小鼠甲状腺过氧化物酶Phe~(473)-Ala~(800)多肽的表达与纯化
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摘要
自身免疫性甲状腺炎(autoimmune thyroditis)又称桥本氏甲状腺炎(Hashimoto’s thyroiditis, HT)是常见的器官特异性自身免疫病。甲状腺过氧化物酶(thyroid peroxidase, TPO)是甲状腺激素合成的关键催化酶,也是建立HT的小鼠模型—实验性自身免疫性甲状腺炎(experimental autoimmune thyroiditis, EAT)的抗原。EAT模型是诱导动物甲状腺的炎症,模拟人的自身免疫性甲状腺炎,最常用的是小鼠模型,尤其是H-2k和H-2s单元型小鼠等有较重的甲状腺浸润,能更好的反应甲状腺炎的状况。甲状腺过氧化物酶(TPO)是一种糖基化血红素蛋白,跨于甲状腺细胞顶缘的细胞膜上,是甲状腺激素合成的关键酶,也是引起自身免疫性甲状腺炎的一个重要自身抗原,TPO表位的异常表达或TPO和TPO抗体(TPOAb)的免疫反应,常是引起HT患者甲状腺细胞损伤的重要机制。本实验旨在通过原核表达系统,表达和纯化小鼠甲状腺过氧化物酶Phe~(473)-Ala~(800)多肽(mouse thyroid peroxidase,mTPOF~(473-A800))。
目的:构建重组小鼠过氧化物酶多肽(mTPOF~(473-A800))原核表达载体,在Rosseta菌中诱导表达,纯化mTPOF~(473-A800)蛋白。
方法:
1提取甲状腺组织RNA。取6只C57BL/6小鼠的甲状腺组织,约60mg,液氮冷冻,研钵研磨组织,提取总RNA。
2扩增目的基因。RT-PCR法扩增mTPOF~(473-A800)基因
3克隆载体的构建。mTPOF~(473-A800)基因与pEASY-T3克隆载体连接,将连接产物转化DH5a感受态细胞,加入含80ug/ml氨苄青霉素的LB培养基,37℃过夜培养,次日取1ul菌液做菌液PCR,初步筛选阳性菌株。将阳性菌液作质粒小提,质粒经EcoRI/XhoI双酶切鉴定,挑选阳性的重组克隆载体10ul送华大基因公司测序。
4重组表达载体的构建。pEASY-T3-mTPOF~(473-A800)载体、pET28a、pET32a和pGEX-6p-1做EcoRI/XhoI双酶切,后胶回收所需要的片段,经DNA连接酶做连接反应,将连接产物转化DH5α感受态细胞,次日取1ul菌液做菌液PCR,初步筛选阳性菌株。将阳性菌液作质粒小提,质粒经EcoRI/XhoI双酶切鉴定,挑选阳性的重组表达载体转化E.coli Rosseta感受态细胞,做诱导表达,筛选表达量高的重组表达载体,后进行表达条件的优化。
5 pGEX-6p-1- mTPO~(F473-A800)的表达和GST柱纯化。在最优化的条件诱导表达GST-mTPO~(F473-A800)融合蛋白,经GST柱纯化,得到mTPO~(F473-A800)蛋白。
结果:
1提取总RNA,RT-PCR PCR扩增目的基因(mTPO~(F473-A800)基因),后连接到pEASY-T3克隆载体,测序结果显示扩增的mTPO~(F473-A800)基因序列正确。
2重组表达载体的构建与表达条件的优化。通过双酶切鉴定,mTPO~(F473-A800)基因成功连接3个表达载体。构建成功的重组表达载体转化E.coli Rosseta感受态细胞,经诱导表达,pGEX-6p-1- mTPO~(F473-A800)高效表达mTPO~(F473-A800)蛋白,且可溶性较高。最佳诱导条件是:30℃、1mM IPTG。
3 pGEX-6p-1- mTPO~(F473-A800)大量表达和纯化。采用Western blotting鉴定蛋白表达产物,经GST柱纯化,SDS-PAGE电泳显示清晰的GST条带和mTPO~(F473-A800)条带。
结论:
构建pGEX-6p-1-mTPO~(F473-A800)原核表达载体,并在Rosseta菌诱导表达了GST- mTPO~(F473-A800)融合蛋白,GST柱纯化获得mTPO~(F473-A800)蛋白。
Autoimmune thyroidits also named Hashimoto’s thyoiditis (HT) is a common organ specific autoimmune disease. Thyroid peroxidase (TPO) is a key catalase on the catalytic reaction of the synthesis of thyroid hormone. And it is also an autoantigen in experimental autoimmune thyroiditis (EAT). EAT has the similar characterisics with autoimmune thyroiditis. So it is usually used to study the pathogenesis of HT. H-2k and H-2s mice have severous infiltration and are the most used animals for EAT model.
TPO is a kind of glycosylated hemoglobin, expressing on the thyroid cell surface. It is a critical catalase on the synthesis of thyroid hormones, also it is a major thyroid autoantigen. Expression disorder of epitopes of TPO or immune responses to TPO and TPOAb ususlly became the immportant damage mechanism of thyrocyte of patients with autoimmune thyroidits.
