重症肌无力抗乙酰胆碱受体单链抗体基因的构建
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摘要
重症肌无力（myasthenia gravis , MG）患者血清中2/3的抗乙酰胆碱受体（acetylcholine receptor, AChR）抗体是针对α亚单位上主要免疫原区（main immunogenic region, MIR）的抗体，因此针对AChR MIR的抗体成为MG致病机制中的主要的致病性抗体。由抗AChR MIR抗体制备的单链抗体（single chain variable fragment, ScFv）做为单价片段既不与AChR交叉结合，也不引起补体经典途径的激活，因此不会引起AChR的丢失。但是，ScFv与MIR结合后能特异性地封闭致病性AChR抗体与AChR MIR结合，从而对AChR起到保护作用。
本文应用PCR从抗AChR MIR抗体扩增重链可变区（heavy chain variable region , VH）基因，应用纯化试剂盒对PCR产物进行纯化。纯化后的VH PCR产物经NcoⅠ和 XhoⅠ酶切、低熔点琼脂糖凝胶电泳回收及纯化后与经同样酶切割并纯化的载体质粒pHEN2连接。将连接物转化E.coli DH5α扩增，分离纯化重组子再经NcoⅠ和XhoⅠ酶切，并用琼脂糖凝胶电泳检查VH基因的正确性。采用同样的方法将轻链可变区（light chain variable region , VL）基因克隆至pHEN2-VH，并转化E.coli HB2151扩增，用ApalⅠ和NotⅠ酶切检查VL基因的正确性。构建的ScFv经测序发现核苷酸序列正确，并且VH和VL基因正确克隆至载体开放读码框架内。已成功地构建抗AChR MIR ScFv基因，为进一步制备基因工程抗体奠定了基础。
Antibody competition experiments suggest that about 2/3 of the antibodies in the sera of myasthenia gravis (MG) patients are directed against the main immunogenic region (MIR) of acetylcholine receptor (AChR). Univalent antibody fragments, such as single chain variable fragment (ScFv), do not cross-link AChR molecules or bind complement, and therefore do not cause AChR loss; on the contrary, such fragments derived from anti-MIR monoclonal antibodies (mAbs) seem capable of protecting the receptor against the loss induced by intact anti-MIR mAb or MG sera.
The heavy chain variable region (VH) gene of a mAb against AChR amplified by polymerase chain reaction (PCR) was purified by Wizard PCR Preps DNA Purification System, then digested with NcoⅠand XhoⅠ. The digested products were run in low gelling temperature agarose eletrophoresis and purified by Wizard PCR Preps DNA Purification System, then ligated into plasmid pHEN2 digested and purified by the same method. There recombinant pHEN2-VH were transformed into E.coli DH5αfor amplification and isolated from E.coli DH5αand digested with NcoⅠand XhoⅠagain. The light chain variable region (VL) gene of the mAb was cloned to pHEN2-VH by the same ways and than the recombinant plasmids were transformed into E.coli HB2151. The VL gene was analysed for an insert of the right size by digestion with ApalⅠand
NotⅠ. The sequencing showed that the nucleotide sequence of cons-
tructed ScFv was correct and cloned into the open reading frame (ORF) in pHEN2. A ScFv gene against the MIR of AChR has been successfully constructed.
本文应用PCR从抗AChR MIR抗体扩增重链可变区（heavy chain variable region , VH）基因，应用纯化试剂盒对PCR产物进行纯化。纯化后的VH PCR产物经NcoⅠ和 XhoⅠ酶切、低熔点琼脂糖凝胶电泳回收及纯化后与经同样酶切割并纯化的载体质粒pHEN2连接。将连接物转化E.coli DH5α扩增，分离纯化重组子再经NcoⅠ和XhoⅠ酶切，并用琼脂糖凝胶电泳检查VH基因的正确性。采用同样的方法将轻链可变区（light chain variable region , VL）基因克隆至pHEN2-VH，并转化E.coli HB2151扩增，用ApalⅠ和NotⅠ酶切检查VL基因的正确性。构建的ScFv经测序发现核苷酸序列正确，并且VH和VL基因正确克隆至载体开放读码框架内。已成功地构建抗AChR MIR ScFv基因，为进一步制备基因工程抗体奠定了基础。
Antibody competition experiments suggest that about 2/3 of the antibodies in the sera of myasthenia gravis (MG) patients are directed against the main immunogenic region (MIR) of acetylcholine receptor (AChR). Univalent antibody fragments, such as single chain variable fragment (ScFv), do not cross-link AChR molecules or bind complement, and therefore do not cause AChR loss; on the contrary, such fragments derived from anti-MIR monoclonal antibodies (mAbs) seem capable of protecting the receptor against the loss induced by intact anti-MIR mAb or MG sera.
