T7噬菌体衣壳蛋白P11的表达纯化及单克隆抗体的制备
|
摘要
T7噬菌体是一种感染大肠杆菌的烈性噬菌体,基因组为线状双链DNA,全长39 936bp。其衣壳蛋白包括头蛋白P10A、次要头蛋白P10B、颈圈蛋白P8、尾蛋白P11、P12和尾丝蛋白P17。T7噬菌体作为一种常见的展示系统,因其具有载体容量大,插入片段稳定,洗涤条件灵活,生长周期短等优点而被广泛使用。T7噬菌体蛋白芯片是近年来发展起来的一种新的蛋白质检测手段,具有高质量、高灵敏度、高特异性且微型化特点的一种蛋白质分析技术,能使外源蛋白表达并展示于T7噬菌体表面,再将这些展示外源蛋白的T7噬菌体固化在芯片上,对其展示的蛋白进行检测。
本试验根据GenBank发表的P11基因序列,设计了一对特异性核苷酸引物,用PCR方法获得598bp大小的基因片段,并将其克隆到表达载体上,测序鉴定,成功构建pET-28a(+)/P11表达载体,转化到大肠杆菌表达菌株BL21(DE3)中,诱导表达重组蛋白P11,并进一步通过Ni-NTA亲合层析柱纯化目的蛋白,纯化率达90%。纯化产物经SDS-PAGE鉴定后免疫Balb/c小鼠,用聚乙二醇PEG介导Balb/c小鼠脾细胞与SP2/0骨髓瘤细胞融合,用纯化的P11重组蛋白筛选阳性杂交瘤细胞株,对阳性克隆细胞株经3次有限稀释法克隆化筛选后,最终获得1株能稳定分泌单克隆抗体的杂交瘤细胞株,命名为2G11。对单克隆抗体亚类鉴定结果表明2G11为IgG2b亚类。ELISA试验结果和Western blot分析表明,单抗能与T7噬菌体蛋白和P11蛋白特异结合。这为进一步研究P11蛋白的结构和功能,提高T7噬菌体蛋白芯片的筛选效率以及建立噬菌体检测方法奠定了基础。
Bacteriophage T7 is a lysogeny phage capable of infecting most strains of Escherichia coli, a double standed DNA of genome molecular is 39 936 bp. The capside protein of T7 include major head protein P10A, head assemble protein P10B, neck protein P8, Tail protein P11, P12 and Tail fibers protein P17. T7 phage display system is easy to use and has the capacity to display peptides up to about 1200 amino acids, including stability of insert gene, varity of elution conditions and short of growth cycle. T7 phage protein chip is fixed the bacteriophage T7 which expressed and displayed the exogenous proteins on the chip, the chip could be used for the detection the exogenous proteins. P11 gene was inserted into plasmid pET-28a(+) to construct recombinant plasmid pET-28a(+)/P11. Recombinant P11 protein was expressed under the introduction of 0.1mol/L IPTG and well purified by nickel ion affinity chromatogagh method. SDS-PAGE analysis showed that the molecular weight of the expressed protein was approximately 27 kDa. The Balb/c mice were immunized with the purified protein P11 mixed adjuvant. The monoclonal antibodys (2P11) were identified with indirect immunized fluresent assay and Western blot assay. Indirect ELISA was used to detect the Ig isotype.
We acquired the recombinant P11 protein and prepared a strain of hybridoma cell secreting the MAbs against P11 protein, the MAbs are useful material in vitro diagnosis.
