PATHOGENICITY AND SEQUENCE ANALYSIS OF A MOUSE MONOCLONAL ANTIBODY AGAINST HUMAN ACETYLCHOLINE RECEPTOR IN MYASTHENIA GRAVIS
作者：Meng Fanping(孟繁平)1，Yang Kangjuan(杨康鹃)1， Zhang Qinggao(张庆镐)1，Y.Graus2，M. de Baets2ayn, http://www.100md.com
单位：ayn, http://www.100md.com
关键词：重症肌无力 单克隆抗体 乙酰胆碱受体 致病性 核苷酸序列ayn, http://www.100md.com
免疫学杂志990101 摘 要 通过对乙酰胆碱受体(AChR)自身抗体分子结构以及与致病性关系的研究探讨重症肌无力(MG)及其动物模型——实验性自身免疫性重症肌无力(EAMG)的发病机理。AChR抗体被动转移至大鼠后诱导出明显的EAMG。全身肌肉AChR损失率和体重减轻率达47.2±15.3%和13.4±2.2%。这株AChR抗体的重链可变区基因由小鼠Q52胚系基因编码，其同源性为94.8%，将这株抗体的重链和轻链可变区、尤其是互补决定区(CDR)的核苷酸和氨基酸序列与其他致病性AChR抗体比较发现，能诱导MG和EAMG的致病性AChR抗体的结构并不是完全一致的。ayn, http://www.100md.com
中图号 R392.1ayn, http://www.100md.com
Abstract The pathogenesis of myasthenia gravis(MG) on the animal model of experimental autoimmune myasthenia gravis (EAME) was investigated. The pathogenicity of a mouse monoclonal antibody against human acetylcholine receptor (AChR) was determined on the rats and the sequences of the variable regions of the antibody analysed. The anti-AChR antibody studied could induce EAMG. AChR and weight loss by 47.2±15.3% and 13.4±2.2% respectively after passively transferred into rats. The heavy chain genetic element of the antibody， which showed a 94.8% homology with the most closely related germline VH， was derived from the Q52 germline family. Comparison of the variable region sequences， especially the complementarity determining regions (CDRs) with the antibody and other pathogenic anti-AChR antibodies indicates that not is the unique structure of pathogenic anti-AChR antibodies needed in mediating MG and EAMG.
Key words Myasthenia gravis， Monoclonal antibodies， Acetylcholine receptor， Pathogenicity， Nucleotide sequencekk5/l, 百拇医药
Myasthenia gravis(MG) is an organ specific autoimmune disease mediated by autoantibodies against the acetylcholine receptor(AChR). Its animal model-experimental autoimmune myasthenia gravis(EAMG)， closely resembling human MG in both immunological and clinical signs， can be induced by immunization with purified AChR or passive transfer of anti-AChR antibodies[1，2]. Binding of anti-AChR antibodies to AChR at the neuromuscular junction leads to AChR loss by antigenic modulation， complement dependent focal lysis and direct functional inhibition of AChR[3]. The AChR is a well characterized autoantigen[4]. The pathogenic antibodies are exclusively found to be those that are directed against a region on the α-subunit， which includes the amino acid sequence 67-76， termed the main immunogenic region(MIR). In the present study we have analysed the sequences of the variable region of a mouse anti-AChR monoclonal antibody (McAb) specifically directed against MIR. This will allow us to understand the pathogenesis of MG and EAMG.
MATERIALS AND METHODS{g], http://www.100md.com
Anti-AChR McAb The anti-AChR McAb D6(a kind gift of Dr. A.Vincent， Neurosciences group， Institute of Molecular Medicine， John Radcliffe Hospital， Oxford， United Kingdom) was made from Balb/c mice immunized with human purified AChR as previously described[5].{g], http://www.100md.com
Passive transfer of anti-AChR McAb D6 The potential of anti-AChR McAb D6 to induce clinical signs of EAMG was determined by passive transfer of McAb D6 to 8～10 week old female Lewis rats. A group of 5 animals were intraperitoneally injec-ted with 5ml 20× concentrated culture supernatant. Control animals were injected with 5 ml PBS. The rats were sacrificed 48 hours after injection of McAb D6. The severity of EAMG was established by a strength duration test performed according to Lennon[2]. Clinical signs of EAMG were expressed as 0(no obvious abnormalities)，+(decreased strength)，++(head down， hunched posture， weak grip)，+++(no grip， moribund). Loss of AChR was confirmed by measurement of the muscle AChR concentration in whole carcasses according to Lindstrom[1].{g], http://www.100md.com
Cloning of the variable regions of anti-AChR McAb D6 Total RNA was isolated from hybridoma cells which secreted McAb D6 using RNAzol (Cinna/Biotecx Laboratories Inc， Houston TX，USA). Oligo dT primed first strand cDNA was synthesized using AMV reverse transcriptase (promega， USA) and used as template for polymerase chain reaction (PCR) amplification. Heavy(H) and light(L) chain PCR products were diges-ted with EcoR 1 and BamH 1， and subsquently li-gated onto vector Bluescript M13 SK plasmid(Stratagene， La Jolla， CA，USA). The recombinant plasmids were transformed into competent E. coli DH5 α bacteria. The positive clones were identified by digestion using EcoR 1 and BalmH 1[6]
Sequencing and sequence comparison The recombinant plasmid DNA were sequenced with Bluescript KS and SK primer sites and a T7 sequencing kit (Pharmacia， Woerden， The Netherlands ) using the dideoxy chain termination sequencing precedure[7]. The homology of McAb D6 with other antibody sequences in the EMBL Data Library (Heidelberg， Germany) was performed by FASTA computer search. The sequences of H and L chain variable regions of McAb D6 have been accepted by the EMBL Data Library (H chain AC No. X80942 and L chain AC No. X80941).iaw, 百拇医药
RESULTSiaw, 百拇医药
In vivo pathogenicity of anti-AChR McAb D6 McAb D6 was able to induce muscular weakness and severe AChR loss. Rats receiving McAb D6 developed grade + ++ muscular weakness 48 hours after injection. At this time point McAb D6 injec-ted rats showed 47.2%±15.3% AChR loss and 13.4%±2.2% weight loss. Whereas， the control rats showed only 0.2%±0.6% weight loss (see Fig1).
iaw, 百拇医药
Fig 1Weight and ACHR loss as meature for EAMG severityiaw, 百拇医药
The relative weight and total ACHR loss in whole carcasses were determined 48 hours after injection of anti-ACHR Mchab D6.Weight loss is expressed as percen-tage of the weight at the start of the experiment.ACHR loss is expressed as percentage of the ACHR concentration(pmol ACHR/100g muscle in order to compare animals of different body weight)in unmanipulated control rats.