The heavy chain variable region (VH) gene of a mAb against AChR amplified by polymerase chain reaction (PCR) was purified by Wizard PCR Preps DNA Purification System, then digested with NcoⅠand XhoⅠ. The digested products were run in low gelling temperature agarose eletrophoresis and purified by Wizard PCR Preps DNA Purification System, then ligated into plasmid pHEN2 digested and purified by the same method. There recombinant pHEN2-VH were transformed into E.coli DH5αfor amplification and isolated from E.coli DH5αand digested with NcoⅠand XhoⅠagain. The light chain variable region (VL) gene of the mAb was cloned to pHEN2-VH by the same ways and than the recombinant plasmids were transformed into E.coli HB2151. The VL gene was analysed for an insert of the right size by digestion with ApalⅠand
NotⅠ. The sequencing showed that the nucleotide sequence of cons-
tructed ScFv was correct and cloned into the open reading frame (ORF) in pHEN2. A ScFv gene against the MIR of AChR has been successfully constructed.
引文
1 Lindstrom J, Shelton D, Fujii Y. Myasthenia gravis. Adv Immunol 1988; 42:
233-84.
2 Oosterhuis H. Clinical aspects. In: De Baets M, Oosterhuis H eds. Myasthenia
Gravis. Boca Ration: CRC Press; 1993.
3 Pordage S. In Willis T: Translation of de Anima Brutorum. 1672; 431.
4 Simpson JA. Myasthenia gravis: a new hypothesis. Scottish Med J 1960; 5: 419.
5 Patrick J, Lindstrom J. Autoimmune response to acetylcholine receptor. Science
1973; 180(88): 871-2.
6 Almon RR, Andrew CG, Appel SH. Serum globulin in myasthenia gravis:
inhibition of alpha-bungarotoxin binding to acetylcholine receptors. Science
1974; 186(4158): 55-7.
7 Lindstrom J, Merlie J, Yogeeswaran G. Biochemical properties of acteylcholine
receptor subunits from Torpedo californica. Biochemistry 1979; 8: 4465-70.
8 Tzartos SJ, Barkas T, Cung MT, et al. Anatomy of the antigenic structure of a
large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor.
Immunol Rev 1998; 163:89-120.
9 Tzartos SJ, Kokla A, Walgrave SL, et al. Localization of the main immunogenic
region of human muscle acetylcholine receptor to residues 67-76 of the alpha
subunit. Proc Natl Acad Sci USA 1988; 85(9): 2899-903.
10 Tzartos SJ, Seybold ME, Lindstrom JM. Specificities of antibodies to acetyl-
choline receptors in sera from myasthenia gravis patients measured by
monoclonal antibodies. Proc Natl Acad Sci USA 1982; 79(1):188-92.
11 Conti-Tronconi B, Tzartos S, Lindstrom J. Monoclonal antibodies as probes of
acetylcholine receptor structure. 2. Binding to native receptor. Biochemistry
1981; 20(8): 2181-91.
12 Engel AG, Lambert EH, Howard FM. Immune complexes (IgG and C3) at
the motor end-plate in myasthenia gravis: ultrastructural and light micro scopic
localization and electrophysiologic correlations. Mayo Clin Proc 1977; 52(5):
267-80.
13 Lennon VA, Lindstrom JM, Seybold ME. Experimental autoimmune
myasthenia: A model of myasthenia gravis in rats and guinea pigs. J Exp Med
1975; 141(6): 1365-75.
14 Baggi F, Andreetta F, Caspani E, et al. Oral administration of immuno-
dominant T-cell epitope downregulates Th1/Th2 cytokines and prevents
experimental myasthenia gravis. J Clin Invest 1999; 104: 1287-95.
15 Im SH, Barchan D, Fuchs S, et al. Suppression of ongoing experimental
myasthenia by oral treatment with an acetylcholine receptor recombinant
fragment. J Clin Invest 1999; 104: 1723-30.
16 Barchan D, Souroujon MC, Im SH, et al. Antigen-specific modulation of
experimental myasthenia gravis: nasal tolerirization with recombinant of the
human acetylcholine receptor alpha-subunit. Natl Acad Sci 1999; 96: 8086-91.
17 Ma CG, Zhang GX, Xiao BG, et al. Celluar mRNA expression of IFN-r、IL-4 and
TGF-β in rat nasally tolerized against EAMG. Clin Exp Immunol 1996; 104:
509-17.