本试验根据GenBank发表的P11基因序列,设计了一对特异性核苷酸引物,用PCR方法获得598bp大小的基因片段,并将其克隆到表达载体上,测序鉴定,成功构建pET-28a(+)/P11表达载体,转化到大肠杆菌表达菌株BL21(DE3)中,诱导表达重组蛋白P11,并进一步通过Ni-NTA亲合层析柱纯化目的蛋白,纯化率达90%。纯化产物经SDS-PAGE鉴定后免疫Balb/c小鼠,用聚乙二醇PEG介导Balb/c小鼠脾细胞与SP2/0骨髓瘤细胞融合,用纯化的P11重组蛋白筛选阳性杂交瘤细胞株,对阳性克隆细胞株经3次有限稀释法克隆化筛选后,最终获得1株能稳定分泌单克隆抗体的杂交瘤细胞株,命名为2G11。对单克隆抗体亚类鉴定结果表明2G11为IgG2b亚类。ELISA试验结果和Western blot分析表明,单抗能与T7噬菌体蛋白和P11蛋白特异结合。这为进一步研究P11蛋白的结构和功能,提高T7噬菌体蛋白芯片的筛选效率以及建立噬菌体检测方法奠定了基础。
Bacteriophage T7 is a lysogeny phage capable of infecting most strains of Escherichia coli, a double standed DNA of genome molecular is 39 936 bp. The capside protein of T7 include major head protein P10A, head assemble protein P10B, neck protein P8, Tail protein P11, P12 and Tail fibers protein P17. T7 phage display system is easy to use and has the capacity to display peptides up to about 1200 amino acids, including stability of insert gene, varity of elution conditions and short of growth cycle. T7 phage protein chip is fixed the bacteriophage T7 which expressed and displayed the exogenous proteins on the chip, the chip could be used for the detection the exogenous proteins. P11 gene was inserted into plasmid pET-28a(+) to construct recombinant plasmid pET-28a(+)/P11. Recombinant P11 protein was expressed under the introduction of 0.1mol/L IPTG and well purified by nickel ion affinity chromatogagh method. SDS-PAGE analysis showed that the molecular weight of the expressed protein was approximately 27 kDa. The Balb/c mice were immunized with the purified protein P11 mixed adjuvant. The monoclonal antibodys (2P11) were identified with indirect immunized fluresent assay and Western blot assay. Indirect ELISA was used to detect the Ig isotype.
We acquired the recombinant P11 protein and prepared a strain of hybridoma cell secreting the MAbs against P11 protein, the MAbs are useful material in vitro diagnosis.
引文
[1] Majlesi,Bordenave G. Role of CD40 in a T cell-mediated negative regulation of Lg production [J]. Immounol, 2001, 166(2) :841-847.
[2] Maloy KJ, Kthdig TM,KeVin J,et al. Intra lymphatic immounization enhances DNA vaccination [J]. Proc Natl Acad Sci USA, 2001, 98(6):3299-3303.
[3] Kilpatrick KE,Wring SA,Walker DH,et al. Rapid development of affinity matured monoclonal antibodies using RIMMOL/LS[J]. Hydrodoma, 1997, 16(4):381-359.
[4] Alligood KJ,Milla M,Rhodes N,et al. Monoclonal antibodies generated against recombi nant ATM support kinase activity[J]. Hybridoma, 2000, 19(4):317-320
[5] Kipatrick KE,Danger DP,Hull-ryde EA,et al. High affinity monoclonal antibodies to PED/pEA215 generated using 5μg DNA[J]. Hybridoma,2000,19(4) :297-302.
[6] Li J,Sai T,Berger M, et al. Human antibodies for immounotherapy development generated via a human B cell hybridoma technology[J].Proc Nat Acad Sci, 2006, 103 (10): 3557-3562.
[7] Smith GP. Filamentous fusion phage: novel expression vectors that displaycloned agains on the virion surface[J]. Science, 1985, 228:1315-1317.
[8] Catherine D. Bacterio phage lambda is a highly stable DNA vaccine delivery vehicle[J]. Vaccine, 2004, (22):2413-2419
[9] Smith GR. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface [J]. Science, 1985, 228(4705):1315-1317.
[10] Jespers LS, Messens JH,Keyser A,et al .Surface expression andligand-based selection of cDNAs used to filamentous phage geneBiotechnology, 1995,13(4):378-382
[11] Maruyama IN, Maruyama HI, Brenner S. Lambda foo:a lambda phage vector for the expression of foreign proteins[J]. Proc Natl Acad Sci USA,1994, 91(17):8273-8277.
[12] Sternberg N, Hoess RH. Display of peptides and proteins on the surface ofBacteriophage lambda[J]. Proc Natl Acad Sci USA .1995, 92(5):1609-1613.
[13] Yang F, Forrer P, Dauter Z, et al. Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD[J]. Nat Struct Biol. 2000,7(3):230-237.
[14] Efimov VP, NePluev IV, Mesyanzhinov VV. Bacterio phage T4 as a surface display vector[J] .Virus Genes, 1995,10(2):173-177
[15] 吴健敏,涂长春,任兆钧. T4噬菌体表面展示技术的研究进展[J]. 病毒学报, 2004, 20 (4):385-388
[16] Ren Z, Black LW. Phage T4 SOC and HOC display of biologically active, full-length proteins on the viral capsid [J]. Gene,1998,215(2):439-444.