This study focuses on the expression and purification of mouse thyroid peroxidase peptide Phe473-Ala800 (mTPO~(F473-A800)).
Objective:
Constructed the recombinant pGEX-6p-1-mTPO~(F473-A800) prokaryotic expressing plasmid, expressed it in E. coli strain Rosseta, and purified the expressed peptide.
Method:
1 Extraction of the total RNA. The thyroid glands of 6 C57BL/6 mice were removed and grinded in liquid nitrogen for the RNA.
2 Amplification of mTPO~(F473-A800) gene. The mTPO~(F473-A800) gene was amplified by RT-PCR.
3 Construction of recombinant cloning vector. mTPO~(F473-A800) gene was induced into pEASY-T3 vector. The productions were induced into DH5a competent cells ,cultivation at 37℃for 12 hours.The next day, screening the \successfully induced pEASY-T3- mTPO~(F473-A800) plasmid by PCR.To choose the positive clones to extract the recombinant plasmids and to identify the correct recombinant plasmid pEASY-T3- mTPO~(F473-A800) by double enzyme digestion of EcRoI/XhoI. Send out 10ul of recombinant plasmids for sequencing.
4 Construction of recombinant expression plasmid. pEASY-T3- mTPO~(F473-A800) plasmid togethe with pET28a, pET32a and pGEX-6p-1 were digested by EcRoI/XhoI. Then induce mTPO~(F473-A800) into pET28a, pET32a and pGEX-6p-1 by DNA ligase. The productions were induced into DH5a competent cells, cultivation at 37℃for 12 hours. The next day, screen the successfully induced pEASY-T3- mTPO~(F473-A800) plasmids by PCR. Choosed the positive clones to extract recombinant plasmids and to identify the correct recombinant expression plasmids by double enzyme digestion of EcRoI/XhoI. Then the positive recombinant plasmids were induced into E.coli Rosseta competent cells. Optimised the expression conditions and screened the best expressed plasmid.
5 The expression of pGEX-6p-1- mTPO~(F473-A800)and purification of GST- mTPO~(F473-A800) recombinant peptides. Express GST-mTPO~(F473-A800) recombinant protein at optimum conditions and obtain mTPO~(F473-A800) peptide by GST column.
Results:
1 Extracted the total RNA of mouse thyroid peroxidase. The mTPO~(F473-A800) gene was induced into pEASY-T3 vector, and the sequencing results shown that we got the correct gene.
2 Constructing recombinant expression vectors and optimizating expression conditions. After mTPO~(F473-A800) gene was induced into 3 vectors successfully identified by double enzyme digestion, Rosseta/pGEX-6p-1- mTPO~(F473-A800) were induced by 1mM IPTG at 30℃SDS-PAGE electrophoresis showed a great production and high solubility under this optumized conditions.
3 Expression and purification of pGEX-6p-1- mTPO~(F473-A800) recombinant peptides. Rosseta/pGEX-6p-1- mTPO~(F473-A800) was nduced by 1mM IPTG at 30℃. Then the bacteia were collected and the products were analyzed by SDS-PAGE and Western blotting. The mTPO~(F473-A800) protein was purified by GST column.
Conclution:
Recombinant vector pGEX-6p-1-mTPO~(F473-A800) was etablished successfully. Large scale expression was made under optimum conditions and to purified mTPO~(F473-A800) peptides were obtained in GST column.
目的:构建重组小鼠过氧化物酶多肽(mTPOF~(473-A800))原核表达载体,在Rosseta菌中诱导表达,纯化mTPOF~(473-A800)蛋白。
方法:
1提取甲状腺组织RNA。取6只C57BL/6小鼠的甲状腺组织,约60mg,液氮冷冻,研钵研磨组织,提取总RNA。
2扩增目的基因。RT-PCR法扩增mTPOF~(473-A800)基因
3克隆载体的构建。mTPOF~(473-A800)基因与pEASY-T3克隆载体连接,将连接产物转化DH5a感受态细胞,加入含80ug/ml氨苄青霉素的LB培养基,37℃过夜培养,次日取1ul菌液做菌液PCR,初步筛选阳性菌株。将阳性菌液作质粒小提,质粒经EcoRI/XhoI双酶切鉴定,挑选阳性的重组克隆载体10ul送华大基因公司测序。
4重组表达载体的构建。pEASY-T3-mTPOF~(473-A800)载体、pET28a、pET32a和pGEX-6p-1做EcoRI/XhoI双酶切,后胶回收所需要的片段,经DNA连接酶做连接反应,将连接产物转化DH5α感受态细胞,次日取1ul菌液做菌液PCR,初步筛选阳性菌株。将阳性菌液作质粒小提,质粒经EcoRI/XhoI双酶切鉴定,挑选阳性的重组表达载体转化E.coli Rosseta感受态细胞,做诱导表达,筛选表达量高的重组表达载体,后进行表达条件的优化。
5 pGEX-6p-1- mTPO~(F473-A800)的表达和GST柱纯化。在最优化的条件诱导表达GST-mTPO~(F473-A800)融合蛋白,经GST柱纯化,得到mTPO~(F473-A800)蛋白。
结果:
1提取总RNA,RT-PCR PCR扩增目的基因(mTPO~(F473-A800)基因),后连接到pEASY-T3克隆载体,测序结果显示扩增的mTPO~(F473-A800)基因序列正确。
2重组表达载体的构建与表达条件的优化。通过双酶切鉴定,mTPO~(F473-A800)基因成功连接3个表达载体。构建成功的重组表达载体转化E.coli Rosseta感受态细胞,经诱导表达,pGEX-6p-1- mTPO~(F473-A800)高效表达mTPO~(F473-A800)蛋白,且可溶性较高。最佳诱导条件是:30℃、1mM IPTG。