18 Shi FD, Bai XF, Li HL, et al. Nasal tolerance in EAMG: induction of
protective tolerance in primed animals. Clin Exp Immunol 1998; 111: 506-12.
19 Araga S, Xu L, Nakashima K, et al. A peptide vaccine that prevents experi-
mental autoimmune myasthenia gravis by specifically blocking T cell help.
FASEB J 2000; 14: 185-96.
20 Wu LK, Villain M, Galin FS, et al. Prevention and reversal of experimental
autoimmune myasthenia gravis by a monoclonal antibody against acetylcholine
receptor-specific T cells. Cellular Immunology 2001; 208: 107-14.
21 Wu JM, Wu B, Miagkov A, et al. Specific immunotherapy of experimental
myasthenia gravis in vitro: the "guided missile" strategy. Cellular Immunology
2001; 208: 137-47.
22 Takamori M, Maruta T. Immunoadsorption in myasthenia gravis based on
specific ligands mimicking the immunogenic sites of the acetylcholine receptor.
Ther Apher 2001; 5: 340-50.
23 Miyahara T, Oka K, Nakaji S. Specific immunoadsorbent for myasthenia
gravis treatment: development of synthetic peptide designed to remove
antiacetylcholine receptor receptor antibody. Ther Apher 1998; 2: 246-48.
24 Agius MA, Yuen E. Effect of anti-idiotopic antibody on the course of
experimental autoimmune myasthenia gravis. Ann NY Acad Sci 1998; 841:
584-6.
25 Papanastasiou D, Poulas K, Kokla A, et al. Prevention of passively transferred
experimental autoimmune myasthenia gravis by Fab of monoclonal antibodies
directed against the main immunogenic region of the acetylcholine
receptor. J Neuroimmunology 2000; 104: 124-32.
26 Meng F, Stassen MH, Schillberg S, et al. Construction and characterrization
of a single-chain antibody fragment derived from thymus of a patient with
myasthenia gravis. Autoimmunity 2002; 35: 125-33.
27 Loutrari H, Kokla A, Tzaros SJ. Passive transfer of experimental myasthenia
gravis via antigenic modulation of acetylcholine receptor. Eur J Immunol 1992;
22: 2449-52.
28 Graus Y, Meng F, Vincent A, et al. Sequence analysis of anti-AChR antibodies
in experimental autoimmune myasthe niagravis. J Immunol 1995; 154: 6382-96.
29 Stassen MH, Meng F, Melgert E, et al. Experimental autoimmune myasthenia
gravis in mice expressing human Immunoglobulin loci. J Neuroimmunol 2003;
135(1-2): 56-61.
30 Tsantili P, Tzartos SJ, Mamalaki A. High affinity single-chain Fv fragments
protecting the human nicotinic acetylcholine receptor. J Neuroimmunol
1999; 94: 15-27.
31 Papanastasiou D, Mamalaki A, Eliopoulos E, et al. Construction and
characterization of a humanized single chain Fv antibody fragment against the
main immunogenic region of the acetylcholine receptor. J Neuroimmunol 1999;
94: 182-95.
32 Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual.
2nd ed. 1992.
33 孟繁平. 布氏菌质粒实验研究初步探讨. 中国地方病防治杂志1988; 3: 327.
34 Kabat E, Wu T, Foeller C, et al. Sequences of proteins of immunological
interest. 5th ed, No 91-3243. National Institute of Health, Bethesda, MD. 1991.
35 Skerra A, Pluckthun A. Assembly of a functional immunoglobulin Fv fragment
in Escherichia coli. Science 1988; 240(4855): 1038-41.
36 Bird RE, Hardaman KD, Jacobson JW, et al. Single chain antigen binding
proteins. Science 1988; 240(4877): 423-26.
37 Huston JS, Levinson D, Mudgett-Hunter M, et al. Protein engineering of
antibody binding sites: recovery of specific activity in an anti digoxin single-
Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA 1988;
85(16): 5879-83.
38 Hu Z, Sun Y, Garen A. Targeting tumor vasculature endothelial cells and
tumor cells for immunotherapy of human melanoma in a mouse xeno graft
model. Proc Natl Acad Sci USA 1999; 96: 8161-66.
39 Goldberg MR, Heimbrook DC, Russo P, et al. Phase I clinical study of the
recombinant oncotoxin Tp40 in superficial bladder cancer. Clin Cancer Res 1995;
1: 57-61.
40 Rodrigues ML, Presta LG, Kotts CE, et al. Development of a humanized
disulfide stabilized anti-p185 HER2 Fv-beta-lactamase fusion protein for
activation of a cephalosporin doxorubicin prodrug. Cancer Res 1995; 55: 63-70.
