DAF的原核表达，重折叠，ELISA检测系统的建立，及其在大鼠体内的代谢和生物学活性的测定
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摘要
本研究的主要目的是表达足量的可溶性促衰变因子(DAF)蛋白用于动物试验,测定DAF在血清中的代谢规律和对补体系统的抑制效果。在本研究中,充分采用了如PCR、载体构建、转化、蛋白诱导表达和液相色谱等技术得到了高度纯化的蛋白质。进而采用单克隆抗体技术,制备了20株抗-大鼠DAF(rDAF)的单克隆抗体,建立了检测可溶性rDAF的ELISA系统(最低浓度下限可达10 ng/ml)和Western blot用单抗株(可检出40 ng rDAF蛋白)。通过大鼠的在体试验证明rDAF在大鼠体内代谢非常快且对大鼠血浆补体抑制程度很小,其本身并不适合作为一种补体抑制剂使用。除此以外,还完成了以下两个工作:建立了新的高效折叠复性DAF蛋白的方案;探索了可溶性表达DAF的可能性并揭示了NusA-DAF融合蛋白形成可溶性包涵体的原因。
The purpose of the present study is to produce enough amount of soluble DAF for in vivo studies, i.e. the metabolization of soluble DAF in the blood and its inhibitive effect to the complement system. In order to achieve this goal, the 4 short concensus repeats (SCR1-4) of rat DAF (rDAF) and human DAF (hDAF) were successfully overexpressed in E. coli. The inclusion body was purified and refolded to obtain soluble and active DAF. Then, the recombinant rDAF was used as an immunongen to produce monoclonal antibodies against rDAF. Finally, an ELISA system was established in which as little as 10 ng/ml of rDAF could be detected. A monoclonal antibody which could detect as little as 40 ng of rDAF was isolated. The results of the in vivo experiment showed that rDAF was metabolized rapidly in the blood. Within 90 min after injection, it could not be detected. Even at the highest concentration, the negative effect of soluble DAF against the complement system was not detected. This result showed that soluble DAF is not a good candidate of complement inhibitor. In the mean time, two other studies were done. First, an efficienct and economic way of refolding DAF was established. Second, the possibility of soluble expression of DAF was explored and a possible reason was given for the soluble inclusion bodies which were formed by the NusA-DAF fusion protein.
The purpose of the present study is to produce enough amount of soluble DAF for in vivo studies, i.e. the metabolization of soluble DAF in the blood and its inhibitive effect to the complement system. In order to achieve this goal, the 4 short concensus repeats (SCR1-4) of rat DAF (rDAF) and human DAF (hDAF) were successfully overexpressed in E. coli. The inclusion body was purified and refolded to obtain soluble and active DAF. Then, the recombinant rDAF was used as an immunongen to produce monoclonal antibodies against rDAF. Finally, an ELISA system was established in which as little as 10 ng/ml of rDAF could be detected. A monoclonal antibody which could detect as little as 40 ng of rDAF was isolated. The results of the in vivo experiment showed that rDAF was metabolized rapidly in the blood. Within 90 min after injection, it could not be detected. Even at the highest concentration, the negative effect of soluble DAF against the complement system was not detected. This result showed that soluble DAF is not a good candidate of complement inhibitor. In the mean time, two other studies were done. First, an efficienct and economic way of refolding DAF was established. Second, the possibility of soluble expression of DAF was explored and a possible reason was given for the soluble inclusion bodies which were formed by the NusA-DAF fusion protein.
引文
1. Abbas AK, Lichtman AH. Cellular and Molecular Immunology北京大学医学出版社; 2003.
2. Huber-Lang M, Sarma JV, Zetoune FS, Rittirsch D, Neff TA, McGuire SR, Lambris JD, Warner RL, Flierl MA, Hoesel LM, Gebhard F, Younger JG, Drouin SM, Wetsel RA, Ward PA. Generation of C5a in the absence of C3: a new complement activation pathway. Nat Med 2006;12(6):682-687.
3. Westwood OMR, Nelson PN, Hay FC. Rheumatoid factors: what's new? Rheumatology 2006;45(4):379-385.
4. Chadban SJ, Atkins RC. Glomerulonephritis. Lancet 2005;365(9473):1797-1806.
5. Urich E, Gutcher I, Prinz M, Becher B. Autoantibody-mediated demyelination depends on complement activation but not activatory Fc-receptors. Proc Natl Acad Sci U S A 2006;103(49):18697-18702.