3 pGEX-6p-1- mTPO~(F473-A800)大量表达和纯化。采用Western blotting鉴定蛋白表达产物,经GST柱纯化,SDS-PAGE电泳显示清晰的GST条带和mTPO~(F473-A800)条带。
结论:
构建pGEX-6p-1-mTPO~(F473-A800)原核表达载体,并在Rosseta菌诱导表达了GST- mTPO~(F473-A800)融合蛋白,GST柱纯化获得mTPO~(F473-A800)蛋白。
Autoimmune thyroidits also named Hashimoto’s thyoiditis (HT) is a common organ specific autoimmune disease. Thyroid peroxidase (TPO) is a key catalase on the catalytic reaction of the synthesis of thyroid hormone. And it is also an autoantigen in experimental autoimmune thyroiditis (EAT). EAT has the similar characterisics with autoimmune thyroiditis. So it is usually used to study the pathogenesis of HT. H-2k and H-2s mice have severous infiltration and are the most used animals for EAT model.
TPO is a kind of glycosylated hemoglobin, expressing on the thyroid cell surface. It is a critical catalase on the synthesis of thyroid hormones, also it is a major thyroid autoantigen. Expression disorder of epitopes of TPO or immune responses to TPO and TPOAb ususlly became the immportant damage mechanism of thyrocyte of patients with autoimmune thyroidits.
This study focuses on the expression and purification of mouse thyroid peroxidase peptide Phe473-Ala800 (mTPO~(F473-A800)).
Objective:
Constructed the recombinant pGEX-6p-1-mTPO~(F473-A800) prokaryotic expressing plasmid, expressed it in E. coli strain Rosseta, and purified the expressed peptide.
Method:
1 Extraction of the total RNA. The thyroid glands of 6 C57BL/6 mice were removed and grinded in liquid nitrogen for the RNA.
2 Amplification of mTPO~(F473-A800) gene. The mTPO~(F473-A800) gene was amplified by RT-PCR.
3 Construction of recombinant cloning vector. mTPO~(F473-A800) gene was induced into pEASY-T3 vector. The productions were induced into DH5a competent cells ,cultivation at 37℃for 12 hours.The next day, screening the \successfully induced pEASY-T3- mTPO~(F473-A800) plasmid by PCR.To choose the positive clones to extract the recombinant plasmids and to identify the correct recombinant plasmid pEASY-T3- mTPO~(F473-A800) by double enzyme digestion of EcRoI/XhoI. Send out 10ul of recombinant plasmids for sequencing.
4 Construction of recombinant expression plasmid. pEASY-T3- mTPO~(F473-A800) plasmid togethe with pET28a, pET32a and pGEX-6p-1 were digested by EcRoI/XhoI. Then induce mTPO~(F473-A800) into pET28a, pET32a and pGEX-6p-1 by DNA ligase. The productions were induced into DH5a competent cells, cultivation at 37℃for 12 hours. The next day, screen the successfully induced pEASY-T3- mTPO~(F473-A800) plasmids by PCR. Choosed the positive clones to extract recombinant plasmids and to identify the correct recombinant expression plasmids by double enzyme digestion of EcRoI/XhoI. Then the positive recombinant plasmids were induced into E.coli Rosseta competent cells. Optimised the expression conditions and screened the best expressed plasmid.
5 The expression of pGEX-6p-1- mTPO~(F473-A800)and purification of GST- mTPO~(F473-A800) recombinant peptides. Express GST-mTPO~(F473-A800) recombinant protein at optimum conditions and obtain mTPO~(F473-A800) peptide by GST column.
Results:
1 Extracted the total RNA of mouse thyroid peroxidase. The mTPO~(F473-A800) gene was induced into pEASY-T3 vector, and the sequencing results shown that we got the correct gene.
2 Constructing recombinant expression vectors and optimizating expression conditions. After mTPO~(F473-A800) gene was induced into 3 vectors successfully identified by double enzyme digestion, Rosseta/pGEX-6p-1- mTPO~(F473-A800) were induced by 1mM IPTG at 30℃SDS-PAGE electrophoresis showed a great production and high solubility under this optumized conditions.
3 Expression and purification of pGEX-6p-1- mTPO~(F473-A800) recombinant peptides. Rosseta/pGEX-6p-1- mTPO~(F473-A800) was nduced by 1mM IPTG at 30℃. Then the bacteia were collected and the products were analyzed by SDS-PAGE and Western blotting. The mTPO~(F473-A800) protein was purified by GST column.