41 Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic
amplification of DNA with a thermostable DNA polymerase. Science 1988; 239:
487-91.
42 Anand NN, Mandal S, Mackenzie CR, et al. Bacterial expression and
secretion of various single-chain Fv genes encoding proteins specific for a
Salmonella serotype B O-antigen. J Biol Chem 1991; 266(32): 21874-9.
43 Pantoliano MW, Bird RE, Johnson S, et al. Conformtaional stability, folding,
and ligand-binding affinity of single-chain Fv immunoglobulin fragments
expressed in Escherichia coli. Biochemistry 1991; 30: 10117-25.
233-84.
2 Oosterhuis H. Clinical aspects. In: De Baets M, Oosterhuis H eds. Myasthenia
Gravis. Boca Ration: CRC Press; 1993.
3 Pordage S. In Willis T: Translation of de Anima Brutorum. 1672; 431.
4 Simpson JA. Myasthenia gravis: a new hypothesis. Scottish Med J 1960; 5: 419.
5 Patrick J, Lindstrom J. Autoimmune response to acetylcholine receptor. Science
1973; 180(88): 871-2.
6 Almon RR, Andrew CG, Appel SH. Serum globulin in myasthenia gravis:
inhibition of alpha-bungarotoxin binding to acetylcholine receptors. Science
1974; 186(4158): 55-7.
7 Lindstrom J, Merlie J, Yogeeswaran G. Biochemical properties of acteylcholine
receptor subunits from Torpedo californica. Biochemistry 1979; 8: 4465-70.
8 Tzartos SJ, Barkas T, Cung MT, et al. Anatomy of the antigenic structure of a
large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor.
Immunol Rev 1998; 163:89-120.
9 Tzartos SJ, Kokla A, Walgrave SL, et al. Localization of the main immunogenic
region of human muscle acetylcholine receptor to residues 67-76 of the alpha
subunit. Proc Natl Acad Sci USA 1988; 85(9): 2899-903.
10 Tzartos SJ, Seybold ME, Lindstrom JM. Specificities of antibodies to acetyl-
choline receptors in sera from myasthenia gravis patients measured by
monoclonal antibodies. Proc Natl Acad Sci USA 1982; 79(1):188-92.
11 Conti-Tronconi B, Tzartos S, Lindstrom J. Monoclonal antibodies as probes of
acetylcholine receptor structure. 2. Binding to native receptor. Biochemistry
1981; 20(8): 2181-91.
12 Engel AG, Lambert EH, Howard FM. Immune complexes (IgG and C3) at
the motor end-plate in myasthenia gravis: ultrastructural and light micro scopic
localization and electrophysiologic correlations. Mayo Clin Proc 1977; 52(5):
267-80.
13 Lennon VA, Lindstrom JM, Seybold ME. Experimental autoimmune
myasthenia: A model of myasthenia gravis in rats and guinea pigs. J Exp Med
1975; 141(6): 1365-75.
14 Baggi F, Andreetta F, Caspani E, et al. Oral administration of immuno-
dominant T-cell epitope downregulates Th1/Th2 cytokines and prevents
experimental myasthenia gravis. J Clin Invest 1999; 104: 1287-95.
15 Im SH, Barchan D, Fuchs S, et al. Suppression of ongoing experimental
myasthenia by oral treatment with an acetylcholine receptor recombinant
fragment. J Clin Invest 1999; 104: 1723-30.
16 Barchan D, Souroujon MC, Im SH, et al. Antigen-specific modulation of
experimental myasthenia gravis: nasal tolerirization with recombinant of the
human acetylcholine receptor alpha-subunit. Natl Acad Sci 1999; 96: 8086-91.
17 Ma CG, Zhang GX, Xiao BG, et al. Celluar mRNA expression of IFN-r、IL-4 and
TGF-β in rat nasally tolerized against EAMG. Clin Exp Immunol 1996; 104:
509-17.
18 Shi FD, Bai XF, Li HL, et al. Nasal tolerance in EAMG: induction of
protective tolerance in primed animals. Clin Exp Immunol 1998; 111: 506-12.
19 Araga S, Xu L, Nakashima K, et al. A peptide vaccine that prevents experi-
mental autoimmune myasthenia gravis by specifically blocking T cell help.
FASEB J 2000; 14: 185-96.
20 Wu LK, Villain M, Galin FS, et al. Prevention and reversal of experimental
autoimmune myasthenia gravis by a monoclonal antibody against acetylcholine
receptor-specific T cells. Cellular Immunology 2001; 208: 107-14.