[17] Jiang J, Abu-Shilbayeh L, Rao VB.Display of a PorA peptide from Neisseria meningitidis on the bacteriophage T4 capsid surface[J]. Infect Immonu, 1997, 65 (11):4770-4777.
[18] Cafferty J, Griffiths AD,Winter G, et al.Phage antibodies:filamentous phage displaying antibody variable domains[J].Nature, 1990, 348(6301):552-554.
[19] Ren Z J , Lewis GK, Black LW, et al . Phage display of intact domains at high copy number: A system based on SOC, the small outer capsid protein of bacteriophage T4[J]. Protein Science,1996,5:1833-1843
[20] Ren Z, Black LW .Phage T4 SOC and HOC display of biologically active, full-length proteins on the viral capsid[J].Gene,1998, 215(2):439-444.
[21] 郭莉, 汤永民.噬菌体展示系统的研究进展[J].医学分子生物学杂志, 2005, 2(3): 205-209
[22] 贾盘兴.《噬菌体分子生物学》[M].北京:科学出版社.2001:95-98.
[23] Cerritelli ME, Studier FW. Assembly of T7 capsids from independently expressed and purified head protein and scafolding protein [J]. J Mol Biol, 1996, 258(2):286-298.
[24] Smith GP, Petrenko VA. Phage Display [J]. Chem Rev, 1997, 97(2):391-410.
[25] Rosenberg A,Griffin K,Studier FW,et al. T7 Select Phage Display System:A powerful new protein display system: A powerful new protein display system based on bacteriophage T7[J]. Invations, 1996, 6:1-6.
[26] Woiwode TF,Haggerty JE,Katz R,et al. Synthetic compound libraries displayed on the surface of bacteriophage [J].Chem Biol, 2003, 10(9):847-858.
[27] Danner S, Belasco JG. T7 phage display: a novel genetic selection system for cloning RNA-binding proteins from cDNA libraries [J]. Proc Natl Acad Sci USA, 2001, 98(23): 12954-12959.
[28] George P,Valery AP. Phage display [J]. Chen Rev, 1997, 97:391-410.
[29] Kay BK, Adey NB, HeYS,et al. M13 phage library displaying randorn 38-amino-acid peptides as a source of novel sequences with affinity to selected targets[J].Gene, 1993, 12(1):59-65.
[30] Scott J K, S mith G P. A family of coneanaval in a-binding peptides from a hexapeptide epitope library. Proc Natl Acad Sci USA,1992,89:5398-5402.
[31] Wang LF, Hertzog P, Galanis M, et al. Structure-function analysis of human IFN-alpha: mapping of a conformational epitope by homologue scanning [J]. J Immounol, 1994, 152( 2):705-715.
[32] 余传信,朱荫昌,殷旭仁,等.用噬菌体肽库筛选SiC-TPI的模拟抗原表位[J].中国寄生虫学与寄生虫病杂志, 2001, 19(1):11-14.
[33]Davies JM,Ohehir RE,SupHioglu C,et al. Use of phage display technology to in vestigate allergen-antibody interactions[J].J Allergy Clin Immounol,2000,105(61): 1085-1092.
[34] Li CM, Haapalainen M, Lee J, et al. Harpin of Pseudomonas syringae pv.Phaseolicola harbors a protein bindingsite[J]. Mol Plant Microbe Interact, 2005, 18(1) :60- 66.
[35] Li CM, Haapalainen M, Lee J. Harpin of Pseudomonas syringae pv. phaseolicola harbors a protein binding site[J]. Mol Plant Microbe Interact, 2005, 18(1):60-66.
[36] Daniel J. Nelson, Lawrence, et al. Research progress of screening in vivo of phage Display[J]. Acad Sci USA, 2001, 98(7):4107-4112.
[37] Kelly KA, Allport JR, Tsourkas A, et al. Detection of vascular adhesionmolecule-1 expression using a novel multimoda nanoparticle[J].Circ Res, 2005, 96(3):327-336.
[38] Cloutier SM, Chagas JR , Mach JP, et al . Substrate specificity of human kallikrein2(hk2 ) as determined by phage display technology[J]. Eur J Biochem, 2002, 269(11): 2747-2754.
[39] Cheadle C, IvashchenkoY, South V,et al. Identification of a SH3 domain binding motif by screening a random phage display library [J]. J Biol Chem, 1994, 269(39) :24034-24039.