Conclution:
Recombinant vector pGEX-6p-1-mTPO~(F473-A800) was etablished successfully. Large scale expression was made under optimum conditions and to purified mTPO~(F473-A800) peptides were obtained in GST column.
引文
1 Wang C, Crapo LM 1997 The epidemiology of thyroid disease and implication for screening[J]. Endocrinol Metab Clin North Am, 26:189-218
2 Vladutiu AO, Rose NR. Autoimmune murine thyroiditis[J]. Science, 1971, 174:1137-1138
3 Banga JP.Thyroid peroxidase: developments in our understanding of its structure and the relevance of these findings to autoimmunity[J]. Curr Opin Endocrinol Diabetes,5:275-281
4 Dayan CM, Daniels GH. Chronic autoimmune thyroiditis[J]. N Engl J Med 335:99-107
5 Calder AE, Penhale WJ, McLeman D, Barnes EW, Irvine WJ 1973 Lymphocyte-dependent antibody-mediated cytotoxicity in Hashimoto thyroiditis[J]. Clin Exp Immunol 14:153-158
6 Suzuki S, Mitsunuga M, Ofuji T,et al. Cytophilic anti-thyroglobulin antibody and antibody-dependent monocyte-mediated cytotoxicity in Hashimoto’s thyroiditis[J]. J Clin Endocrinol Metab, 1980, 51:446-453
7 Rodien P, Madec AM, Ruf J, Rajas F, Bornet H, Carayon P, Orgiazzi J Antibody-dependent cell-mediated cytotoxicity in autoimmune thyroid disease: relationship to antithyroperoxidase antibodies[J]. J Clin Endocrinol Metab, 1996, 81:2595-2600
8 Jin G, Juan CJ, Rapoport B, McLachlan S,et al. Recombinant thyroid peroxidase-specific Fab converted to immunoglobulin G (IgG) molecules: evidence for thyroid cell damage by IgG1, but not IgG4, autoantibodies[J]. J Clin Endocrinol Metab, 1997, 82:925-931
9 Weetman AP, McGregor AM, Hall R,et al. Thyroid antibodies are produced by thyroid-derived lymphocytes[J]. Clin Exp Immunol, 1982, 48:196-200
10 Esquivel PS, Rose NR, Kong YM,et al. Induction of autoimmunity in good and poor responder mice with mouse thyroglobulin and lipopolysaccharide[J]. J Exp Med, 1977, 145:1250-1262
11 EIRehewy M, Kong YM, Giraldo AA, Rose NR. Syngeneic thyroglobulin is immunogenic in good responder mice[J]. Eur J Immunol, 1981, 11:146-51
12 Tomazic V, Rose NR. Role of different thyroid antigens in the induction of experimental autoimmune thyroiditis[J]. Immunology, 1976, 30:63–8.
13 Wucherpfennig K W, Eisenbarth G S. Type 1 diabetes[J].Nature Immunol, 2001, 2:767-768
14 Steinman, L. Multiple sclerosis: a two-stage disease[J]. NatureImmunol, 2001, 2:762-764
15 Weetman, A. P. & McGregor, A. M. Autoimmune thyroid disease: further developments in our understanding[J]. Endocr.Rev, 1994, 15:788-830
16 Guo J,McLachlan SM, Rapo port B, et al.Profization of the thyroid peroxidase autoantibcdy immunedominant region to a junctional region containing portions of the domains homologous to complement control protein and myelopemxldase[J].J Biol Chem, 2002, 277(43):40l89-40l95
17 Xiong Z, Farilla L,Guo J. et al.Does the autoantibody immunodominant on thyroid pomxidase include amino acid residues 742-771[J].Thyroid,2001, 11(3):227-231
18 McLachlan SM, Rapoport B.Thyroid peroxidase as an autoantigen[J]. Thyroid, 2007, 17(10):939-948
19 Holiowell J G,Staehling N W ,Flan ders W D et al.Serum TSHL and thyroid antibodies in the united states population(1988 to 1994):national health and nutrition examination survey [J].J Clin Endocrlnol Metab, 2002, 87(2):489-499
20 Blanchin S,Estlenne V, Durand Gorde J M et a1.Complement activation by direct c4 binding to thyroporoxidase in Hashimoto’s thyroiditis[J].Endocrinology, 2003, 144(12):5422-5429
21 Czarnocka B, Ruf J, Ferrand M, Carayon P, Lissitzky S, et al. Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases[J]. FEBS Lett, 1985, 190:147-152
22 Seto P, Hirayu H, Magnusson RP, Gestautas J, Portmann L, DeGroot LJ,Rapoport B. Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase[J]. J Clin Invest, 1987, 80:1205-1208
23 Libert F, Ruel J, Lufgate M, Swillens S, Alexander N, Vassart G, Dinsart C Thyroperoxidase, an auto-antigen with a mosaic structure made of nuclear and mitochondrial gene modules[J]. EMBO J, 1987, 6: 4193-4196
24 Rapoport B, McLachlan SThyroid peroxidase as an autoantigen in autoimmune thyroid disease: update[J]. Endocr Rev , 1994, 3:96-102