21 Wu JM, Wu B, Miagkov A, et al. Specific immunotherapy of experimental
myasthenia gravis in vitro: the "guided missile" strategy. Cellular Immunology
2001; 208: 137-47.
22 Takamori M, Maruta T. Immunoadsorption in myasthenia gravis based on
specific ligands mimicking the immunogenic sites of the acetylcholine receptor.
Ther Apher 2001; 5: 340-50.
23 Miyahara T, Oka K, Nakaji S. Specific immunoadsorbent for myasthenia
gravis treatment: development of synthetic peptide designed to remove
antiacetylcholine receptor receptor antibody. Ther Apher 1998; 2: 246-48.
24 Agius MA, Yuen E. Effect of anti-idiotopic antibody on the course of
experimental autoimmune myasthenia gravis. Ann NY Acad Sci 1998; 841:
584-6.
25 Papanastasiou D, Poulas K, Kokla A, et al. Prevention of passively transferred
experimental autoimmune myasthenia gravis by Fab of monoclonal antibodies
directed against the main immunogenic region of the acetylcholine
receptor. J Neuroimmunology 2000; 104: 124-32.
26 Meng F, Stassen MH, Schillberg S, et al. Construction and characterrization
of a single-chain antibody fragment derived from thymus of a patient with
myasthenia gravis. Autoimmunity 2002; 35: 125-33.
27 Loutrari H, Kokla A, Tzaros SJ. Passive transfer of experimental myasthenia
gravis via antigenic modulation of acetylcholine receptor. Eur J Immunol 1992;
22: 2449-52.
28 Graus Y, Meng F, Vincent A, et al. Sequence analysis of anti-AChR antibodies
in experimental autoimmune myasthe niagravis. J Immunol 1995; 154: 6382-96.
29 Stassen MH, Meng F, Melgert E, et al. Experimental autoimmune myasthenia
gravis in mice expressing human Immunoglobulin loci. J Neuroimmunol 2003;
135(1-2): 56-61.
30 Tsantili P, Tzartos SJ, Mamalaki A. High affinity single-chain Fv fragments
protecting the human nicotinic acetylcholine receptor. J Neuroimmunol
1999; 94: 15-27.
31 Papanastasiou D, Mamalaki A, Eliopoulos E, et al. Construction and
characterization of a humanized single chain Fv antibody fragment against the
main immunogenic region of the acetylcholine receptor. J Neuroimmunol 1999;
94: 182-95.
32 Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual.
2nd ed. 1992.
33 孟繁平. 布氏菌质粒实验研究初步探讨. 中国地方病防治杂志1988; 3: 327.
34 Kabat E, Wu T, Foeller C, et al. Sequences of proteins of immunological
interest. 5th ed, No 91-3243. National Institute of Health, Bethesda, MD. 1991.
35 Skerra A, Pluckthun A. Assembly of a functional immunoglobulin Fv fragment
in Escherichia coli. Science 1988; 240(4855): 1038-41.
36 Bird RE, Hardaman KD, Jacobson JW, et al. Single chain antigen binding
proteins. Science 1988; 240(4877): 423-26.
37 Huston JS, Levinson D, Mudgett-Hunter M, et al. Protein engineering of
antibody binding sites: recovery of specific activity in an anti digoxin single-
Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA 1988;
85(16): 5879-83.
38 Hu Z, Sun Y, Garen A. Targeting tumor vasculature endothelial cells and
tumor cells for immunotherapy of human melanoma in a mouse xeno graft
model. Proc Natl Acad Sci USA 1999; 96: 8161-66.
39 Goldberg MR, Heimbrook DC, Russo P, et al. Phase I clinical study of the
recombinant oncotoxin Tp40 in superficial bladder cancer. Clin Cancer Res 1995;
1: 57-61.
40 Rodrigues ML, Presta LG, Kotts CE, et al. Development of a humanized
disulfide stabilized anti-p185 HER2 Fv-beta-lactamase fusion protein for
activation of a cephalosporin doxorubicin prodrug. Cancer Res 1995; 55: 63-70.
41 Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic
amplification of DNA with a thermostable DNA polymerase. Science 1988; 239:
487-91.
42 Anand NN, Mandal S, Mackenzie CR, et al. Bacterial expression and
secretion of various single-chain Fv genes encoding proteins specific for a
Salmonella serotype B O-antigen. J Biol Chem 1991; 266(32): 21874-9.
43 Pantoliano MW, Bird RE, Johnson S, et al. Conformtaional stability, folding,
and ligand-binding affinity of single-chain Fv immunoglobulin fragments
expressed in Escherichia coli. Biochemistry 1991; 30: 10117-25.