6. Berger T, Rubner P, Schautzer F, Egg R, Ulmer H, Mayringer I, Dilitz E, Deisenhammer F, Reindl M. Antimyelin antibodies as a predictor of clinically definite multiple sclerosis after a first demyelinating event. N Engl J Med 2003;349(2):139-145.
7. Lindstrom JM, Einarson BL, Lennon VA, Seybold ME. Pathological mechanisms in experimental autoimmune myasthenia gravis. I. Immunogenicity of syngeneic muscle acetylcholine receptor and quantitative extraction of receptor and antibody-receptor complexes from muscles of rats with experimental automimmune myasthenia gravis. J Exp Med 1976;144(3):726-738.
8. Maselli RA, Richman DP, Wollmann RL. Inflammation at the neuromuscular junction in myasthenia gravis. Neurology 1991;41(9):1497-1504.
9. Fazekas A, Komoly S, Bozsik B, Szobor A. Myasthenia gravis: demonstration of membrane attack complex in muscle end-plates. Clin Neuropathol 1986;5(2):78-83.
10. Sahashi K, Engel AG, Lambert EH, Howard FM, Jr. Ultrastructural localization of the terminal and lytic ninth complement component (C9) at the motor end-plate in myasthenia gravis. J Neuropathol Exp Neurol 1980;39(2):160-172.
11. Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW, Piette JC, Brey R, Derksen R, Harris EN, Hughes GR, Triplett DA, Khamashta MA. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome: report of an international workshop. Arthritis Rheum 1999;42(7):1309-1311.
12. Girardi G, Berman J, Redecha P, Spruce L, Thurman JM, Kraus D, Hollmann TJ, Casali P, Caroll MC, Wetsel RA, Lambris JD, Holers VM, Salmon JE. Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome. J Clin Invest 2003;112(11):1644-1654.
13. Lindstrom JM, Seybold ME, Lennon VA, Whittingham S, Duane DD. Antibody to acetylcholine receptor in myasthenia gravis: Prevalence, clinical correlates, and diagnostic value. Neurology 1976;26(11):1054-.
14. Vincent A. Unravelling the pathogenesis of myasthenia gravis. Nat Rev Immunol 2002;2(10):797-804.
15. Evoli A, Tonali PA, Padua L, Monaco ML, Scuderi F, Batocchi AP, Marino M, Bartoccioni E. Clinical correlates with anti-MuSK antibodies in generalized seronegative myasthenia gravis. Brain 2003;126(Pt 10):2304-2311.
16. Hayashi A, Shiono H, Ohta M, Ohta K, Okumura M, Sawa Y. Heterogeneity of immunopathological features of AChR/MuSK autoantibody-negative myasthenia gravis. J Neuroimmunol 2007;189(1-2):163-168.
17. Sitaru C, Chiriac MT, Mihai S, Buning J, Gebert A, Ishiko A, Zillikens D. Induction of complement-fixing autoantibodies against type VII collagen results in subepidermal blistering in mice. J Immunol 2006;177(5):3461-3468.
18. Newsom-Davis J. Lambert-Eaton myasthenic syndrome. Rev Neurol (Paris) 2004;160(2):177-180.
19. Wang Y, Rollins SA, Madri JA, Matis LA. Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease. Proc Natl Acad Sci U S A 1995;92(19):8955-8959.
20. Zhou Y, Gong B, Lin F, Rother RP, Medof ME, Kaminski HJ. Anti-C5 antibody teatment ameliorates weakness in experimentally acquired Myasthenia Gravis. J Immunol 2007;179(12):8562-8567.
21. Ballow M, Cochrane CG. Two Anticomplementary Factors in Cobra Venom: Hemolysis of Guinea Pig Erythrocytes by One of Them. J Immunol 1969;103(5):944-952.
22. Alper CA, Balavitch D. Cobra venom factor: evidence for its being altered cobra C3 (the third component of complement). Science 1976;191(4233):1275-1276.
23. Leventhal JR, Dalmasso AP, Cromwell JW, Platt JL, Manivel CJ, Bolman RM, 3rd, Matas AJ. Prolongation of cardiac xenograft survival by depletion of complement. Transplantation 1993;55(4):857-865; discussion 865-856.
24. Kolln J, Spillner E, Andra J, Klensang K, Bredehorst R. Complement inactivation by recombinant human C3 derivatives. J Immunol 2004;173(9):5540-5545.