25 Banga JP. Thyroid peroxidase: developments in our understanding of its structure and the relevance of these findings to autoimmunity[J]. Curr Opin Endocrinol Diabetes, 1998, 5:275-281
26 Dayan CM, Daniels GH. Chronic autoimmune thyroiditis[J]. N Engl J Med, 1996, 335:99-107
27 Bogner U, Schleusener H, Wall JR, et al. Antibody-dependent cell-mediated cytotoxicity against human thyroid cells in Hashimoto’s thyroiditis but not Graves’disease[J]. J Clin Endocrinol Metab, 1984, 59:734-738
28 McLachlan SM, Pegg CAS, Rees Smith B, et al. The thyroid microenvironment in autoimmune thyroid disease: effects of TSH and lymphokines on thyroid lymphocytes and thyroid cells[J]. Acta Endocrinol (Copenh), 1987, 281:125-132
29 Rodien P, Madec. A-M, Orgiazzi J, et al. Antibody-dependent cell- mediated cytotoxicity in autoimmune thyroid disease:relationship to antithyroid antibodies[J]. J Clin Endocrinol Metab,1996, 81:2595-2600
30 Guo J, Jaume JC, Rapoport B, McLachlan SM. Recombinant thyroid peroxidase-specific Fab converted to immunoglobulin (IgG) molecules: evidence for thyroid cell damage by IgG1 but not IgG4 autoantibodies[J]. J Clin Endocrinol Metab, 1997, 82:925-931
31 Dietrich, H. M., Oliveira-dos-Santos, A. J. & Wick, G. Development of spontaneous autoimmune thyroiditis in Obese strain (OS) chickens. Vet. Immunol[J]. Immunopathol, 1997, 57:141-146
32 Wright J R, J Senhauser, D A Yates, et al. Spontaneous thyroiditis in BB Wistar diabeticrats[J]. Vet Pathol, 1983, 20:522-530
33 Rasooly L, Burek C L, Rose N R,et al. Iodine-induced autoimmune thyroiditis in NOD-H-2h4 mice[J]. Clin Immunol, 1996, 81:287-292
34 Tomazic V, Rose N R. Role of different thyroid antigens in the induction of experimental autoimmune thyroiditis[J]. Immunology, 1976, 30:63-68
35 Knight S C. Induction of autoimmunity with dendritic cells: studies on thyroiditis in mice[J]. Clin Immunol, 1988, 48:277-289
36 Flynn J C, Conaway D H, Cobbold S, et al. Depletion of L3T4+ and Lyt-2+ cells by rat monoclonal antibodies alters the development of adoptively transferred experimental autoimmune thyroiditis[J]. Cell Immunol, 1989, 122:377-390
37 Ewins DL, Barnett PS, Tomlinson RW, et al. Mapping epitope specificities of monoclonal antibodies to thyroid peroxidase using recombinant antigen preparations[J]. Autoimmunity, 1992,11:1141-1149
38 H P Ng, Annie W C, Kung,et al. Induction of Autoimmune Thyroiditis and Hypothyroidism by Immunization of Immunoactive T Cell Epitope of Thyroid Peroxidase[J]. Endocrinology, 2006, 147(6):3085-3092
1 Flynn JC,Wan Q,Panos JC,et al. Coexpression of susceptible and resistant HLA class II transgenes in murine experimental autoimmune thyroiditis: DQ8 molecules downregulate DR3-mediatedthyroiditis.[J] J Autoimmun, 2002, 18 (3) :213-220
2 Imaizumi M, Pritsker A,Kita M,et al. Non-MHC driven exacerbation of experimental thyroiditis in the postpartum period.[J] .Autoimmunity, 2001,34(2):95-105
3 Imaizumi M, Pritsker A, unger P et al. Intrathyroidal fetal microchimerism in pregnancy and postpartum [J]. Endocrinology, 2002, 143 (1):247-253
4 Kong YC ,David CS. New revelations in susceptibility to autoimmune thyroiditis by the use of H2 and HLA class II transgenic models [J] . Int Rev Immunol, 2000, 19:573-585
5 Verginis P,Stanford MM,et al. Delineation of fivethyroglobulin T cell epitopes with pathogenic potential in experimental autoimmune thyroiditis [J]. J Immunol, 2002, 169(9):5332-5337
6 Yan Y,Panos JC ,Mc Cormick DJ ,et al. Characterization of a novel H2A(-)E+ transgenic model susceptible to heterologous but not self thyroglobulin in autoimmune thyroiditis: thyroiditis transfer with Vbeta8+ T cells[J]. Cell Immunol, 2001, 212(1) :63-70
7 Costagliola S, Rodien P, Many MC, et al. Genetic immunization against the human thyrotropin receptor causes thyroiditis and allows production of monoclonal antibodies recognizing the native receptor [J].J Immunol , 1998, 160:1458-1465
8 Esquivel PS, Rose NR, Kong YM. Induction of autoimmunity in good and poor responder mice with mouse thyroglobulin and lipopolysaccharide[J]. J Exp Med, 1977, 145:1250-62