Sequences of variable region of anti-AChR McAb D6 The rearranged gene of the variable region of McAb D6 H chain is 324 bp in length (the first 24 bp in the H chain upstream primer not included)， determining the codons 9-113 of V domain (see Fig 2). McAb D6 uses a VH gene derived from mouse Q52 germline family (EMBL Data Library， AC No. X14515). The McAb D6 VH is joined with D and JH gene belonging to JH3 segment[8]. The rearranged gene of the variable region of McAb D6 L chain is 297 bp in length (the first 24 bp in the L chain upstream primer not included)， determining the codons 9-107 of V domain(see Fig 3). The VL gene used by McAb D6 is a member of mouse Vk5 subgroup[9] and associated with Jk belonging to Jk1 segment[10].|'\, http://www.100md.com
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ctg3, 百拇医药
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Fig 2 Comparison of the nucleotide and amino acid sequences of heavy chain variable regions of anti-AChR McAb D6 with the most closely related germline gene gegments.5, 百拇医药
CDR regions are indicated A
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gtc2, 百拇医药
acc2, 百拇医药
atc2, 百拇医药
aca2, 百拇医药
tgt2, 百拇医药
cga2, 百拇医药
gca
agtj6, 百拇医药
gag
j6, 百拇医药
Wj6, 百拇医药
Yj6, 百拇医药
Qj6, 百拇医药
Qj6, 百拇医药
Kj6, 百拇医药
Qj6, 百拇医药
Gj6, 百拇医药
Kj6, 百拇医药
Sj6, 百拇医药
Pj6, 百拇医药
Qj6, 百拇医药
Fj6, 百拇医药
aatj6, 百拇医药
attj6, 百拇医药
aacj6, 百拇医药
agtj6, 百拇医药
tacj6, 百拇医药
ttaj6, 百拇医药
gcaj6, 百拇医药
tggj6, 百拇医药
tatj6, 百拇医药
cagj6, 百拇医药
cagj6, 百拇医药
aaaj6, 百拇医药
cagj6, 百拇医药
ggaj6, 百拇医药
aaaj6, 百拇医药
tctj6, 百拇医药
cct
cag;[px^hv, 百拇医药
ttc;[px^hv, 百拇医药
L;[px^hv, 百拇医药
V;[px^hv, 百拇医药
Y
;[px^hv, 百拇医药
G;[px^hv, 百拇医药
V;[px^hv, 百拇医药
P;[px^hv, 百拇医药
S;[px^hv, 百拇医药
R;[px^hv, 百拇医药
F;[px^hv, 百拇医药
S;[px^hv, 百拇医药
G;[px^hv, 百拇医药
S;[px^hv, 百拇医药
ctg;[px^hv, 百拇医药
gtc;[px^hv, 百拇医药
tat;[px^hv, 百拇医药
aat;[px^hv, 百拇医药
gca;[px^hv, 百拇医药
aaa;[px^hv, 百拇医药
acc;[px^hv, 百拇医药
tta;[px^hv, 百拇医药
gca;[px^hv, 百拇医药
gaa;[px^hv, 百拇医药
gaa;[px^hv, 百拇医药
gta;[px^hv, 百拇医药
cca;[px^hv, 百拇医药
tca;[px^hv, 百拇医药
agg;[px^hv, 百拇医药
ttc;[px^hv, 百拇医药
agt
ggc{+gxp6), 百拇医药
agt{+gxp6), 百拇医药
G{+gxp6), 百拇医药
S{+gxp6), 百拇医药
G{+gxp6), 百拇医药
T{+gxp6), 百拇医药
Q{+gxp6), 百拇医药
F{+gxp6), 百拇医药
S{+gxp6), 百拇医药
L{+gxp6), 百拇医药
K{+gxp6), 百拇医药
I{+gxp6), 百拇医药
N{+gxp6), 百拇医药
S{+gxp6), 百拇医药
L{+gxp6), 百拇医药
Q{+gxp6), 百拇医药
P{+gxp6), 百拇医药
E{+gxp6), 百拇医药
D{+gxp6), 百拇医药
F{+gxp6), 百拇医药
G{+gxp6), 百拇医药
gga{+gxp6), 百拇医药
tca{+gxp6), 百拇医药
ggc{+gxp6), 百拇医药
aca{+gxp6), 百拇医药
cag{+gxp6), 百拇医药
ttt{+gxp6), 百拇医药
tct{+gxp6), 百拇医药
ctg{+gxp6), 百拇医药
aag{+gxp6), 百拇医药
atc{+gxp6), 百拇医药
aac
agc\62&}r, 百拇医药
ctg\62&}r, 百拇医药
cag\62&}r, 百拇医药
cct\62&}r, 百拇医药
gaa\62&}r, 百拇医药
gat\62&}r, 百拇医药
ttt\62&}r, 百拇医药
ggg\62&}r, 百拇医药
T\62&}r, 百拇医药
Y\62&}r, 百拇医药
Y\62&}r, 百拇医药
C
\62&}r, 百拇医药
F\62&}r, 百拇医药
G\62&}r, 百拇医药
G\62&}r, 百拇医药
G\62&}r, 百拇医药
T\62&}r, 百拇医药
K\62&}r, 百拇医药
cat\62&}r, 百拇医药
tat\62&}r, 百拇医药
tac\62&}r, 百拇医药
tgt\62&}r, 百拇医药
caa\62&}r, 百拇医药
cat\62&}r, 百拇医药
cat\62&}r, 百拇医药
tat\62&}r, 百拇医药
ggt\62&}r, 百拇医药
cct\62&}r, 百拇医药
ccg\62&}r, 百拇医药
tgg\62&}r, 百拇医药
acg
ttcn, 百拇医药
ggtn, 百拇医药
ggan, 百拇医药
ggcn, 百拇医药
accn, 百拇医药
aaan, 百拇医药
Ln, 百拇医药
Qn, 百拇医药
In, 百拇医药
Kn, 百拇医药
ctan, 百拇医药
caan, 百拇医药
atcn, 百拇医药
aaan, 百拇医药
Fig 3 Nucleotide and deduced amino acid sequences of light chain variable region of anti-ACHR MCAB D6n, 百拇医药
CDR regions are indicatedn, 百拇医药
DISCUSSIONn, 百拇医药
A mouse anti-AChR McAb was analysed for structural properties in relation to its potential to cause AChR loss and muscular weakness in the passive transfer EAMG model. McAb D6 is direc-ted against the MIR which was confirmed by complete inhibition with McAb ＃35[11] and by binding to the sequence α 65～78 of human AChR[12]. In this study McAb D6 was demonstrated to be able to induce EAMG in rats， indicating that McAb D6 is a pathogenic anti-AChR antibody. This finding is in concordance with previous study in which pathogenicity of anti-AChR MIR McAb was confirmed[13].
