AChR-IgGFc段融合蛋白真核表达载体的构建及表达
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摘要
重症肌无力(myasthenia gravis ,MG)是一种典型的抗体介导、补体参与的器官特异性自身免疫性疾病,烟碱型乙酰胆碱受体(nicotinic Acetylcholine receptor ,nAChR)抗体是其发病的重要效应因子。临床表现主要为横纹肌无力,严重时可累及呼吸肌,危及生命。
现有治疗手段主要为胆碱酯酶抑制剂、皮质激素、免疫抑制剂、血浆置换、静脉注射免疫球蛋白、胸腺切除术等,能一定程度控制病情,但不能抑制病程发展,且会影响全身免疫系统。致病性B细胞分泌产生AChR抗体,是MG发病的关键因子,若能从源头上清除这类B细胞则有望控制MG病情。
本研究希望通过真核表达载体转染CHOk1细胞以获得一种AChR‐ IgG Fc融合蛋白,该融合蛋白可与分泌AChR抗体的致病B细胞表面抑制性受体FcγRⅡB或单核‐巨噬细胞表面的Fcγ受体特异性结合,抑制B细胞活化并促进其凋亡。
以人nAChRα1亚基全序列为模板,以PCR法扩增AChRα1亚基胞外段主要免疫区基因片段Hα1-121 ,将其插入真核表达载体pAN1782。将构建的新载体转染至CHOk1细胞,建立稳定表达AChR‐ IgG Fc融合蛋白的CHOk1细胞株,以Western blot法检测AChR‐ IgG Fc融合蛋白表达。
结果显示Hα1-121经PCR法扩增后所获428 bp基因片段大小符合预计结果,测序所得核苷酸序列与人类基因库中AChRα1亚基胞外段基因片段Hα1-121序列完全一致,未出现点突变或移码突变。所构建的新载体经酶切鉴定证实片段插入正确,载体构建成功。Western blot法检测结果显示转染新载体的CHOk1细胞培养上清液中有AChR‐ IgG Fc融合蛋白表达。
综上所述,成功构建人AChR‐ IgG Fc段融合蛋白真核表达载体,且该融合蛋白可在转染新载体的CHOk1细胞中稳定表达,为下一步靶向B细胞治疗重症肌无力奠定了初步基础。
EAMG是MG的动物模型,是研究MG发病机制、治疗策略的重要手段。我们采用重组表达的人肌肉AChR的α亚基(α1-210)ECD蛋白免疫雌性Lewis大鼠,观察免疫后大鼠的体重变化、行走、进食情况,抽取大鼠尾静脉血液,用ELISA法检测血清中AChR抗体滴度变化。
该方法较传统建模方法简便快捷,EAMG模型的成功建立可为功能蛋白的检测以及MG发病机制、治疗方法的研究奠定基础。
Myasthenia gravis (MG) is a disorder of neuromuscular transmission mediated by autoantibodies to the nicotinic acetylcholine receptor (AChR) and involved in activation of complement. It is characterised by fatigable muscle weakness. The most common treatment options used for MG are acetylcholinesterase inhibitors, corticosteroids, immunosuppressants, plasmapheresis, intravenous immunoglobulin, thymectomy, which can relieve the conditions but can not stop course of the disease, and may affect the whole immune system.The pathogenic B cell which produces AChR antibodies, is a key factor in the pathogenesis of MG.
Our study is to construct an AChR‐IgG Fc fusion protein which can specificly bind to the FcγRⅡB of pathogenic B cells or FcγR of mononuclear macrophage to inhibit the activation of pathogenic B cell and accelerate its apoptosis.
The genomic fragment Hα1-121 of the human skeletal muscle Acetylcholine receptor (AChR)α1 subunit was amplified by PCR and inserted into the eukaryotic expression vector pAN1782.The recombinant plasmid was transfected into CHOk1 cells with screening culture by G418 for stable expression.The expression of the AChR‐IgG Fc fusion protein was detected by Western blot.
A 428 bp band was amplified as expected and the sequencing results showed no mutation or frame shift .The recombinant plasmid was confirmed by enzyme digestion and transfected into CHOk1 cells.The stable expression of the AChR‐IgG Fc fusion protein was demonstrated by Western blot .
To conclude, the eukaryotic expression vector of AChR‐IgG Fc/pAN1782 was successfully constructed and the AChR‐IgG Fc fusion protein was stably expressed in CHOk1 cells,providing basis for the B cell targeted therapy of myasthenia gravis .
Experimental autoimmune myasthenia gravis (EAMG) in mouse is the animal model of MG and helps a lot for finding the pathogenesis and therapy of the disease. The ECD (N-terminal extracellular domain (amino acids 1-210) of theαsubunit of AChR) protein was used to immunize the female Lewis rats and the clinical manifestation , the change of body weight were evaluated. The blood serum of the vena caudalis was extracted every week to test the titer of AChR-ab by ELISA.