9 EIRehewy M, Kong YM, Giraldo AA, Rose NR. Syngeneic thyroglobulin is immunogenic in good responder mice[J]. Eur J Immunol, 1981, 11:146-151
10 Tomazic V, Rose NR. Autoimmune murine thyroiditis. VIII. Role of different thyroid antigens in the induction of experimental autoimmune thyroiditis[J]. Immunology, 1976, 30:63-68
11 Hutchings PR,Verma , Jenny MP,et al. Both CD4(+)T cells and CD8(+) T cells are required for iodine accelerated thyroiditis in NOD mice[J]. Cell Immunol, 1999, 192 :113-121
12 Dai YD ,Rao VP ,Carayanniotis G. Enhanced iodination of thyroglobulin facilitates processing and presentation of a cryptic pathogenic peptide[J]. J Immunol, 2002, 168 (11) :5907-5911
13 Salmaso, C., Bagnasco, M., Pesce, G., Montagna, P., Brizzolara, R., Altrinetti, V. et al. Regulation of apoptosis in endocrine autoimmunity: insights from Hashimoto’s thyroiditis and Graves’disease[J]. Annals of the New York Academy of Sciences, 2002, 966:496-501
14 Ajjan ,Weetman AP. Techniques to quantify TSH receptor antibodies[J]. Nat Clin Pract Endocrinol Metab, 2008, 4(8):461-468
15 Paschke R, Van Sande J, Parma J, Vassart G. The TSH receptor and thyroid diseases[J]. Baillieres Clin Endocrinol Metab, 1996, 10(1):9-27
16 Tomer Y, Davies TF. Infections and autoimmune endocrine disease[J]. Baillieres Clin Endocrinol Metab, 1995, 9(1):47-70
17 Arscott PL, Baker JR Jr. Apoptosis and thyroiditis[J]. Clin Immunol Immunopathol, 1998, 87(3):207-217
18 Koh LK, Greenspan FS, Yeo PP. Interferon-alpha induced thyroid dysfunction: three clinical presentations and a review of the literature[J]. Thyroid, 1997, 7:891-896
19 Youde SJ, Mower J, Moore DP, Parkes AB. Stress protein expression in primary and immortalized cultures of human thyroid cells: a model system for the study of stress proteins in the pathogenesis of autoimmune thyroid disease[J]. Cell Stress Chaperones, 1998, 3(2):89-93
20 Fukata S, Kuma K, Sugawara M. Relationship between cigarette smoking and hypothyroidism in patients with Hashimoto's thyroiditis[J]. J Endocrinol Invest, 1996, 19(9):607-12
21 Fantz CR, Dagogo-Jack S, Ladenson JH, Gronowski AM. Thyroid function during pregnancy[J]. Clin Chem, 1999, 45(12):2250-2258
2 Vladutiu AO, Rose NR. Autoimmune murine thyroiditis[J]. Science, 1971, 174:1137-1138
3 Banga JP.Thyroid peroxidase: developments in our understanding of its structure and the relevance of these findings to autoimmunity[J]. Curr Opin Endocrinol Diabetes,5:275-281
4 Dayan CM, Daniels GH. Chronic autoimmune thyroiditis[J]. N Engl J Med 335:99-107
5 Calder AE, Penhale WJ, McLeman D, Barnes EW, Irvine WJ 1973 Lymphocyte-dependent antibody-mediated cytotoxicity in Hashimoto thyroiditis[J]. Clin Exp Immunol 14:153-158
6 Suzuki S, Mitsunuga M, Ofuji T,et al. Cytophilic anti-thyroglobulin antibody and antibody-dependent monocyte-mediated cytotoxicity in Hashimoto’s thyroiditis[J]. J Clin Endocrinol Metab, 1980, 51:446-453
7 Rodien P, Madec AM, Ruf J, Rajas F, Bornet H, Carayon P, Orgiazzi J Antibody-dependent cell-mediated cytotoxicity in autoimmune thyroid disease: relationship to antithyroperoxidase antibodies[J]. J Clin Endocrinol Metab, 1996, 81:2595-2600
8 Jin G, Juan CJ, Rapoport B, McLachlan S,et al. Recombinant thyroid peroxidase-specific Fab converted to immunoglobulin G (IgG) molecules: evidence for thyroid cell damage by IgG1, but not IgG4, autoantibodies[J]. J Clin Endocrinol Metab, 1997, 82:925-931
9 Weetman AP, McGregor AM, Hall R,et al. Thyroid antibodies are produced by thyroid-derived lymphocytes[J]. Clin Exp Immunol, 1982, 48:196-200
10 Esquivel PS, Rose NR, Kong YM,et al. Induction of autoimmunity in good and poor responder mice with mouse thyroglobulin and lipopolysaccharide[J]. J Exp Med, 1977, 145:1250-1262
11 EIRehewy M, Kong YM, Giraldo AA, Rose NR. Syngeneic thyroglobulin is immunogenic in good responder mice[J]. Eur J Immunol, 1981, 11:146-51
12 Tomazic V, Rose NR. Role of different thyroid antigens in the induction of experimental autoimmune thyroiditis[J]. Immunology, 1976, 30:63–8.