Sequence analysis of anti-AChR McAb D6 reveals the characterization at the molecular level of the VH and VL gene segment usage. The comparison between the expressed VH gene and its most closely related germline counter parts shows some nucleotide differences. The VH sequence of McAb D6 shares 94.8% homology at the nucleotide level with the germline Q52 family. Alignment of the VH nucleotide sequence of McAb D6 with that of Q52 germline gene segment reveals 14 nucleotide substitutions， comprised in 13 codons， 9 of which give rise to amino acid replacement， 6 of them are located in the CDRs (complemetarity determining regions)， 2 in the CDR1 and 4 in the CDR2. The nucleotide substitutions seen in the VH gene of McAb D6 may result from somatic mutations driven by antigen AChR. The D gene sequence used in McAb D6 shows less than 60% homology with the germline D gene segments so far described[9]. This probably is the result of extensive deletion or insertion during VH-D-JH recombination events. Alternatively， the D gene may be derived from a different， not yet characterized germline gene.m&d*'/9, 百拇医药
One of the key question in the pathogenesis of MG is the contribution of individual antibody specificities to AChR destruction. Comparison of the variable regions between anti-AChR McAb D6 and A7[6] or G10[14]， which are pathogenic anti-AChR McAbs directed against MIR， shows that the overall sequence homology of both H and L chains is less than 65% and 40% respectively both at nucleotide and amino acid level. However， all the three anti-AChR MIR McAbs above could bind to AChR at the neuromuscular junction in human muscle cryosections (results not shown)， sugges-ting that not is the unique structure of pathogenic anti-AChR antibodies needed in mediating MG and EAMG.More knowledge about the structure of anti-AChR antibodies in relation to their pathogenicity will lead us to the understanding of the question.
作者单位：Meng Fanping(孟繁平)Yang Kangjuan(杨康鹃) Zhang Qinggao(张庆镐) Department of Microbiology and Immunology，Yanbian University Colleye of Medicine，Yanji 133000ahdq-2c, http://www.100md.com
Y.Graus M. de Baets Department of Immunology，Faculty of Medicine ，Univerity of Limburg，The Netherlandsahdq-2c, http://www.100md.com
第一作者：男，40岁，硕士，教授ahdq-2c, http://www.100md.com
REFERENCESahdq-2c, http://www.100md.com
[1] Lindstrom JM， Einarson BL，Lennon VA， et al. Pathological mechanisms in experimental myasthenia gravis. I.Immunogenicity of syngeneic muscle acetylcholine receptor and quantitative extraction of receptor and antibody-receptor complexes from muscles of rats with experimental autoimmune myasthenia gravis. J Exp Med， 1976， 144：726ahdq-2c, http://www.100md.com
[2] Lennon VA， Lindstrom JM，Seybold ME.Experimental autoimmune myasthenia： a model of myasthenia gravis in rats and guinea pigs. J Exp Med， 1975， 141：1365ahdq-2c, http://www.100md.com
[3] Graus Y， de Baets M.Myasthenia gravis： an autoimmune response against the acetylcholine receptor. Immunol Res， 1993， 12：78ahdq-2c, http://www.100md.com
[4] Gomez C，Richman D.Anti-acetylcholine receptor antibodies directed against the α-BT binding site induce a unique form of experimental myasthenia. Proc Natl Acad Sci USA，1983，80：4089
[5] Whiting P，Vincent A， Schluep M， et al. Monoclonal antibodies that distinguish between normal and denervated human acetylcholine receptor. J Neuroimmunol， 1986，11：223)km]6t#, 百拇医药
[6] 孟繁平，杨康鹃，Graus Y，等.AChR单抗(A7)诱导大鼠的实验性重症肌无力的分子基础.中华微生物学和免疫学杂志，1996，16(1)：45)km]6t#, 百拇医药
[7] Sanger F，Nicklen S，Coulson AR.DNA sequencing with chain-terminating inhibitors. Proc Nat1 Acad Sci USA，1977，74：5463)km]6t#, 百拇医药