[40] Arap W, HaedickeT, Bernasconi M, et al. Targeting the prostate for destruction through a vascular address[J]. Proc Natl Acad Sci USA, 2002, 99(3):1527-1531.
[41] Ghosh AK, Moreira LA, Jacobs-Lorena M, et al. Plasmodium-mosquitointeractions, phage displaylibraries and transgenic mosquitoes impaired formalariat ransmission[J]. Insect Biochem Mol Biol, 2002, 32(10):1325-1331
[42] Fossa A, Alsoe L, Crameri R, et al.Serological cloning of cancer/testis antigens expressed in prostate cancer using cDNA phage surface display[J]. Cancer Immunol, 2002, 2(1):10-18.
[43] Wang X, Yu J, Sreekumar A, et al. Autoantibody signatures in prostate cancer[J]. N Engl J Med. 2005, 353(12):1224-1235.
[44] Fodor SP,Rava RP,Huang XC,et al. Multiplexed biochemical assays with biological chips[J].Nature, 1993,(23): 364-556.
[45] Weinberger SR, Morris TS, Palak M. Recent trends in protein biochip technology [J].Phamacogenomics, 2000, 1(4):395-416.
[46] Von-Eggeling F, Davies H, Lomas L, et al. Tissue-specific miciodissection coupled with protein chip array technologies: applications in cancer research[J]. Biotechniques, 2000, 29:1066-1070.
[47] Vlahou A,M endrinos S, Kondylis F,et al. Identification of protein changes in bladder cancer patient urines by protein chip SELDI affinity mass spectrometry[J]. Proc Amer Assoc Cancer Res, 2000, 41:852-853.
[48] Kodadek, T. Protein microarrays: prospects and problems[J]. Chemistry & Biology, 8, 105-115
[49] Zhu H, Snyder M. Protein chip technology[J]. Curr Opin Chem Biol,2003,7(1):55-63.
[50] Smith MG, Jona G, Ptacek J, et al. Global analysis of protein function using protein nicroarrys[J]. Mach Ageing Dev,2005,126(1):171-175.
[51] Zhu H, Hu S, Jona G,et al. Severe acute respiratory syndrome diagnostica using a coronavirus protein microarray[J]. Proc Natl Acad Sci U S A.2006 ,103(11):4011-40166.
[52] Ptacek J, Devgan G, Michaud G, et al. Global analysis of protein phosphorylation in yeast[J]. Nature, 2005, 438(7068):679-684
[53] Chen CS, Zhu H. Protein microarrays[J].Biotechniques, 2006,40(4):423,425,427.
[54] Tao SC, Zhu H. Protein chip fabrication by capture of nascent polypeptides[J].Nat Biotechnol.2006, 24(10):1253-1254.
[55] Tenenbaum SA, L ager PJ, Carson CC,et al. Ribonomics:identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays[J]. Methods, 2002, 26(2):191-198.
[56] Bothmann H, Pluckthun A. Selection for a periplasmic factor improving phage display and functional periplasmic expression[J]. Nat Biotech, 1998, 16:376-380.
[2] Maloy KJ, Kthdig TM,KeVin J,et al. Intra lymphatic immounization enhances DNA vaccination [J]. Proc Natl Acad Sci USA, 2001, 98(6):3299-3303.
[3] Kilpatrick KE,Wring SA,Walker DH,et al. Rapid development of affinity matured monoclonal antibodies using RIMMOL/LS[J]. Hydrodoma, 1997, 16(4):381-359.
[4] Alligood KJ,Milla M,Rhodes N,et al. Monoclonal antibodies generated against recombi nant ATM support kinase activity[J]. Hybridoma, 2000, 19(4):317-320
[5] Kipatrick KE,Danger DP,Hull-ryde EA,et al. High affinity monoclonal antibodies to PED/pEA215 generated using 5μg DNA[J]. Hybridoma,2000,19(4) :297-302.
[6] Li J,Sai T,Berger M, et al. Human antibodies for immounotherapy development generated via a human B cell hybridoma technology[J].Proc Nat Acad Sci, 2006, 103 (10): 3557-3562.
[7] Smith GP. Filamentous fusion phage: novel expression vectors that displaycloned agains on the virion surface[J]. Science, 1985, 228:1315-1317.
