CD86人—鼠嵌合抗体的构建、表达及其生物学活性的初步研究
发布时间：2018-10-21 10:29
【摘要】： B7分子(包括B7-1和B7-2,又称CD80和CD86)是表达在APC表面的共刺激分子,其与表达于T细胞表面的相应受体CD28/CTLA-4结合产生共刺激信号,对T细胞的活化和耐受起关键性调节作用。CD86为一个由1120bp的基因所编码,其基因序列与B7-1有26%的同源性,整个蛋白由306个氨基酸组成,属于I型跨膜糖蛋白。本研究在已获得稳定分泌鼠抗人CD86单克隆抗体杂交瘤细胞株的基础上,采用基因重组技术及细胞与分子生物学方法等进行了以下研究:1.CD86人-鼠嵌合抗体表达载体的构建及在CHO细胞中的表达 首先采用RT-PCR从特异性分泌抗人CD86单克隆抗体的杂交瘤细胞株1D1中提取总RNA,常规逆转录,应用简并引物进行PCR扩增,并根据重、轻链基因序列分析结果设计引物,应用SMART-PCR方法扩增含信号肽序列的V_H和V_L基因,进行测序鉴定。在本科室构建的表达质粒PIRES/ch5C11基础上,通过PCR获得人IgG1γ链、κ链恒定区基因。将含信号肽序列的V_H序列和人的IgG1重链恒定区序列进行拼接获得嵌合重链,将含信号肽序列的V_L序列和人的κ链恒定区序列进行拼接获得嵌合轻链,拼接产物测序鉴定,构建共表达嵌合重、轻链的重组质粒pIRES/ch1D1。采用脂质体法将重组质粒转染293T细胞。以CD86-L929基因转染细胞株作为检测细胞,经流式细胞术对293T细胞培养上清鉴定后转染CHO细胞,再行鉴定后大量收集无血清培养上清,经Protein G亲和层析纯化及Lowry法定量。结果显示,本研究成功构建了含人-鼠嵌合重链和嵌合轻链基因的CD86人-鼠嵌合抗体真核表达载体pIRES/ch1D1。选择中国仓鼠卵巢细胞(CHO)作为表达宿主,经试剂盒抽提的pIRES/ch1D1嵌合抗体重组表达质粒用脂质体法转染CHO细胞,经G418加压筛选后进行亚克隆,获得持续稳定分泌抗人CD86人-鼠嵌合抗体(ch1D1)的基因转染细胞株(ch1D1-CHO)。RT-PCR鉴定结果表明ch1D1表达基因成功整合在ch1D1-CHO细胞中;FCM结果表明,转染pIRES/ch1D1质粒的CHO培养上清中的嵌合抗体与L929-CD86基因转染细胞膜型CD86分子的阳性结合率为98.5%。从CHO细胞培养上清中获取嵌合抗体纯化品的量约为3.0mg/L。 2.CD86人—鼠嵌合抗体的生物学活性的初步研究 采用间接免疫荧光及流式细胞术方法,对多株肿瘤细胞株Daudi、Sub-T、U937、Jurkat、HO8910、H1299及WERI-RB1细胞膜性CD86分子进行分析。在此基础上,选取天然高表达CD86及CD80分子的Daudi细胞为靶细胞,观察和分析了抗CD86嵌合抗体单独及联合抗CD80嵌合抗体对细胞生长和增殖的影响作用。将嵌合抗体(终浓度为5μg/mL)加入到Daudi细胞的培养体系中,经显微镜观察及MTT法分析,嵌合抗体对Daudi细胞的生长具有抑制作用(p＜0.05),且CD86与CD80嵌合抗体具有协同抑制作用。将两个健康人外周血单个核细胞(PBMC)加入96孔板中,分别加入CD86嵌合抗体及联合CD80嵌合抗体(终浓度为5μg/mL),于培养72小时采用MTT法检测增殖情况。结果显示在双向混合淋巴细胞反应中,CD86嵌合抗体能抑制混合淋巴细胞的增殖效应,且CD86与CD80嵌合抗体具有协同抑制作用。 综上所述,本实验成功构建了CD86人-鼠嵌合抗体(ch1D1)真核表达质粒,并在CHO细胞中实现了稳定表达,表达的嵌合抗体对B淋巴瘤细胞株Daudi细胞的生长增殖具有抑制作用,并且在混合淋巴细胞反应中,能抑制混合淋巴细胞的增殖效应。
[Abstract]:B7 molecules (including B7-1 and B7-2, also known as CDR3 and CD86) are co-stimulatory molecules expressed on the surface of the APC, which bind to the corresponding receptors CCR5/ CTLA-4, which are expressed on the surface of T cells, to produce co-stimulatory signals that play a pivotal role in the activation and tolerance of T cells. CD86 is encoded by 1120bp gene, its gene sequence has 26% homology with B7-1, and the whole protein consists of 306 amino acids, belonging to type I transmembrane glycoprotein. Based on the hybridoma cell lines of anti-human CD86 monoclonal antibody which stably secrete murine anti-human CD86, the following studies were carried out by using gene recombination technology and cell and molecular biology methods. Construction of CD86 human-mouse chimeric antibody expression vector and its expression in CHO cells firstly extracting total RNA from hybridoma cell strain 1D1 specifically secreting anti-human CD86 monoclonal antibody by RT-PCR, performing PCR amplification by using degenerate primer, and The V _ H and V _ L genes of signal peptide sequence were amplified by SMART-PCR according to the results of heavy and light chain gene sequence analysis. On the basis of the expression plasmid PIRES/ ch5C11 constructed in the undergraduate room, the human IgG1 chain was obtained by PCR and the chain constant was constant. splicing a V _ H sequence containing a signal peptide sequence and a human IgG1 heavy chain constant region sequence to obtain a chimeric heavy chain, splicing the V _ L sequence containing the signal peptide sequence and the sequence of the constant region of the chain constant region of a human, obtaining the chimeric light chain, sequencing and identifying the spliced product, and constructing the co-expression Recombinant plasmid pIRES/ ch of chimeric heavy and light chain 1D1. transfection of recombinant plasmid using liposome method 293 T cells were transfected with CD86-L929 gene and transfected into CHO cells by flow cytometry. After identification, the cells were transfected into CHO cells. After re-identification, no serum culture supernatant was collected and purified by ProSphere G affinity chromatography. The results showed that the eukaryotic expression vector pIRES/ ch of CD86 human-mouse chimeric antibody containing human-mouse chimeric heavy chain and chimeric light chain gene was successfully constructed in this study. 1D1. Select Chinese hamster ovary cell (CHO) as the expression host, and the recombinant expression plasmid of pIRES/ ch1D1 chimeric antibody extracted by the kit is transfected into CHO cells by liposome method, and then subjected to high pressure screening. Subcloning, a gene transfected cell strain (ch1D1-CHO) for stably secreting anti-human CD86 human-mouse chimeric antibody (ch1D1) was obtained. The results of RT-PCR showed that the ch1D1 expression gene was successfully integrated in ch1D1-CHO cells; FCM junctions The results showed that the positive binding rate of chimeric antibody in CHO culture supernatant transfected with pIRES/ ch1D1 plasmid and L929-CD86 gene into cell membrane type CD86 was 98.. 