25. Hepburn NJ, Williams AS, Nunn MA, Chamberlain-Banoub JC, Hamer J, Morgan BP, Harris CL. In vivo characterization and therapeutic efficacy of a C5-specific inhibitor from the soft tick ornithodoros moubata. J Biol Chem 2007;282(11):8292-8299.
26. Piddlesden SJ, Jiang S, Levin JL, Vincent A, Morgan BP. Soluble complement receptor 1 (sCR1) protects against experimental autoimmune myasthenia gravis. J Neuroimmunol 1996;71(1-2):173-177.
27. Harris CL, Williams AS, Linton SM, Morgan BP. Coupling complement regulatorsto immunoglobulin domains generates effective anti-complement reagents with extended half-life in vivo. Clin Exp Immunol 2002;129(2):198-207.
28. Hepburn NJ, Chamberlain-Banoub JL, Williams AS, Morgan BP, Harris CL. Prevention of experimental autoimmune myasthenia gravis by rat Crry-Ig: A model agent for long-term complement inhibition in vivo. Mol Immunol 2008;45:395-405.
29. Higgins PJ, Ko JL, Lobell R, Sardonini C, Alessi MK, Yeh CG. A soluble chimeric complement inhibitory protein that possesses both decay-accelerating and factor I cofactor activities. J Immunol 1997;158(6):2872-2881.
30. Harris CL, Hughes CE, Williams AS, Goodfellow I, Evans DJ, Caterson B, Morgan BP. Generation of anti-complement "prodrugs": cleavable reagents for specific delivery of complement regulators to disease sites. J Biol Chem 2003;278(38):36068-36076.
31. Morgan BP. Complement Methods and Protocols. Totowa: Humana Press; 2000.
32. Sambrook J, Russell DW. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 2001.
33. Miwa T, Okada N, Okada H. Alternative exon usage in the 3' region of a single gene generates glycosylphosphatidylinositol-anchored and transmembrane forms of rat decay-accelerating factor. Immunogenetics 2000;1:129-137.
34. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680-685.
35. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254.
36. White J, Lukacik P, Esser D, Steward M, Giddings N, Bright JR, Fritchley SJ, Morgan BP, Lea SM, Smith GP, Smith RA. Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli. Protein Sci 2004;13(9):2406-2415.
37. Brodbeck WG, Liu D, Sperry J, Mold C, Medof ME. Localization of classical and alternative pathway regulatory activity within the decay-accelerating factor. J Immunol 1996;156(7):2528-2533.
38. Hinchliffe SJ, Spiller OB, Morgan BP. Molecular cloning and functional characterization of the rat analogue of human decay-accelerating factor (CD55) J Immunol 1998(161 ):5695-5703.
39. Brodbeck WG, Kuttner-Kondo L, Medof ME. Structure/function studies of human decay-accelerating factor. Immunology 2000;101:104-111.
40. Kuttner-Kondo LA, Mitchell L, Hourcade DE, Medof ME. Characterization of the active sites in decay-accelerating factor. J Immunol 2001;167(4):2164-2171.
41. Lin F, Immormino RM, Shoham M. Bulk production and functional analyses of mouse CD55's native and deglycosylated active domains. Archives of Biochemistry and Biophysic, 2001 93:67-72.
42. Williams P, Chaudhry Y, Goodfellow IG, Billington J, Powell R, Spiller OB, Evans DJ, Lea S. Mapping CD55 function. the structure of two pathogen-binding domains at 1.7 A. J Biol Chem 2003;278(12):10691-10696.
43. Dodd I, Mossakowska DE, Camilleri P, Haran M, Hensley P, Lawlor EJ, McBay DL, Pindar W, Smith RA. Overexpression in Escherichia coli, folding, purification, and characterization of the first three short consensus repeat modules of human complement receptor type 1. Protein Expr Purif 1995;6(6):727-736.
44.马歇克DR,门永JT,布格斯RR,克努特MW,布伦南WA, H.林S.蛋白质纯化与鉴定实验指南.科学出版社; 2000.
45. Tsumoto K, Umetsu M, Kumagai I, Ejima D, Philo JS, Arakawa T. Role of arginine in protein refolding, solubilization, and purification. Biotechnol Prog 2004;20(5):1301-1308.
46. Quaggio RB, Ferro JA, Monteiro PB, Reinach FC. Cloning and expression of chicken skeletal muscle troponin I in Escherichia coli: The role of rare codons on the expression level. Protein Sci 1993;2(6):1053-1056.