[8] Sakano H， Maki R，Kurosawa Y， et al. Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy-chain genes. Nature， 1980，286：676)km]6t#, 百拇医药
[9] Kabat E，Wu T，Perry H， et al. Sequences of proteins of immunological interest. 5th ed. NIH publication， 1991)km]6t#, 百拇医药
[10] Sakano H，Huppi K， Heinrich G， et al. Sequences at the somatic recombination sites of immunoglobulin light-chain genes. Nature， 1979，280：288)km]6t#, 百拇医药
[11] Kordossi A，Tzartos S.Monoclonal antibodies against the main immunogenic region of the acetylcholine receptor. Mapping on the intact molecle. J Neuroimmunol， 1989，23：35)km]6t#, 百拇医药
[12] Wood H，Beeson D，Vincent A， et al. Epitopes on human acetylcholine receptor α-subunit：binding of monoclonal antibodies to recombinant and synthetic peptides. Biochem Soc Trans， 1989，17：220)km]6t#, 百拇医药
[13] Tzartos S，Hochschwender S，Vasquez P， et al. Passive transfer of experimental autoimmune myasthenia gravis by monoclonal antibodies to the main immunogenic region of the acetylcholine receptor. J Neuroimmunol， 1987，15：185)km]6t#, 百拇医药
[14] 孟繁平，杨康鹃，Graus Y， et al.抗人乙酰胆碱受体单抗的致病性和可变区序列分析.中华神经科杂志，1996，29(5)：283)km]6t#, 百拇医药
(1997-12-03收稿；1998-03-24修回)(Meng Fanping(孟繁平)1，Yang Kangjuan(杨康鹃)1， Zhang Qinggao(张庆镐)1，Y.Graus2，M. de Baets2)
单位：ayn, http://www.100md.com
关键词：重症肌无力 单克隆抗体 乙酰胆碱受体 致病性 核苷酸序列ayn, http://www.100md.com
免疫学杂志990101 摘 要 通过对乙酰胆碱受体(AChR)自身抗体分子结构以及与致病性关系的研究探讨重症肌无力(MG)及其动物模型——实验性自身免疫性重症肌无力(EAMG)的发病机理。AChR抗体被动转移至大鼠后诱导出明显的EAMG。全身肌肉AChR损失率和体重减轻率达47.2±15.3%和13.4±2.2%。这株AChR抗体的重链可变区基因由小鼠Q52胚系基因编码，其同源性为94.8%，将这株抗体的重链和轻链可变区、尤其是互补决定区(CDR)的核苷酸和氨基酸序列与其他致病性AChR抗体比较发现，能诱导MG和EAMG的致病性AChR抗体的结构并不是完全一致的。ayn, http://www.100md.com
中图号 R392.1ayn, http://www.100md.com
Abstract The pathogenesis of myasthenia gravis(MG) on the animal model of experimental autoimmune myasthenia gravis (EAME) was investigated. The pathogenicity of a mouse monoclonal antibody against human acetylcholine receptor (AChR) was determined on the rats and the sequences of the variable regions of the antibody analysed. The anti-AChR antibody studied could induce EAMG. AChR and weight loss by 47.2±15.3% and 13.4±2.2% respectively after passively transferred into rats. The heavy chain genetic element of the antibody， which showed a 94.8% homology with the most closely related germline VH， was derived from the Q52 germline family. Comparison of the variable region sequences， especially the complementarity determining regions (CDRs) with the antibody and other pathogenic anti-AChR antibodies indicates that not is the unique structure of pathogenic anti-AChR antibodies needed in mediating MG and EAMG.
Key words Myasthenia gravis， Monoclonal antibodies， Acetylcholine receptor， Pathogenicity， Nucleotide sequencekk5/l, 百拇医药
Myasthenia gravis(MG) is an organ specific autoimmune disease mediated by autoantibodies against the acetylcholine receptor(AChR). Its animal model-experimental autoimmune myasthenia gravis(EAMG)， closely resembling human MG in both immunological and clinical signs， can be induced by immunization with purified AChR or passive transfer of anti-AChR antibodies[1，2]. Binding of anti-AChR antibodies to AChR at the neuromuscular junction leads to AChR loss by antigenic modulation， complement dependent focal lysis and direct functional inhibition of AChR[3]. The AChR is a well characterized autoantigen[4]. The pathogenic antibodies are exclusively found to be those that are directed against a region on the α-subunit， which includes the amino acid sequence 67-76， termed the main immunogenic region(MIR). In the present study we have analysed the sequences of the variable region of a mouse anti-AChR monoclonal antibody (McAb) specifically directed against MIR. This will allow us to understand the pathogenesis of MG and EAMG.
