抗人CD86双价抗体的表达及与抗原的结合特性
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摘要
目的:用中国仓鼠卵巢(CHO)细胞表达抗人CD86双价抗体(CD86-diabody),鉴定该抗体对表达CD86分子肿瘤细胞的识别及介导的生物学效应。方法:从分泌抗人CD86小鼠源抗体的杂交瘤1D1中克隆抗体重、轻链可变区基因VH及VL。SOE-PCR构建VH-(GGGGS)-VL双价抗体基因,整合到真核表达载体pIRES2-EGFP中,脂质体法转染CHO细胞,FCM筛选G418加压培养的阳性克隆。IMAC纯化并定量抗体。免疫荧光法分析抗体对人B系淋巴瘤细胞株Raji及Daudi膜型CD86分子的阳性结合率,将抗体加入到Raji细胞的培养体系中,培养72 h,MTT法分析抗体对细胞体外增殖的影响。结果:获得了稳定分泌抗人CD86-diabody的CHO细胞株。培养上清中抗体纯品的得率为5.24 mg/L。抗体与Raji及Daudi细胞的阳性结合率分别为77.2%及70.6%。经抗体孵育72 h,Raji细胞体外增殖抑制率为37%。结论:成功制备了抗人CD86-diabody,可特异性结合细胞膜型CD86分子,并对表达该分子的肿瘤细胞体外增殖具有抑制效应。
AIM: To construct a recombinant eukaryotic expression vector of anti-human CD86 diabody gene and express anti-CD86 diabody through Chinese hamster ovary(CHO) cells,then analyze the capability of the diabody to recognize the tumor cells expressing CD86 and its biological effect.METHODS: The antibody heavy and light chain viable region gene(VH and VL) were cloned from hybridoma cell 1D1 which secreted anti-human CD86 monoclonal antibody.Anti-human CD86 diabody gene VH-(GGGGS)-VL was constructed by SOE-PCR.Then we inserted it into eukaryotic expression vector to construct a recombinant vector pIRES2-EGFP/CD86-diabody.The recombinant vector was transfected into CHO cells with LipofectamineTM 2000,and the cell clones secreting CD86 diabody were screened by G418.We used IMAC to purify CD86 diabody and quantified its concentration by BCA method.The capability of the diabody to recognize the CD86 expressed on Raji and Daudi was analyzed through flow cytometry.After Raji cells were treated with CD86 diabody for 72 h,its proliferation inhibiting effect was investigated by MTT assay.RESULTS: We have obtained one CHO cell line that stably secreted CD86 diabody.The concentration of CD86 diabody after purification was 5.24 mg/L.The positive rates of CD86 diabody to recognize Raji and Daudi were 77.2% and 70.6%,respectively.After CD86 diabody treatment for 72 h,the inhibition rate of Raji cells was 37%.CONCLUSION: The anti-human CD86 diabody which we obtained successfully could recognize CD86 expressed on tumor cells specifically,and inhibit the proliferation of these tumor cells effectively.
AIM: To construct a recombinant eukaryotic expression vector of anti-human CD86 diabody gene and express anti-CD86 diabody through Chinese hamster ovary(CHO) cells,then analyze the capability of the diabody to recognize the tumor cells expressing CD86 and its biological effect.METHODS: The antibody heavy and light chain viable region gene(VH and VL) were cloned from hybridoma cell 1D1 which secreted anti-human CD86 monoclonal antibody.Anti-human CD86 diabody gene VH-(GGGGS)-VL was constructed by SOE-PCR.Then we inserted it into eukaryotic expression vector to construct a recombinant vector pIRES2-EGFP/CD86-diabody.The recombinant vector was transfected into CHO cells with LipofectamineTM 2000,and the cell clones secreting CD86 diabody were screened by G418.We used IMAC to purify CD86 diabody and quantified its concentration by BCA method.The capability of the diabody to recognize the CD86 expressed on Raji and Daudi was analyzed through flow cytometry.After Raji cells were treated with CD86 diabody for 72 h,its proliferation inhibiting effect was investigated by MTT assay.RESULTS: We have obtained one CHO cell line that stably secreted CD86 diabody.The concentration of CD86 diabody after purification was 5.24 mg/L.The positive rates of CD86 diabody to recognize Raji and Daudi were 77.2% and 70.6%,respectively.After CD86 diabody treatment for 72 h,the inhibition rate of Raji cells was 37%.CONCLUSION: The anti-human CD86 diabody which we obtained successfully could recognize CD86 expressed on tumor cells specifically,and inhibit the proliferation of these tumor cells effectively.
引文
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[10]胡静平,董海辉,邱玉华,等.鼠抗人B7-2分子单链抗体的合成及其在家蚕中的表达[J].蚕业科学,2010,36(2):171-181.
[11]Bühler P,Molnar E,Dopfer EP,et al.Target-dependent T-cellactivation by coligation with a PSMA x CD3 diabody induces lysis ofprostate cancer cells[J].J Immunother,2009,32(6):565-573.
[2]王艳茹,马泓冰,陈永井,等.CD80人2鼠嵌合抗体对B淋巴瘤细胞株Raji与Daudi的生长抑制及杀伤作用[J].细胞与分子免疫学杂志,2009,25(7):615-618.
[3]Perisic O,Webb PA,Holliger P,et al.Crystal structure of a dia-body,a bivalent antibody fragment[J].Structure,1994,2(12):1217-1226.
[4]陶然,石云杰,孙中文,等.鼠抗人B7-2单克隆抗体的研制及生物学特性研究[J].现代免疫学杂志,2006,26(3):203-207.
[5]郭静雅,陈昌友,王志强,等.人CXCR3B基因转染细胞株的构建、鉴定及生物学功能研究[J].细胞与分子免疫学杂志,2011,27(12):1288-1290.
[6]Jeannin P,Delneste Y,Lecoanet-Henchoz S,et al.CD86(B7-2)onhuman B cells.A functional role in proliferation and selective differen-tiation into IgE-and IgG4-producing cells[J].J Biol Chem,1997,272(25):15613-15619.
[7]Rau FC,Dieter J,Luo Z,et al.B7-1/2(CD80/CD86)directsignaling to B cells enhances IgG secretion[J].J Immunol,2009,183(12):7661-7671.
[8]Campanero MR.Mechanisms involved in Burkitt’s tumor formation[J].Clin Transl Oncol,2008,10(5):250-255.
[9]刘玉华,孙杰,郭静雅,等.B7-2基因工程抗体的制备及体内外抗B淋巴瘤作用研究[J].中山大学学报,2010,49(3):107-112.
[10]胡静平,董海辉,邱玉华,等.鼠抗人B7-2分子单链抗体的合成及其在家蚕中的表达[J].蚕业科学,2010,36(2):171-181.
[11]Bühler P,Molnar E,Dopfer EP,et al.Target-dependent T-cellactivation by coligation with a PSMA x CD3 diabody induces lysis ofprostate cancer cells[J].J Immunother,2009,32(6):565-573.