47. Lu D, Liu Z. Dynamic Redox Environment-intensified disulfide bond shuffling for protein refolding in vitro: molecular simulation and experimental validation. J Phys Chem B 2008;112(47):15127-15133.
48. Garvey JS, Cremer NE, Sussdorf DH. Methods In Immunology; 1977. 207-209.
49. Fearon DT, Austen KF. Activation of the alternative complement pathway with rabbit erythrocytes by circumvention of the regulatory action of endogenous control proteins. J Exp Med 1977;146(1):22-33.
50. Harris CL, Spiller OB, Morgan BP. Human and rodent decay-accelerating factors (CD55) are not species restricted in their complement-inhibiting activities. Immunology 2000;100:462-470.
51. Lane LC. A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis. Anal Biochem 1978;86(2):655-664.
52. Derman AI, Prinz WA, Belin D, Beckwith J. Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science 1993;262(5140):1744-1747.
53. Prinz WA, Aslund F, Holmgren A, Beckwith J. The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J Biol Chem 1997;272(25):15661-15667.
54. Bessette PH, Aslund F, Beckwith J, Georgiou G. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proceedings of the National Academy of Sciences 1999;96(24):13703-13708.
55.徐志凯.实用单克隆抗体技术.西安:陕西科学技术出版社; 1991.
56.金伯泉.细胞和分子免疫学实验技术.西安:第四军医大学出版社; 2002.
57. Zhang H-f, Lu S, Morrison SL, Tomlinson S. Targeting of functional antibody-decay accelerating factor (DAF) fusion proteins to a cell surface. J Biol Chem 2001:M100436200.
58. Zhang H-f, Yu J, Bajwa E, Morrison SL, Tomlinson S. Targeting of functional antibody-CD59 fusion proteins to a cell surface. J Clin Invest 1999;103(1):55-61.
2. Huber-Lang M, Sarma JV, Zetoune FS, Rittirsch D, Neff TA, McGuire SR, Lambris JD, Warner RL, Flierl MA, Hoesel LM, Gebhard F, Younger JG, Drouin SM, Wetsel RA, Ward PA. Generation of C5a in the absence of C3: a new complement activation pathway. Nat Med 2006;12(6):682-687.
3. Westwood OMR, Nelson PN, Hay FC. Rheumatoid factors: what's new? Rheumatology 2006;45(4):379-385.
4. Chadban SJ, Atkins RC. Glomerulonephritis. Lancet 2005;365(9473):1797-1806.
5. Urich E, Gutcher I, Prinz M, Becher B. Autoantibody-mediated demyelination depends on complement activation but not activatory Fc-receptors. Proc Natl Acad Sci U S A 2006;103(49):18697-18702.
6. Berger T, Rubner P, Schautzer F, Egg R, Ulmer H, Mayringer I, Dilitz E, Deisenhammer F, Reindl M. Antimyelin antibodies as a predictor of clinically definite multiple sclerosis after a first demyelinating event. N Engl J Med 2003;349(2):139-145.
7. Lindstrom JM, Einarson BL, Lennon VA, Seybold ME. Pathological mechanisms in experimental autoimmune myasthenia gravis. I. Immunogenicity of syngeneic muscle acetylcholine receptor and quantitative extraction of receptor and antibody-receptor complexes from muscles of rats with experimental automimmune myasthenia gravis. J Exp Med 1976;144(3):726-738.
8. Maselli RA, Richman DP, Wollmann RL. Inflammation at the neuromuscular junction in myasthenia gravis. Neurology 1991;41(9):1497-1504.
9. Fazekas A, Komoly S, Bozsik B, Szobor A. Myasthenia gravis: demonstration of membrane attack complex in muscle end-plates. Clin Neuropathol 1986;5(2):78-83.
10. Sahashi K, Engel AG, Lambert EH, Howard FM, Jr. Ultrastructural localization of the terminal and lytic ninth complement component (C9) at the motor end-plate in myasthenia gravis. J Neuropathol Exp Neurol 1980;39(2):160-172.
11. Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW, Piette JC, Brey R, Derksen R, Harris EN, Hughes GR, Triplett DA, Khamashta MA. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome: report of an international workshop. Arthritis Rheum 1999;42(7):1309-1311.
12. Girardi G, Berman J, Redecha P, Spruce L, Thurman JM, Kraus D, Hollmann TJ, Casali P, Caroll MC, Wetsel RA, Lambris JD, Holers VM, Salmon JE. Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome. J Clin Invest 2003;112(11):1644-1654.