MATERIALS AND METHODS{g], http://www.100md.com
Anti-AChR McAb The anti-AChR McAb D6(a kind gift of Dr. A.Vincent， Neurosciences group， Institute of Molecular Medicine， John Radcliffe Hospital， Oxford， United Kingdom) was made from Balb/c mice immunized with human purified AChR as previously described[5].{g], http://www.100md.com
Passive transfer of anti-AChR McAb D6 The potential of anti-AChR McAb D6 to induce clinical signs of EAMG was determined by passive transfer of McAb D6 to 8～10 week old female Lewis rats. A group of 5 animals were intraperitoneally injec-ted with 5ml 20× concentrated culture supernatant. Control animals were injected with 5 ml PBS. The rats were sacrificed 48 hours after injection of McAb D6. The severity of EAMG was established by a strength duration test performed according to Lennon[2]. Clinical signs of EAMG were expressed as 0(no obvious abnormalities)，+(decreased strength)，++(head down， hunched posture， weak grip)，+++(no grip， moribund). Loss of AChR was confirmed by measurement of the muscle AChR concentration in whole carcasses according to Lindstrom[1].{g], http://www.100md.com
Cloning of the variable regions of anti-AChR McAb D6 Total RNA was isolated from hybridoma cells which secreted McAb D6 using RNAzol (Cinna/Biotecx Laboratories Inc， Houston TX，USA). Oligo dT primed first strand cDNA was synthesized using AMV reverse transcriptase (promega， USA) and used as template for polymerase chain reaction (PCR) amplification. Heavy(H) and light(L) chain PCR products were diges-ted with EcoR 1 and BamH 1， and subsquently li-gated onto vector Bluescript M13 SK plasmid(Stratagene， La Jolla， CA，USA). The recombinant plasmids were transformed into competent E. coli DH5 α bacteria. The positive clones were identified by digestion using EcoR 1 and BalmH 1[6]
Sequencing and sequence comparison The recombinant plasmid DNA were sequenced with Bluescript KS and SK primer sites and a T7 sequencing kit (Pharmacia， Woerden， The Netherlands ) using the dideoxy chain termination sequencing precedure[7]. The homology of McAb D6 with other antibody sequences in the EMBL Data Library (Heidelberg， Germany) was performed by FASTA computer search. The sequences of H and L chain variable regions of McAb D6 have been accepted by the EMBL Data Library (H chain AC No. X80942 and L chain AC No. X80941).iaw, 百拇医药
RESULTSiaw, 百拇医药
In vivo pathogenicity of anti-AChR McAb D6 McAb D6 was able to induce muscular weakness and severe AChR loss. Rats receiving McAb D6 developed grade + ++ muscular weakness 48 hours after injection. At this time point McAb D6 injec-ted rats showed 47.2%±15.3% AChR loss and 13.4%±2.2% weight loss. Whereas， the control rats showed only 0.2%±0.6% weight loss (see Fig1).
iaw, 百拇医药Fig 1Weight and ACHR loss as meature for EAMG severityiaw, 百拇医药
The relative weight and total ACHR loss in whole carcasses were determined 48 hours after injection of anti-ACHR Mchab D6.Weight loss is expressed as percen-tage of the weight at the start of the experiment.ACHR loss is expressed as percentage of the ACHR concentration(pmol ACHR/100g muscle in order to compare animals of different body weight)in unmanipulated control rats.
Sequences of variable region of anti-AChR McAb D6 The rearranged gene of the variable region of McAb D6 H chain is 324 bp in length (the first 24 bp in the H chain upstream primer not included)， determining the codons 9-113 of V domain (see Fig 2). McAb D6 uses a VH gene derived from mouse Q52 germline family (EMBL Data Library， AC No. X14515). The McAb D6 VH is joined with D and JH gene belonging to JH3 segment[8]. The rearranged gene of the variable region of McAb D6 L chain is 297 bp in length (the first 24 bp in the L chain upstream primer not included)， determining the codons 9-107 of V domain(see Fig 3). The VL gene used by McAb D6 is a member of mouse Vk5 subgroup[9] and associated with Jk belonging to Jk1 segment[10].|'\, http://www.100md.com
