Tn5细胞中单链抗体基因抗人重组质粒分子的表达分析
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摘要
目的研究Tn5细胞中单抗抗人重组质粒(B7-2)基因的表达及其活性。方法基于具备信号肽单抗抗人B7-2构建,构建系统Bac-to-Bac并含单抗抗人B7-2杆状重组病毒AcBacmid单抗B7-2,对Tn5细胞采用不同感染复数(MOI)实施感染,进行24、48、72h不同时点细胞蛋白活性的分泌表达,并分析人外周血中单个核细胞受Raji刺激呈增殖表达,予以单抗抑制的效果。结果应用不同MOI对Tn5细胞感染,达72h后进行细胞收集,予以聚丙烯酰氨凝胶电泳(PAGE-SDS)分析,其MOI为1~10表达目的蛋白的量,确定最佳MOI为5;MTT法下自动酶标仪波长570nm位置吸光度,其吸光值的大小同细胞PBMC增殖可呈正相关;各组吸光值显示细胞Raji刺激外周血单个核细胞(PBMC)呈增殖表达,单抗抗人B7-2可以抑制Raji细胞刺激PBMC出现的增殖效应。结论单抗B7-2抗原抗体相结合的活性,以及对PBMC产生的抑制效应均存在,但未见单抗B7-2效应明显表达,有待进行更进一步的试验研究。
OBJECTIVE To study the expression of monoclonal antibody against human B7-2gene in Tn5 cells and its activity.METHODS Bac-to-Bac and monoclonal antibody against human B7-2containing rod-shaped recombinant virus AcBacmid monoclonal antibody B7-2was constructed based on the construction of monoclonal antibody of anti human B7-2with signal peptide,Tn5 cells were infected by using different MOI(multiplicity of infection),the optimal MOI was obtained,the expression and secretion of cell protein activity were conducted at different time points(24hours/48hours/72hours),the proliferation expression of mononuclear cells in peripheral blood,which was stimulated by Raji,was analyzed,and the inhibiting effect of monoclonal antibody was observed.RESULTS The cells were collected after the Tn5 cells were infected for 72 hours by using different MOI and were analyzed with the use of PAGE-SDS,the MOI was the content of expression of target protein from 1to 10,and the optimal MOI was determined as 5.As for the absorbance at the position of 570 nm wavelength of automatic enzyme mark instrument under MTT,the absorbance value was positively correlated with the proliferation of PBMC cells.The absorbance value indicated that the peripheral blood mononuclear cells(PBMC)stimulated by Raji showed proliferation expression and that the monoclonal antibody of anti human B7-2could inhibit the proliferation effect of the PBMC stimulated by Raji.CONCLUSIONB7-2monoclonal antibody combined with antigen antibody activity and the inhibiting effect on proliferation of PBMC exist,however,the expression of monoclonal antibody B7-2is not obvious,which needs to be further proved by experimental study.
OBJECTIVE To study the expression of monoclonal antibody against human B7-2gene in Tn5 cells and its activity.METHODS Bac-to-Bac and monoclonal antibody against human B7-2containing rod-shaped recombinant virus AcBacmid monoclonal antibody B7-2was constructed based on the construction of monoclonal antibody of anti human B7-2with signal peptide,Tn5 cells were infected by using different MOI(multiplicity of infection),the optimal MOI was obtained,the expression and secretion of cell protein activity were conducted at different time points(24hours/48hours/72hours),the proliferation expression of mononuclear cells in peripheral blood,which was stimulated by Raji,was analyzed,and the inhibiting effect of monoclonal antibody was observed.RESULTS The cells were collected after the Tn5 cells were infected for 72 hours by using different MOI and were analyzed with the use of PAGE-SDS,the MOI was the content of expression of target protein from 1to 10,and the optimal MOI was determined as 5.As for the absorbance at the position of 570 nm wavelength of automatic enzyme mark instrument under MTT,the absorbance value was positively correlated with the proliferation of PBMC cells.The absorbance value indicated that the peripheral blood mononuclear cells(PBMC)stimulated by Raji showed proliferation expression and that the monoclonal antibody of anti human B7-2could inhibit the proliferation effect of the PBMC stimulated by Raji.CONCLUSIONB7-2monoclonal antibody combined with antigen antibody activity and the inhibiting effect on proliferation of PBMC exist,however,the expression of monoclonal antibody B7-2is not obvious,which needs to be further proved by experimental study.
引文
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[8]徐重新,张霄,刘媛,等.抗苏云金芽孢杆菌Cry1B毒蛋白质单链抗体的筛选与鉴定[J].江苏农业学报,2012,28(4):886-890.
[9]阚方明,任桂萍,郭茉,等.抗IL-1βscfv抗体与TNF-α可溶性受体融合蛋白的构建及其生物学活性分析[J].中华微生物学和免疫学杂志,2012,32(10):855-860.
[10]李小鸥,黄巍,李莉,等.封闭N-cadherin解整合素金属蛋白酶水解位点的单链抗体的制备及鉴定[J].细胞与分子免疫学杂志,2013,29(9):949-952.
[11]王朝阳,任学群,刘瑞敏,等.抗人CTGF单链抗体噬菌体展示文库的构建[J].中国实用医刊,2013,40(11):1-3.
[2]董海辉,邱玉华,陈永井,等.抗人B7-2单链抗体基因的构建及其在大肠埃希菌中的表达[J].科技通报,2010,26(3):367-373.
[3]陈昌友,郭静雅,陈永井,等.抗人CD80单链抗体的构建、表达及与抗原结合的特性分析[J].中国免疫学杂志,2011,27(2):153-157.
[4]李天鹤,任桂萍,徐黎明,等.抗人IL-1β单链抗体的构建及其中和活性的检测[J].中国免疫学杂志,2013,29(3):304-308.
[5]周松峰,廖文军,覃绍敏,等.抗人红细胞单链抗体与狂犬病毒G蛋白双功能融合蛋白的构建及生物学活性检测[J].中国人兽共患病学报,2012,28(6):597-601.
[6]张中林,艾勇彪,张吉发,等.利用噬菌体展示技术制备人血管生成素2单链抗体[J].中华实验外科杂志,2012,29(11):2159-2161.
[7]黄巍,周丽荣,周婷,等.APP-β分泌酶切割位点特异性单链抗体的制备和鉴定[J].中国药理学通报,2012,28(3):337-342.
[8]徐重新,张霄,刘媛,等.抗苏云金芽孢杆菌Cry1B毒蛋白质单链抗体的筛选与鉴定[J].江苏农业学报,2012,28(4):886-890.
[9]阚方明,任桂萍,郭茉,等.抗IL-1βscfv抗体与TNF-α可溶性受体融合蛋白的构建及其生物学活性分析[J].中华微生物学和免疫学杂志,2012,32(10):855-860.
[10]李小鸥,黄巍,李莉,等.封闭N-cadherin解整合素金属蛋白酶水解位点的单链抗体的制备及鉴定[J].细胞与分子免疫学杂志,2013,29(9):949-952.
[11]王朝阳,任学群,刘瑞敏,等.抗人CTGF单链抗体噬菌体展示文库的构建[J].中国实用医刊,2013,40(11):1-3.