[8] Catherine D. Bacterio phage lambda is a highly stable DNA vaccine delivery vehicle[J]. Vaccine, 2004, (22):2413-2419
[9] Smith GR. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface [J]. Science, 1985, 228(4705):1315-1317.
[10] Jespers LS, Messens JH,Keyser A,et al .Surface expression andligand-based selection of cDNAs used to filamentous phage geneBiotechnology, 1995,13(4):378-382
[11] Maruyama IN, Maruyama HI, Brenner S. Lambda foo:a lambda phage vector for the expression of foreign proteins[J]. Proc Natl Acad Sci USA,1994, 91(17):8273-8277.
[12] Sternberg N, Hoess RH. Display of peptides and proteins on the surface ofBacteriophage lambda[J]. Proc Natl Acad Sci USA .1995, 92(5):1609-1613.
[13] Yang F, Forrer P, Dauter Z, et al. Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD[J]. Nat Struct Biol. 2000,7(3):230-237.
[14] Efimov VP, NePluev IV, Mesyanzhinov VV. Bacterio phage T4 as a surface display vector[J] .Virus Genes, 1995,10(2):173-177
[15] 吴健敏,涂长春,任兆钧. T4噬菌体表面展示技术的研究进展[J]. 病毒学报, 2004, 20 (4):385-388
[16] Ren Z, Black LW. Phage T4 SOC and HOC display of biologically active, full-length proteins on the viral capsid [J]. Gene,1998,215(2):439-444.
[17] Jiang J, Abu-Shilbayeh L, Rao VB.Display of a PorA peptide from Neisseria meningitidis on the bacteriophage T4 capsid surface[J]. Infect Immonu, 1997, 65 (11):4770-4777.
[18] Cafferty J, Griffiths AD,Winter G, et al.Phage antibodies:filamentous phage displaying antibody variable domains[J].Nature, 1990, 348(6301):552-554.
[19] Ren Z J , Lewis GK, Black LW, et al . Phage display of intact domains at high copy number: A system based on SOC, the small outer capsid protein of bacteriophage T4[J]. Protein Science,1996,5:1833-1843
[20] Ren Z, Black LW .Phage T4 SOC and HOC display of biologically active, full-length proteins on the viral capsid[J].Gene,1998, 215(2):439-444.
[21] 郭莉, 汤永民.噬菌体展示系统的研究进展[J].医学分子生物学杂志, 2005, 2(3): 205-209
[22] 贾盘兴.《噬菌体分子生物学》[M].北京:科学出版社.2001:95-98.
[23] Cerritelli ME, Studier FW. Assembly of T7 capsids from independently expressed and purified head protein and scafolding protein [J]. J Mol Biol, 1996, 258(2):286-298.
[24] Smith GP, Petrenko VA. Phage Display [J]. Chem Rev, 1997, 97(2):391-410.
[25] Rosenberg A,Griffin K,Studier FW,et al. T7 Select Phage Display System:A powerful new protein display system: A powerful new protein display system based on bacteriophage T7[J]. Invations, 1996, 6:1-6.
[26] Woiwode TF,Haggerty JE,Katz R,et al. Synthetic compound libraries displayed on the surface of bacteriophage [J].Chem Biol, 2003, 10(9):847-858.
[27] Danner S, Belasco JG. T7 phage display: a novel genetic selection system for cloning RNA-binding proteins from cDNA libraries [J]. Proc Natl Acad Sci USA, 2001, 98(23): 12954-12959.
[28] George P,Valery AP. Phage display [J]. Chen Rev, 1997, 97:391-410.
[29] Kay BK, Adey NB, HeYS,et al. M13 phage library displaying randorn 38-amino-acid peptides as a source of novel sequences with affinity to selected targets[J].Gene, 1993, 12(1):59-65.
[30] Scott J K, S mith G P. A family of coneanaval in a-binding peptides from a hexapeptide epitope library. Proc Natl Acad Sci USA,1992,89:5398-5402.
[31] Wang LF, Hertzog P, Galanis M, et al. Structure-function analysis of human IFN-alpha: mapping of a conformational epitope by homologue scanning [J]. J Immounol, 1994, 152( 2):705-715.
[32] 余传信,朱荫昌,殷旭仁,等.用噬菌体肽库筛选SiC-TPI的模拟抗原表位[J].中国寄生虫学与寄生虫病杂志, 2001, 19(1):11-14.
[33]Davies JM,Ohehir RE,SupHioglu C,et al. Use of phage display technology to in vestigate allergen-antibody interactions[J].J Allergy Clin Immounol,2000,105(61): 1085-1092.