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P|'\, http://www.100md.com
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S|'\, http://www.100md.com
L|'\, http://www.100md.com
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I/:2, http://www.100md.com
T/:2, http://www.100md.com
C/:2, http://www.100md.com
T/:2, http://www.100md.com
V/:2, http://www.100md.com
S/:2, http://www.100md.com
G/:2, http://www.100md.com
F/:2, http://www.100md.com
D6/:2, http://www.100md.com
cct/:2, http://www.100md.com
ggc/:2, http://www.100md.com
ctg/:2, http://www.100md.com
gtg/:2, http://www.100md.com
gcg/:2, http://www.100md.com
ccc/:2, http://www.100md.com
tca/:2, http://www.100md.com
cag/:2, http://www.100md.com
agc/:2, http://www.100md.com
ctg/:2, http://www.100md.com
tcc/:2, http://www.100md.com
atc/:2, http://www.100md.com
aca/:2, http://www.100md.com
tgc/:2, http://www.100md.com
acc/:2, http://www.100md.com
gtc/:2, http://www.100md.com
tca/:2, http://www.100md.com
gga/:2, http://www.100md.com
ttc/:2, http://www.100md.com
Q52/:2, http://www.100md.com
-/:2, http://www.100md.com
-/:2, http://www.100md.com
-a
-]m(60, 百拇医药
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-]m(60, 百拇医药
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-]m(60, 百拇医药
-]m(60, 百拇医药
-]m(60, 百拇医药
-]m(60, 百拇医药
-]m(60, 百拇医药
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-]m(60, 百拇医药
-]m(60, 百拇医药
-]m(60, 百拇医药
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S]m(60, 百拇医药
L]m(60, 百拇医药
T]m(60, 百拇医药
G]m(60, 百拇医药
Y]m(60, 百拇医药
G]m(60, 百拇医药
V]m(60, 百拇医药
N]m(60, 百拇医药
W]m(60, 百拇医药
V]m(60, 百拇医药
R]m(60, 百拇医药
Q]m(60, 百拇医药
P]m(60, 百拇医药
P]m(60, 百拇医药
G]m(60, 百拇医药
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tca!y$me, 百拇医药
tta!y$me, 百拇医药
acc!y$me, 百拇医药
ggc!y$me, 百拇医药
tat!y$me, 百拇医药
ggt!y$me, 百拇医药
gta!y$me, 百拇医药
aac!y$me, 百拇医药
tgg!y$me, 百拇医药
gtt!y$me, 百拇医药
cgc!y$me, 百拇医药
cag!y$me, 百拇医药
cct!y$me, 百拇医药
cca!y$me, 百拇医药
gga!y$me, 百拇医药
aag!y$me, 百拇医药
ggt!y$me, 百拇医药
ctg!y$me, 百拇医药
gag!y$me, 百拇医药
S!y$me, 百拇医药
H!y$me, 百拇医药
Q52!y$me, 百拇医药
-!y$me, 百拇医药
-!y$me, 百拇医药
-!y$me, 百拇医药
-!y$me, 百拇医药
a-!y$me, 百拇医药
-
-cu, 百拇医药
c-cu, 百拇医药
-cu, 百拇医药
-cu, 百拇医药
-cu, 百拇医药
-cu, 百拇医药
-cu, 百拇医药
-cu, 百拇医药
-cu, 百拇医药
-cu, 百拇医药
-cu, 百拇医药
-cu, 百拇医药
-cu, 百拇医药
─cu, 百拇医药
Wcu, 百拇医药
Lcu, 百拇医药
Gcu, 百拇医药
Vcu, 百拇医药
Icu, 百拇医药
Wcu, 百拇医药
Gcu, 百拇医药
Dcu, 百拇医药
Gcu, 百拇医药
Scu, 百拇医药
Tcu, 百拇医药
Dcu, 百拇医药
Ycu, 百拇医药
Kcu, 百拇医药
Scu, 百拇医药
Acu, 百拇医药
Lcu, 百拇医药
Kcu, 百拇医药
S
D6{f5+, 百拇医药
ttg{f5+, 百拇医药
ctg{f5+, 百拇医药
gga{f5+, 百拇医药
gtg{f5+, 百拇医药
ata{f5+, 百拇医药
tgg{f5+, 百拇医药
ggt{f5+, 百拇医药
gat{f5+, 百拇医药
gga{f5+, 百拇医药
agc{f5+, 百拇医药
aca{f5+, 百拇医药
gac{f5+, 百拇医药
tat{f5+, 百拇医药
aaa{f5+, 百拇医药
tac{f5+, 百拇医药
gct{f5+, 百拇医药
ctc{f5+, 百拇医药
aaa{f5+, 百拇医药
tcc{f5+, 百拇医药
V{f5+, 百拇医药
S{f5+, 百拇医药
T{f5+, 百拇医药
N{f5+, 百拇医药
Q52{f5+, 百拇医药
-{f5+, 百拇医药
-{f5+, 百拇医药
-t-{f5+, 百拇医药
-{f5+, 百拇医药
-{f5+, 百拇医药
-{f5+, 百拇医药
a-
-qv+]n$o, http://www.100md.com
-qv+]n$o, http://www.100md.com
-qv+]n$o, http://www.100md.com
-qv+]n$o, http://www.100md.com
ac-qv+]n$o, http://www.100md.com
-qv+]n$o, http://www.100md.com
-tqv+]n$o, http://www.100md.com
-qv+]n$o, http://www.100md.com
-qv+]n$o, http://www.100md.com
-qv+]n$o, http://www.100md.com
-qv+]n$o, http://www.100md.com
-qv+]n$o, http://www.100md.com
Rqv+]n$o, http://www.100md.com
Lqv+]n$o, http://www.100md.com
Sqv+]n$o, http://www.100md.com
Iqv+]n$o, http://www.100md.com
Sqv+]n$o, http://www.100md.com
Rqv+]n$o, http://www.100md.com
Dqv+]n$o, http://www.100md.com
Nqv+]n$o, http://www.100md.com
Sqv+]n$o, http://www.100md.com
Kqv+]n$o, http://www.100md.com
Rqv+]n$o, http://www.100md.com
Qqv+]n$o, http://www.100md.com
Vqv+]n$o, http://www.100md.com
Fqv+]n$o, http://www.100md.com
Lqv+]n$o, http://www.100md.com
Kqv+]n$o, http://www.100md.com
Mqv+]n$o, http://www.100md.com
Nqv+]n$o, http://www.100md.com
Sqv+]n$o, http://www.100md.com
D6qv+]n$o, http://www.100md.com
aga
ctg3, 百拇医药
agc3, 百拇医药
atc3, 百拇医药
agc3, 百拇医药
agg3, 百拇医药
gac3, 百拇医药
aac3, 百拇医药
tcc3, 百拇医药
aag3, 百拇医药
aga3, 百拇医药
caa3, 百拇医药
gtt3, 百拇医药
