger than prokaryotic protein,indicating that the glycosylation modification may affect theefficiency of the interaction between the target protein and the cell,which provides a reference for further exploring the effect of glycosylation on P39 protein and candidate antigen of Brucella glycosylated vaccine.
引文
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[2]Cannella AP, Tsolis RM, Li L, et al. Antigen-specific acquiredimmunity in human brucellosis:implications for diagnosis,prognosis, and vaccine development[J]. Frontiers in Cellular&Infection Microbiology, 2012, 2(2):1.
[3]Scharp DW, al Khalaf SA, al Muhanna MW, et al. Use of massvaccination with a reduced dose of REV 1 vaccine for Brucella melitensis control in a population of small ruminants[J]. TropicalAnimal Health&Production, 1999, 31(3):135.
[4]王真, HAN Gilsu,吴清民.动物布鲁菌病疫苗的来历及亚单位疫苗的研究概况[J].中国农业大学学报, 2014, 19(3):169-174.
[5]Denoel PA, Vo TK, Weynants VE, et al. Identification of themajor T-cell antigens present in the Brucella melitensis B115protein preparation, Brucellergene OCB[J]. Journal of MedicalMicrobiology, 1997, 46(9):801-806.
[6]仲娜,郝林华,王小如.糖蛋白药物的研究进展[J].中国新药杂志, 2005, 14(12):1400-1403.
[7]严钦,俞慧清,成国祥.蛋白糖基化与免疫研究进展[J].现代免疫学, 2008, 28(2):168-168.
[8]Durocher Y, Butler M. Expression systems for therapeutic glycoprotein production[J]. Current Opinion in Biotechnology, 2009, 20(6):700.
[9]许博然. TLR2和TLR4受体封闭肽对小鼠腹腔巨噬细胞细胞因子分泌的影响[D].呼和浩特:内蒙古农业大学, 2017.
[10]Love KR, Shah KA, Whittaker CA, et al. Comparative genomicsand transcriptomics of Pichia pastoris[J]. BMC Genomics, 2016,17(1):550.
[11]Chaffey PK, Guan X, Chen C, et al. Structural Insight into thestabilizing effect of O-glycosylation[J]. Biochemistry, 2017, 56(23):2897.
[12]田媛,张尊建.糖类结构的核磁共振波谱及质谱分析[J].药学进展, 2003, 27(2):78-80.
[13]蒋春霞. Rab6在小鼠巨噬细胞RAW264. 7吞噬过程中的作用[D].杭州:浙江大学, 2013.
[14]毛景东,王景龙,杨艳玲.布鲁菌病的研究进展[J].中国畜牧兽医, 2011, 38(1):222-227.
[15]Christopher S, Umapathy BL, Ravikumar KL. Brucellosis:Reviewon the recent trends in pathogenicity and laboratory diagnosis[J].Journal of Laboratory Physicians, 2010, 2(2):55.
[16]Blasco JM, Molina-Flores B. Control and Eradication of Brucella melitensis infection in sheep and goats[J]. Macedonian Academyof Sciences and Arts, Section of Biological and Medical Sciences,2010, 31(1):145-65.
[17]Hao J, Guo Y, Song X, et al. Elimination of N-glycosylation by sitemutation further prolongs the half-life of IFN-α/Fc fusion proteinsexpressed in Pichia pastoris[J]. Microbial Cell Factories, 2016,15(1):209.
[18]WangW,LuB,ZhouH,etal.Glycosylationat158Nofthe hemagglutinin protein and receptor binding specificitysynergistically affect the antigenicity and immunogenicity of alive attenuated H5N1 A/Vietnam/1203/2004 vaccine virus inferrets[J]. Journal of Virology, 2010, 84(13):6570.
[19]HosslerP,ChumsaeC,RacicotC, et al.Arabinosylationofrecombinanthumanimmunoglobulin-basedproteintherapeutics[J]. Mabs, 2017, 9(4):715.
[20]许春晓.糖基化修饰对蛋白Rsα、AhcY免疫原性影响的研究[D].长春:吉林农业大学, 2016.
[21]Ratanji KD, Derrick JP, Dearman RJ, et al. Immunogenicity oftherapeutic proteins:Influence of aggregation[J]. Journal ofImmunotoxicology, 2014, 11(2):99.
[22]Mahan AE, Jennewein MF, Suscovich T, et al. Antigen-specificantibody glycosylation is regulated via vaccination[J]. PLoSPathogens, 2016, 12(3):e1005456.