[34] Li CM, Haapalainen M, Lee J, et al. Harpin of Pseudomonas syringae pv.Phaseolicola harbors a protein bindingsite[J]. Mol Plant Microbe Interact, 2005, 18(1) :60- 66.
[35] Li CM, Haapalainen M, Lee J. Harpin of Pseudomonas syringae pv. phaseolicola harbors a protein binding site[J]. Mol Plant Microbe Interact, 2005, 18(1):60-66.
[36] Daniel J. Nelson, Lawrence, et al. Research progress of screening in vivo of phage Display[J]. Acad Sci USA, 2001, 98(7):4107-4112.
[37] Kelly KA, Allport JR, Tsourkas A, et al. Detection of vascular adhesionmolecule-1 expression using a novel multimoda nanoparticle[J].Circ Res, 2005, 96(3):327-336.
[38] Cloutier SM, Chagas JR , Mach JP, et al . Substrate specificity of human kallikrein2(hk2 ) as determined by phage display technology[J]. Eur J Biochem, 2002, 269(11): 2747-2754.
[39] Cheadle C, IvashchenkoY, South V,et al. Identification of a SH3 domain binding motif by screening a random phage display library [J]. J Biol Chem, 1994, 269(39) :24034-24039.
[40] Arap W, HaedickeT, Bernasconi M, et al. Targeting the prostate for destruction through a vascular address[J]. Proc Natl Acad Sci USA, 2002, 99(3):1527-1531.
[41] Ghosh AK, Moreira LA, Jacobs-Lorena M, et al. Plasmodium-mosquitointeractions, phage displaylibraries and transgenic mosquitoes impaired formalariat ransmission[J]. Insect Biochem Mol Biol, 2002, 32(10):1325-1331
[42] Fossa A, Alsoe L, Crameri R, et al.Serological cloning of cancer/testis antigens expressed in prostate cancer using cDNA phage surface display[J]. Cancer Immunol, 2002, 2(1):10-18.
[43] Wang X, Yu J, Sreekumar A, et al. Autoantibody signatures in prostate cancer[J]. N Engl J Med. 2005, 353(12):1224-1235.
[44] Fodor SP,Rava RP,Huang XC,et al. Multiplexed biochemical assays with biological chips[J].Nature, 1993,(23): 364-556.
[45] Weinberger SR, Morris TS, Palak M. Recent trends in protein biochip technology [J].Phamacogenomics, 2000, 1(4):395-416.
[46] Von-Eggeling F, Davies H, Lomas L, et al. Tissue-specific miciodissection coupled with protein chip array technologies: applications in cancer research[J]. Biotechniques, 2000, 29:1066-1070.
[47] Vlahou A,M endrinos S, Kondylis F,et al. Identification of protein changes in bladder cancer patient urines by protein chip SELDI affinity mass spectrometry[J]. Proc Amer Assoc Cancer Res, 2000, 41:852-853.
[48] Kodadek, T. Protein microarrays: prospects and problems[J]. Chemistry & Biology, 8, 105-115
[49] Zhu H, Snyder M. Protein chip technology[J]. Curr Opin Chem Biol,2003,7(1):55-63.
[50] Smith MG, Jona G, Ptacek J, et al. Global analysis of protein function using protein nicroarrys[J]. Mach Ageing Dev,2005,126(1):171-175.
[51] Zhu H, Hu S, Jona G,et al. Severe acute respiratory syndrome diagnostica using a coronavirus protein microarray[J]. Proc Natl Acad Sci U S A.2006 ,103(11):4011-40166.
[52] Ptacek J, Devgan G, Michaud G, et al. Global analysis of protein phosphorylation in yeast[J]. Nature, 2005, 438(7068):679-684
[53] Chen CS, Zhu H. Protein microarrays[J].Biotechniques, 2006,40(4):423,425,427.
[54] Tao SC, Zhu H. Protein chip fabrication by capture of nascent polypeptides[J].Nat Biotechnol.2006, 24(10):1253-1254.
[55] Tenenbaum SA, L ager PJ, Carson CC,et al. Ribonomics:identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays[J]. Methods, 2002, 26(2):191-198.
[56] Bothmann H, Pluckthun A. Selection for a periplasmic factor improving phage display and functional periplasmic expression[J]. Nat Biotech, 1998, 16:376-380.