13 Wucherpfennig K W, Eisenbarth G S. Type 1 diabetes[J].Nature Immunol, 2001, 2:767-768
14 Steinman, L. Multiple sclerosis: a two-stage disease[J]. NatureImmunol, 2001, 2:762-764
15 Weetman, A. P. & McGregor, A. M. Autoimmune thyroid disease: further developments in our understanding[J]. Endocr.Rev, 1994, 15:788-830
16 Guo J,McLachlan SM, Rapo port B, et al.Profization of the thyroid peroxidase autoantibcdy immunedominant region to a junctional region containing portions of the domains homologous to complement control protein and myelopemxldase[J].J Biol Chem, 2002, 277(43):40l89-40l95
17 Xiong Z, Farilla L,Guo J. et al.Does the autoantibody immunodominant on thyroid pomxidase include amino acid residues 742-771[J].Thyroid,2001, 11(3):227-231
18 McLachlan SM, Rapoport B.Thyroid peroxidase as an autoantigen[J]. Thyroid, 2007, 17(10):939-948
19 Holiowell J G,Staehling N W ,Flan ders W D et al.Serum TSHL and thyroid antibodies in the united states population(1988 to 1994):national health and nutrition examination survey [J].J Clin Endocrlnol Metab, 2002, 87(2):489-499
20 Blanchin S,Estlenne V, Durand Gorde J M et a1.Complement activation by direct c4 binding to thyroporoxidase in Hashimoto’s thyroiditis[J].Endocrinology, 2003, 144(12):5422-5429
21 Czarnocka B, Ruf J, Ferrand M, Carayon P, Lissitzky S, et al. Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases[J]. FEBS Lett, 1985, 190:147-152
22 Seto P, Hirayu H, Magnusson RP, Gestautas J, Portmann L, DeGroot LJ,Rapoport B. Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase[J]. J Clin Invest, 1987, 80:1205-1208
23 Libert F, Ruel J, Lufgate M, Swillens S, Alexander N, Vassart G, Dinsart C Thyroperoxidase, an auto-antigen with a mosaic structure made of nuclear and mitochondrial gene modules[J]. EMBO J, 1987, 6: 4193-4196
24 Rapoport B, McLachlan SThyroid peroxidase as an autoantigen in autoimmune thyroid disease: update[J]. Endocr Rev , 1994, 3:96-102
25 Banga JP. Thyroid peroxidase: developments in our understanding of its structure and the relevance of these findings to autoimmunity[J]. Curr Opin Endocrinol Diabetes, 1998, 5:275-281
26 Dayan CM, Daniels GH. Chronic autoimmune thyroiditis[J]. N Engl J Med, 1996, 335:99-107
27 Bogner U, Schleusener H, Wall JR, et al. Antibody-dependent cell-mediated cytotoxicity against human thyroid cells in Hashimoto’s thyroiditis but not Graves’disease[J]. J Clin Endocrinol Metab, 1984, 59:734-738
28 McLachlan SM, Pegg CAS, Rees Smith B, et al. The thyroid microenvironment in autoimmune thyroid disease: effects of TSH and lymphokines on thyroid lymphocytes and thyroid cells[J]. Acta Endocrinol (Copenh), 1987, 281:125-132
29 Rodien P, Madec. A-M, Orgiazzi J, et al. Antibody-dependent cell- mediated cytotoxicity in autoimmune thyroid disease:relationship to antithyroid antibodies[J]. J Clin Endocrinol Metab,1996, 81:2595-2600
30 Guo J, Jaume JC, Rapoport B, McLachlan SM. Recombinant thyroid peroxidase-specific Fab converted to immunoglobulin (IgG) molecules: evidence for thyroid cell damage by IgG1 but not IgG4 autoantibodies[J]. J Clin Endocrinol Metab, 1997, 82:925-931
31 Dietrich, H. M., Oliveira-dos-Santos, A. J. & Wick, G. Development of spontaneous autoimmune thyroiditis in Obese strain (OS) chickens. Vet. Immunol[J]. Immunopathol, 1997, 57:141-146
32 Wright J R, J Senhauser, D A Yates, et al. Spontaneous thyroiditis in BB Wistar diabeticrats[J]. Vet Pathol, 1983, 20:522-530
33 Rasooly L, Burek C L, Rose N R,et al. Iodine-induced autoimmune thyroiditis in NOD-H-2h4 mice[J]. Clin Immunol, 1996, 81:287-292
34 Tomazic V, Rose N R. Role of different thyroid antigens in the induction of experimental autoimmune thyroiditis[J]. Immunology, 1976, 30:63-68
35 Knight S C. Induction of autoimmunity with dendritic cells: studies on thyroiditis in mice[J]. Clin Immunol, 1988, 48:277-289
36 Flynn J C, Conaway D H, Cobbold S, et al. Depletion of L3T4+ and Lyt-2+ cells by rat monoclonal antibodies alters the development of adoptively transferred experimental autoimmune thyroiditis[J]. Cell Immunol, 1989, 122:377-390