ttc3, 百拇医药
tta3, 百拇医药
aaa3, 百拇医药
atg3, 百拇医药
aac3, 百拇医药
agt3, 百拇医药
K3, 百拇医药
S3, 百拇医药
Q523, 百拇医药
-3, 百拇医药
-3, 百拇医药
-3, 百拇医药
-3, 百拇医药
-3, 百拇医药
-a-3, 百拇医药
-3, 百拇医药
-3, 百拇医药
-3, 百拇医药
-3, 百拇医药
-c
-jo?o@, 百拇医药
-jo?o@, 百拇医药
-jo?o@, 百拇医药
-jo?o@, 百拇医药
-jo?o@, 百拇医药
-jo?o@, 百拇医药
-jo?o@, 百拇医药
-jo?o@, 百拇医药
─jo?o@, 百拇医药
Ljo?o@, 百拇医药
Qjo?o@, 百拇医药
Tjo?o@, 百拇医药
Djo?o@, 百拇医药
Djo?o@, 百拇医药
Tjo?o@, 百拇医药
Ajo?o@, 百拇医药
Rjo?o@, 百拇医药
Yjo?o@, 百拇医药
Yjo?o@, 百拇医药
Cjo?o@, 百拇医药
Ajo?o@, 百拇医药
Rjo?o@, 百拇医药
Gjo?o@, 百拇医药
Gjo?o@, 百拇医药
Yjo?o@, 百拇医药
Rjo?o@, 百拇医药
Ajo?o@, 百拇医药
Mjo?o@, 百拇医药
D6jo?o@, 百拇医药
ctgjo?o@, 百拇医药
caajo?o@, 百拇医药
actjo?o@, 百拇医药
gat
gace$7[?7, 百拇医药
acae$7[?7, 百拇医药
gcce$7[?7, 百拇医药
agge$7[?7, 百拇医药
tate$7[?7, 百拇医药
tace$7[?7, 百拇医药
tgte$7[?7, 百拇医药
gcce$7[?7, 百拇医药
agae$7[?7, 百拇医药
ggge$7[?7, 百拇医药
ggce$7[?7, 百拇医药
tace$7[?7, 百拇医药
agge$7[?7, 百拇医药
gcte$7[?7, 百拇医药
atge$7[?7, 百拇医药
Me$7[?7, 百拇医药
Q52e$7[?7, 百拇医药
-ce$7[?7, 百拇医药
-e$7[?7, 百拇医药
-e$7[?7, 百拇医药
-e$7[?7, 百拇医药
-e$7[?7, 百拇医药
-e$7[?7, 百拇医药
-e$7[?7, 百拇医药
-t-e$7[?7, 百拇医药
-ce$7[?7, 百拇医药
-e$7[?7, 百拇医药
-e$7[?7, 百拇医药
-e$7[?7, 百拇医药
-e$7[?7, 百拇医药
─e$7[?7, 百拇医药
D
F.5, 百拇医药
W.5, 百拇医药
G.5, 百拇医药
Q.5, 百拇医药
G.5, 百拇医药
T.5, 百拇医药
L.5, 百拇医药
V.5, 百拇医药
T.5, 百拇医药
V.5, 百拇医药
S.5, 百拇医药
A.5, 百拇医药
D6.5, 百拇医药
gac.5, 百拇医药
ttc.5, 百拇医药
tgg.5, 百拇医药
ggt.5, 百拇医药
caa.5, 百拇医药
ggg.5, 百拇医药
act.5, 百拇医药
ctg.5, 百拇医药
g6tc.5, 百拇医药
act.5, 百拇医药
g6tc.5, 百拇医药
tct.5, 百拇医药
gca.5, 百拇医药
Fig 2 Comparison of the nucleotide and amino acid sequences of heavy chain variable regions of anti-AChR McAb D6 with the most closely related germline gene gegments.5, 百拇医药
CDR regions are indicated A
S2, 百拇医药
L2, 百拇医药
S2, 百拇医药
A2, 百拇医药
S2, 百拇医药
V2, 百拇医药
G2, 百拇医药
E2, 百拇医药
T2, 百拇医药
V2, 百拇医药
T2, 百拇医药
I2, 百拇医药
T2, 百拇医药
C
2, 百拇医药gcc2, 百拇医药
tcc2, 百拇医药
cta2, 百拇医药
tct2, 百拇医药
gca2, 百拇医药
tct2, 百拇医药
gtg2, 百拇医药
gga2, 百拇医药
gaa2, 百拇医药
act2, 百拇医药
gtc2, 百拇医药
acc2, 百拇医药
atc2, 百拇医药
aca2, 百拇医药
tgt2, 百拇医药
cga2, 百拇医药
gca
agtj6, 百拇医药
gag
j6, 百拇医药Wj6, 百拇医药
Yj6, 百拇医药
Qj6, 百拇医药
Qj6, 百拇医药
Kj6, 百拇医药
Qj6, 百拇医药
Gj6, 百拇医药
Kj6, 百拇医药
Sj6, 百拇医药
Pj6, 百拇医药
Qj6, 百拇医药
Fj6, 百拇医药
aatj6, 百拇医药
attj6, 百拇医药
aacj6, 百拇医药
agtj6, 百拇医药
tacj6, 百拇医药
ttaj6, 百拇医药
gcaj6, 百拇医药
tggj6, 百拇医药
tatj6, 百拇医药
cagj6, 百拇医药
cagj6, 百拇医药
aaaj6, 百拇医药
cagj6, 百拇医药
ggaj6, 百拇医药
aaaj6, 百拇医药
tctj6, 百拇医药
cct
cag;[px^hv, 百拇医药
ttc;[px^hv, 百拇医药
L;[px^hv, 百拇医药
V;[px^hv, 百拇医药
Y
;[px^hv, 百拇医药G;[px^hv, 百拇医药
V;[px^hv, 百拇医药
P;[px^hv, 百拇医药
S;[px^hv, 百拇医药
R;[px^hv, 百拇医药
F;[px^hv, 百拇医药
S;[px^hv, 百拇医药
G;[px^hv, 百拇医药
S;[px^hv, 百拇医药
ctg;[px^hv, 百拇医药
gtc;[px^hv, 百拇医药
tat;[px^hv, 百拇医药
aat;[px^hv, 百拇医药
gca;[px^hv, 百拇医药
aaa;[px^hv, 百拇医药
acc;[px^hv, 百拇医药
tta;[px^hv, 百拇医药
gca;[px^hv, 百拇医药
gaa;[px^hv, 百拇医药
gaa;[px^hv, 百拇医药
gta;[px^hv, 百拇医药
cca;[px^hv, 百拇医药
tca;[px^hv, 百拇医药
agg;[px^hv, 百拇医药
ttc;[px^hv, 百拇医药
agt
ggc{+gxp6), 百拇医药
agt{+gxp6), 百拇医药
G{+gxp6), 百拇医药
S{+gxp6), 百拇医药
G{+gxp6), 百拇医药
T{+gxp6), 百拇医药
Q{+gxp6), 百拇医药
F{+gxp6), 百拇医药
S{+gxp6), 百拇医药
L{+gxp6), 百拇医药
K{+gxp6), 百拇医药
I{+gxp6), 百拇医药
N{+gxp6), 百拇医药
S{+gxp6), 百拇医药
L{+gxp6), 百拇医药
Q{+gxp6), 百拇医药
P{+gxp6), 百拇医药
E{+gxp6), 百拇医药
D{+gxp6), 百拇医药
F{+gxp6), 百拇医药
G{+gxp6), 百拇医药
gga{+gxp6), 百拇医药
tca{+gxp6), 百拇医药
ggc{+gxp6), 百拇医药
aca{+gxp6), 百拇医药
cag{+gxp6), 百拇医药
ttt{+gxp6), 百拇医药
tct{+gxp6), 百拇医药
ctg{+gxp6), 百拇医药
aag{+gxp6), 百拇医药
atc{+gxp6), 百拇医药
aac
agc\62&}r, 百拇医药
ctg\62&}r, 百拇医药
cag\62&}r, 百拇医药
cct\62&}r, 百拇医药
gaa\62&}r, 百拇医药
gat\62&}r, 百拇医药
ttt\62&}r, 百拇医药
ggg\62&}r, 百拇医药
T\62&}r, 百拇医药
Y\62&}r, 百拇医药
Y\62&}r, 百拇医药
C
\62&}r, 百拇医药F\62&}r, 百拇医药
G\62&}r, 百拇医药
G\62&}r, 百拇医药
G\62&}r, 百拇医药
T\62&}r, 百拇医药
K\62&}r, 百拇医药
cat\62&}r, 百拇医药
tat\62&}r, 百拇医药
tac\62&}r, 百拇医药
tgt\62&}r, 百拇医药
caa\62&}r, 百拇医药
cat\62&}r, 百拇医药
cat\62&}r, 百拇医药
tat\62&}r, 百拇医药
ggt\62&}r, 百拇医药
cct\62&}r, 百拇医药
ccg\62&}r, 百拇医药
tgg\62&}r, 百拇医药
acg
ttcn, 百拇医药
ggtn, 百拇医药
ggan, 百拇医药
ggcn, 百拇医药
accn, 百拇医药
aaan, 百拇医药
Ln, 百拇医药
Qn, 百拇医药
In, 百拇医药
Kn, 百拇医药
ctan, 百拇医药
caan, 百拇医药
atcn, 百拇医药
aaan, 百拇医药
Fig 3 Nucleotide and deduced amino acid sequences of light chain variable region of anti-ACHR MCAB D6n, 百拇医药
CDR regions are indicatedn, 百拇医药