13. Lindstrom JM, Seybold ME, Lennon VA, Whittingham S, Duane DD. Antibody to acetylcholine receptor in myasthenia gravis: Prevalence, clinical correlates, and diagnostic value. Neurology 1976;26(11):1054-.
14. Vincent A. Unravelling the pathogenesis of myasthenia gravis. Nat Rev Immunol 2002;2(10):797-804.
15. Evoli A, Tonali PA, Padua L, Monaco ML, Scuderi F, Batocchi AP, Marino M, Bartoccioni E. Clinical correlates with anti-MuSK antibodies in generalized seronegative myasthenia gravis. Brain 2003;126(Pt 10):2304-2311.
16. Hayashi A, Shiono H, Ohta M, Ohta K, Okumura M, Sawa Y. Heterogeneity of immunopathological features of AChR/MuSK autoantibody-negative myasthenia gravis. J Neuroimmunol 2007;189(1-2):163-168.
17. Sitaru C, Chiriac MT, Mihai S, Buning J, Gebert A, Ishiko A, Zillikens D. Induction of complement-fixing autoantibodies against type VII collagen results in subepidermal blistering in mice. J Immunol 2006;177(5):3461-3468.
18. Newsom-Davis J. Lambert-Eaton myasthenic syndrome. Rev Neurol (Paris) 2004;160(2):177-180.
19. Wang Y, Rollins SA, Madri JA, Matis LA. Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease. Proc Natl Acad Sci U S A 1995;92(19):8955-8959.
20. Zhou Y, Gong B, Lin F, Rother RP, Medof ME, Kaminski HJ. Anti-C5 antibody teatment ameliorates weakness in experimentally acquired Myasthenia Gravis. J Immunol 2007;179(12):8562-8567.
21. Ballow M, Cochrane CG. Two Anticomplementary Factors in Cobra Venom: Hemolysis of Guinea Pig Erythrocytes by One of Them. J Immunol 1969;103(5):944-952.
22. Alper CA, Balavitch D. Cobra venom factor: evidence for its being altered cobra C3 (the third component of complement). Science 1976;191(4233):1275-1276.
23. Leventhal JR, Dalmasso AP, Cromwell JW, Platt JL, Manivel CJ, Bolman RM, 3rd, Matas AJ. Prolongation of cardiac xenograft survival by depletion of complement. Transplantation 1993;55(4):857-865; discussion 865-856.
24. Kolln J, Spillner E, Andra J, Klensang K, Bredehorst R. Complement inactivation by recombinant human C3 derivatives. J Immunol 2004;173(9):5540-5545.
25. Hepburn NJ, Williams AS, Nunn MA, Chamberlain-Banoub JC, Hamer J, Morgan BP, Harris CL. In vivo characterization and therapeutic efficacy of a C5-specific inhibitor from the soft tick ornithodoros moubata. J Biol Chem 2007;282(11):8292-8299.
26. Piddlesden SJ, Jiang S, Levin JL, Vincent A, Morgan BP. Soluble complement receptor 1 (sCR1) protects against experimental autoimmune myasthenia gravis. J Neuroimmunol 1996;71(1-2):173-177.
27. Harris CL, Williams AS, Linton SM, Morgan BP. Coupling complement regulatorsto immunoglobulin domains generates effective anti-complement reagents with extended half-life in vivo. Clin Exp Immunol 2002;129(2):198-207.
28. Hepburn NJ, Chamberlain-Banoub JL, Williams AS, Morgan BP, Harris CL. Prevention of experimental autoimmune myasthenia gravis by rat Crry-Ig: A model agent for long-term complement inhibition in vivo. Mol Immunol 2008;45:395-405.
29. Higgins PJ, Ko JL, Lobell R, Sardonini C, Alessi MK, Yeh CG. A soluble chimeric complement inhibitory protein that possesses both decay-accelerating and factor I cofactor activities. J Immunol 1997;158(6):2872-2881.
30. Harris CL, Hughes CE, Williams AS, Goodfellow I, Evans DJ, Caterson B, Morgan BP. Generation of anti-complement "prodrugs": cleavable reagents for specific delivery of complement regulators to disease sites. J Biol Chem 2003;278(38):36068-36076.
31. Morgan BP. Complement Methods and Protocols. Totowa: Humana Press; 2000.
32. Sambrook J, Russell DW. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 2001.
33. Miwa T, Okada N, Okada H. Alternative exon usage in the 3' region of a single gene generates glycosylphosphatidylinositol-anchored and transmembrane forms of rat decay-accelerating factor. Immunogenetics 2000;1:129-137.
34. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680-685.
35. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254.
36. White J, Lukacik P, Esser D, Steward M, Giddings N, Bright JR, Fritchley SJ, Morgan BP, Lea SM, Smith GP, Smith RA. Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli. Protein Sci 2004;13(9):2406-2415.
37. Brodbeck WG, Liu D, Sperry J, Mold C, Medof ME. Localization of classical and alternative pathway regulatory activity within the decay-accelerating factor. J Immunol 1996;156(7):2528-2533.
38. Hinchliffe SJ, Spiller OB, Morgan BP. Molecular cloning and functional characterization of the rat analogue of human decay-accelerating factor (CD55) J Immunol 1998(161 ):5695-5703.
39. Brodbeck WG, Kuttner-Kondo L, Medof ME. Structure/function studies of human decay-accelerating factor. Immunology 2000;101:104-111.
40. Kuttner-Kondo LA, Mitchell L, Hourcade DE, Medof ME. Characterization of the active sites in decay-accelerating factor. J Immunol 2001;167(4):2164-2171.
41. Lin F, Immormino RM, Shoham M. Bulk production and functional analyses of mouse CD55's native and deglycosylated active domains. Archives of Biochemistry and Biophysic, 2001 93:67-72.
42. Williams P, Chaudhry Y, Goodfellow IG, Billington J, Powell R, Spiller OB, Evans DJ, Lea S. Mapping CD55 function. the structure of two pathogen-binding domains at 1.7 A. J Biol Chem 2003;278(12):10691-10696.
43. Dodd I, Mossakowska DE, Camilleri P, Haran M, Hensley P, Lawlor EJ, McBay DL, Pindar W, Smith RA. Overexpression in Escherichia coli, folding, purification, and characterization of the first three short consensus repeat modules of human complement receptor type 1. Protein Expr Purif 1995;6(6):727-736.
44.马歇克DR,门永JT,布格斯RR,克努特MW,布伦南WA, H.林S.蛋白质纯化与鉴定实验指南.科学出版社; 2000.
45. Tsumoto K, Umetsu M, Kumagai I, Ejima D, Philo JS, Arakawa T. Role of arginine in protein refolding, solubilization, and purification. Biotechnol Prog 2004;20(5):1301-1308.
46. Quaggio RB, Ferro JA, Monteiro PB, Reinach FC. Cloning and expression of chicken skeletal muscle troponin I in Escherichia coli: The role of rare codons on the expression level. Protein Sci 1993;2(6):1053-1056.
47. Lu D, Liu Z. Dynamic Redox Environment-intensified disulfide bond shuffling for protein refolding in vitro: molecular simulation and experimental validation. J Phys Chem B 2008;112(47):15127-15133.
48. Garvey JS, Cremer NE, Sussdorf DH. Methods In Immunology; 1977. 207-209.
49. Fearon DT, Austen KF. Activation of the alternative complement pathway with rabbit erythrocytes by circumvention of the regulatory action of endogenous control proteins. J Exp Med 1977;146(1):22-33.
50. Harris CL, Spiller OB, Morgan BP. Human and rodent decay-accelerating factors (CD55) are not species restricted in their complement-inhibiting activities. Immunology 2000;100:462-470.
51. Lane LC. A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis. Anal Biochem 1978;86(2):655-664.
52. Derman AI, Prinz WA, Belin D, Beckwith J. Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science 1993;262(5140):1744-1747.
53. Prinz WA, Aslund F, Holmgren A, Beckwith J. The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J Biol Chem 1997;272(25):15661-15667.
54. Bessette PH, Aslund F, Beckwith J, Georgiou G. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proceedings of the National Academy of Sciences 1999;96(24):13703-13708.
55.徐志凯.实用单克隆抗体技术.西安:陕西科学技术出版社; 1991.
56.金伯泉.细胞和分子免疫学实验技术.西安:第四军医大学出版社; 2002.
57. Zhang H-f, Lu S, Morrison SL, Tomlinson S. Targeting of functional antibody-decay accelerating factor (DAF) fusion proteins to a cell surface. J Biol Chem 2001:M100436200.
58. Zhang H-f, Yu J, Bajwa E, Morrison SL, Tomlinson S. Targeting of functional antibody-CD59 fusion proteins to a cell surface. J Clin Invest 1999;103(1):55-61.
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