37 Ewins DL, Barnett PS, Tomlinson RW, et al. Mapping epitope specificities of monoclonal antibodies to thyroid peroxidase using recombinant antigen preparations[J]. Autoimmunity, 1992,11:1141-1149
38 H P Ng, Annie W C, Kung,et al. Induction of Autoimmune Thyroiditis and Hypothyroidism by Immunization of Immunoactive T Cell Epitope of Thyroid Peroxidase[J]. Endocrinology, 2006, 147(6):3085-3092
1 Flynn JC,Wan Q,Panos JC,et al. Coexpression of susceptible and resistant HLA class II transgenes in murine experimental autoimmune thyroiditis: DQ8 molecules downregulate DR3-mediatedthyroiditis.[J] J Autoimmun, 2002, 18 (3) :213-220
2 Imaizumi M, Pritsker A,Kita M,et al. Non-MHC driven exacerbation of experimental thyroiditis in the postpartum period.[J] .Autoimmunity, 2001,34(2):95-105
3 Imaizumi M, Pritsker A, unger P et al. Intrathyroidal fetal microchimerism in pregnancy and postpartum [J]. Endocrinology, 2002, 143 (1):247-253
4 Kong YC ,David CS. New revelations in susceptibility to autoimmune thyroiditis by the use of H2 and HLA class II transgenic models [J] . Int Rev Immunol, 2000, 19:573-585
5 Verginis P,Stanford MM,et al. Delineation of fivethyroglobulin T cell epitopes with pathogenic potential in experimental autoimmune thyroiditis [J]. J Immunol, 2002, 169(9):5332-5337
6 Yan Y,Panos JC ,Mc Cormick DJ ,et al. Characterization of a novel H2A(-)E+ transgenic model susceptible to heterologous but not self thyroglobulin in autoimmune thyroiditis: thyroiditis transfer with Vbeta8+ T cells[J]. Cell Immunol, 2001, 212(1) :63-70
7 Costagliola S, Rodien P, Many MC, et al. Genetic immunization against the human thyrotropin receptor causes thyroiditis and allows production of monoclonal antibodies recognizing the native receptor [J].J Immunol , 1998, 160:1458-1465
8 Esquivel PS, Rose NR, Kong YM. Induction of autoimmunity in good and poor responder mice with mouse thyroglobulin and lipopolysaccharide[J]. J Exp Med, 1977, 145:1250-62
9 EIRehewy M, Kong YM, Giraldo AA, Rose NR. Syngeneic thyroglobulin is immunogenic in good responder mice[J]. Eur J Immunol, 1981, 11:146-151
10 Tomazic V, Rose NR. Autoimmune murine thyroiditis. VIII. Role of different thyroid antigens in the induction of experimental autoimmune thyroiditis[J]. Immunology, 1976, 30:63-68
11 Hutchings PR,Verma , Jenny MP,et al. Both CD4(+)T cells and CD8(+) T cells are required for iodine accelerated thyroiditis in NOD mice[J]. Cell Immunol, 1999, 192 :113-121
12 Dai YD ,Rao VP ,Carayanniotis G. Enhanced iodination of thyroglobulin facilitates processing and presentation of a cryptic pathogenic peptide[J]. J Immunol, 2002, 168 (11) :5907-5911
13 Salmaso, C., Bagnasco, M., Pesce, G., Montagna, P., Brizzolara, R., Altrinetti, V. et al. Regulation of apoptosis in endocrine autoimmunity: insights from Hashimoto’s thyroiditis and Graves’disease[J]. Annals of the New York Academy of Sciences, 2002, 966:496-501
14 Ajjan ,Weetman AP. Techniques to quantify TSH receptor antibodies[J]. Nat Clin Pract Endocrinol Metab, 2008, 4(8):461-468
15 Paschke R, Van Sande J, Parma J, Vassart G. The TSH receptor and thyroid diseases[J]. Baillieres Clin Endocrinol Metab, 1996, 10(1):9-27
16 Tomer Y, Davies TF. Infections and autoimmune endocrine disease[J]. Baillieres Clin Endocrinol Metab, 1995, 9(1):47-70
17 Arscott PL, Baker JR Jr. Apoptosis and thyroiditis[J]. Clin Immunol Immunopathol, 1998, 87(3):207-217
18 Koh LK, Greenspan FS, Yeo PP. Interferon-alpha induced thyroid dysfunction: three clinical presentations and a review of the literature[J]. Thyroid, 1997, 7:891-896
19 Youde SJ, Mower J, Moore DP, Parkes AB. Stress protein expression in primary and immortalized cultures of human thyroid cells: a model system for the study of stress proteins in the pathogenesis of autoimmune thyroid disease[J]. Cell Stress Chaperones, 1998, 3(2):89-93
20 Fukata S, Kuma K, Sugawara M. Relationship between cigarette smoking and hypothyroidism in patients with Hashimoto's thyroiditis[J]. J Endocrinol Invest, 1996, 19(9):607-12
21 Fantz CR, Dagogo-Jack S, Ladenson JH, Gronowski AM. Thyroid function during pregnancy[J]. Clin Chem, 1999, 45(12):2250-2258