DISCUSSIONn, 百拇医药
A mouse anti-AChR McAb was analysed for structural properties in relation to its potential to cause AChR loss and muscular weakness in the passive transfer EAMG model. McAb D6 is direc-ted against the MIR which was confirmed by complete inhibition with McAb ＃35[11] and by binding to the sequence α 65～78 of human AChR[12]. In this study McAb D6 was demonstrated to be able to induce EAMG in rats， indicating that McAb D6 is a pathogenic anti-AChR antibody. This finding is in concordance with previous study in which pathogenicity of anti-AChR MIR McAb was confirmed[13].
Sequence analysis of anti-AChR McAb D6 reveals the characterization at the molecular level of the VH and VL gene segment usage. The comparison between the expressed VH gene and its most closely related germline counter parts shows some nucleotide differences. The VH sequence of McAb D6 shares 94.8% homology at the nucleotide level with the germline Q52 family. Alignment of the VH nucleotide sequence of McAb D6 with that of Q52 germline gene segment reveals 14 nucleotide substitutions， comprised in 13 codons， 9 of which give rise to amino acid replacement， 6 of them are located in the CDRs (complemetarity determining regions)， 2 in the CDR1 and 4 in the CDR2. The nucleotide substitutions seen in the VH gene of McAb D6 may result from somatic mutations driven by antigen AChR. The D gene sequence used in McAb D6 shows less than 60% homology with the germline D gene segments so far described[9]. This probably is the result of extensive deletion or insertion during VH-D-JH recombination events. Alternatively， the D gene may be derived from a different， not yet characterized germline gene.m&d*'/9, 百拇医药
One of the key question in the pathogenesis of MG is the contribution of individual antibody specificities to AChR destruction. Comparison of the variable regions between anti-AChR McAb D6 and A7[6] or G10[14]， which are pathogenic anti-AChR McAbs directed against MIR， shows that the overall sequence homology of both H and L chains is less than 65% and 40% respectively both at nucleotide and amino acid level. However， all the three anti-AChR MIR McAbs above could bind to AChR at the neuromuscular junction in human muscle cryosections (results not shown)， sugges-ting that not is the unique structure of pathogenic anti-AChR antibodies needed in mediating MG and EAMG.More knowledge about the structure of anti-AChR antibodies in relation to their pathogenicity will lead us to the understanding of the question.
作者单位：Meng Fanping(孟繁平)Yang Kangjuan(杨康鹃) Zhang Qinggao(张庆镐) Department of Microbiology and Immunology，Yanbian University Colleye of Medicine，Yanji 133000ahdq-2c, http://www.100md.com
Y.Graus M. de Baets Department of Immunology，Faculty of Medicine ，Univerity of Limburg，The Netherlandsahdq-2c, http://www.100md.com
第一作者：男，40岁，硕士，教授ahdq-2c, http://www.100md.com
REFERENCESahdq-2c, http://www.100md.com
[1] Lindstrom JM， Einarson BL，Lennon VA， et al. Pathological mechanisms in experimental myasthenia gravis. I.Immunogenicity of syngeneic muscle acetylcholine receptor and quantitative extraction of receptor and antibody-receptor complexes from muscles of rats with experimental autoimmune myasthenia gravis. J Exp Med， 1976， 144：726ahdq-2c, http://www.100md.com
[2] Lennon VA， Lindstrom JM，Seybold ME.Experimental autoimmune myasthenia： a model of myasthenia gravis in rats and guinea pigs. J Exp Med， 1975， 141：1365ahdq-2c, http://www.100md.com
[3] Graus Y， de Baets M.Myasthenia gravis： an autoimmune response against the acetylcholine receptor. Immunol Res， 1993， 12：78ahdq-2c, http://www.100md.com
[4] Gomez C，Richman D.Anti-acetylcholine receptor antibodies directed against the α-BT binding site induce a unique form of experimental myasthenia. Proc Natl Acad Sci USA，1983，80：4089
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(1997-12-03收稿；1998-03-24修回)(Meng Fanping(孟繁平)1，Yang Kangjuan(杨康鹃)1， Zhang Qinggao(张庆镐)1，Y.Graus2，M. de Baets2)