This method is simpler with a lower cost compared with the traditional EAMG models. The EAMG model can be used to detect the function of the therapeutic protein and research the pathogenesis of MG..
现有治疗手段主要为胆碱酯酶抑制剂、皮质激素、免疫抑制剂、血浆置换、静脉注射免疫球蛋白、胸腺切除术等,能一定程度控制病情,但不能抑制病程发展,且会影响全身免疫系统。致病性B细胞分泌产生AChR抗体,是MG发病的关键因子,若能从源头上清除这类B细胞则有望控制MG病情。
本研究希望通过真核表达载体转染CHOk1细胞以获得一种AChR‐ IgG Fc融合蛋白,该融合蛋白可与分泌AChR抗体的致病B细胞表面抑制性受体FcγRⅡB或单核‐巨噬细胞表面的Fcγ受体特异性结合,抑制B细胞活化并促进其凋亡。
以人nAChRα1亚基全序列为模板,以PCR法扩增AChRα1亚基胞外段主要免疫区基因片段Hα1-121 ,将其插入真核表达载体pAN1782。将构建的新载体转染至CHOk1细胞,建立稳定表达AChR‐ IgG Fc融合蛋白的CHOk1细胞株,以Western blot法检测AChR‐ IgG Fc融合蛋白表达。
结果显示Hα1-121经PCR法扩增后所获428 bp基因片段大小符合预计结果,测序所得核苷酸序列与人类基因库中AChRα1亚基胞外段基因片段Hα1-121序列完全一致,未出现点突变或移码突变。所构建的新载体经酶切鉴定证实片段插入正确,载体构建成功。Western blot法检测结果显示转染新载体的CHOk1细胞培养上清液中有AChR‐ IgG Fc融合蛋白表达。
综上所述,成功构建人AChR‐ IgG Fc段融合蛋白真核表达载体,且该融合蛋白可在转染新载体的CHOk1细胞中稳定表达,为下一步靶向B细胞治疗重症肌无力奠定了初步基础。
EAMG是MG的动物模型,是研究MG发病机制、治疗策略的重要手段。我们采用重组表达的人肌肉AChR的α亚基(α1-210)ECD蛋白免疫雌性Lewis大鼠,观察免疫后大鼠的体重变化、行走、进食情况,抽取大鼠尾静脉血液,用ELISA法检测血清中AChR抗体滴度变化。
该方法较传统建模方法简便快捷,EAMG模型的成功建立可为功能蛋白的检测以及MG发病机制、治疗方法的研究奠定基础。
Myasthenia gravis (MG) is a disorder of neuromuscular transmission mediated by autoantibodies to the nicotinic acetylcholine receptor (AChR) and involved in activation of complement. It is characterised by fatigable muscle weakness. The most common treatment options used for MG are acetylcholinesterase inhibitors, corticosteroids, immunosuppressants, plasmapheresis, intravenous immunoglobulin, thymectomy, which can relieve the conditions but can not stop course of the disease, and may affect the whole immune system.The pathogenic B cell which produces AChR antibodies, is a key factor in the pathogenesis of MG.
Our study is to construct an AChR‐IgG Fc fusion protein which can specificly bind to the FcγRⅡB of pathogenic B cells or FcγR of mononuclear macrophage to inhibit the activation of pathogenic B cell and accelerate its apoptosis.
The genomic fragment Hα1-121 of the human skeletal muscle Acetylcholine receptor (AChR)α1 subunit was amplified by PCR and inserted into the eukaryotic expression vector pAN1782.The recombinant plasmid was transfected into CHOk1 cells with screening culture by G418 for stable expression.The expression of the AChR‐IgG Fc fusion protein was detected by Western blot.
A 428 bp band was amplified as expected and the sequencing results showed no mutation or frame shift .The recombinant plasmid was confirmed by enzyme digestion and transfected into CHOk1 cells.The stable expression of the AChR‐IgG Fc fusion protein was demonstrated by Western blot .
To conclude, the eukaryotic expression vector of AChR‐IgG Fc/pAN1782 was successfully constructed and the AChR‐IgG Fc fusion protein was stably expressed in CHOk1 cells,providing basis for the B cell targeted therapy of myasthenia gravis .
Experimental autoimmune myasthenia gravis (EAMG) in mouse is the animal model of MG and helps a lot for finding the pathogenesis and therapy of the disease. The ECD (N-terminal extracellular domain (amino acids 1-210) of theαsubunit of AChR) protein was used to immunize the female Lewis rats and the clinical manifestation , the change of body weight were evaluated. The blood serum of the vena caudalis was extracted every week to test the titer of AChR-ab by ELISA.
This method is simpler with a lower cost compared with the traditional EAMG models. The EAMG model can be used to detect the function of the therapeutic protein and research the pathogenesis of MG..
引文
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