5%. The amount of purified product purified from CHO cell culture was about 3.0m Bioassay of Chimeric Antibody in g/ L. 2.CD86 Human A preliminary study on the activity of the study was carried out by indirect immunofluorescence and flow cytometry. The cell membranes of Daudi, Sub-T, U937, HO8910, HO8910, H1299 and WERI-RB1 were studied by indirect immunofluorescence and flow cytometry. On this basis, Daudi cells with high expression of CD86 and CD86 were selected as target cells, and anti-CD86 chimeric antibody alone and anti-CD86 chimeric antibody were observed and analyzed. In the culture system of Daudi cells, the chimeric antibody (final concentration of 5. mu.g/ mL) was added to the culture system of Daudi cells, and the inhibition of the chimeric antibody on the growth of Daudi cells was observed by microscopic observation and MTT assay (p <0.05), and CD86 and CD86 were embedded in the culture system of Daudi cells. Two healthy human peripheral blood mononuclear cells (PBMC) were added to 96-well plates, CD86 chimeric antibody and combined anti-CD86 chimeric antibody (final concentration of 5. mu.g/ mL) were added to the 96-well plates. The results showed that CD86 chimeric antibody could inhibit the proliferation effect of mixed lymphocyte in the two-way mixed lymphocyte reaction, and CD86 and CD86 were embedded in the mixed lymphocyte. In conclusion, the expression plasmid of CD86 human-mouse chimeric antibody (ch1D1) was successfully constructed in this experiment, and stable expression was realized in CHO cells. The expression of chimeric antibody has inhibitory effect on the growth and proliferation of Daudi cells of B lymphoma cell line. Effect and in mixed lymphocyte reaction
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2284820
[Abstract]:B7 molecules (including B7-1 and B7-2, also known as CDR3 and CD86) are co-stimulatory molecules expressed on the surface of the APC, which bind to the corresponding receptors CCR5/ CTLA-4, which are expressed on the surface of T cells, to produce co-stimulatory signals that play a pivotal role in the activation and tolerance of T cells. CD86 is encoded by 1120bp gene, its gene sequence has 26% homology with B7-1, and the whole protein consists of 306 amino acids, belonging to type I transmembrane glycoprotein. Based on the hybridoma cell lines of anti-human CD86 monoclonal antibody which stably secrete murine anti-human CD86, the following studies were carried out by using gene recombination technology and cell and molecular biology methods. Construction of CD86 human-mouse chimeric antibody expression vector and its expression in CHO cells firstly extracting total RNA from hybridoma cell strain 1D1 specifically secreting anti-human CD86 monoclonal antibody by RT-PCR, performing PCR amplification by using degenerate primer, and The V _ H and V _ L genes of signal peptide sequence were amplified by SMART-PCR according to the results of heavy and light chain gene sequence analysis. On the basis of the expression plasmid PIRES/ ch5C11 constructed in the undergraduate room, the human IgG1 chain was obtained by PCR and the chain constant was constant. splicing a V _ H sequence containing a signal peptide sequence and a human IgG1 heavy chain constant region sequence to obtain a chimeric heavy chain, splicing the V _ L sequence containing the signal peptide sequence and the sequence of the constant region of the chain constant region of a human, obtaining the chimeric light chain, sequencing and identifying the spliced product, and constructing the co-expression Recombinant plasmid pIRES/ ch of chimeric heavy and light chain 1D1. transfection of recombinant plasmid using liposome method 293 T cells were transfected with CD86-L929 gene and transfected into CHO cells by flow cytometry. After identification, the cells were transfected into CHO cells. After re-identification, no serum culture supernatant was collected and purified by ProSphere G affinity chromatography. The results showed that the eukaryotic expression vector pIRES/ ch of CD86 human-mouse chimeric antibody containing human-mouse chimeric heavy chain and chimeric light chain gene was successfully constructed in this study. 1D1. Select Chinese hamster ovary cell (CHO) as the expression host, and the recombinant expression plasmid of pIRES/ ch1D1 chimeric antibody extracted by the kit is transfected into CHO cells by liposome method, and then subjected to high pressure screening. Subcloning, a gene transfected cell strain (ch1D1-CHO) for stably secreting anti-human CD86 human-mouse chimeric antibody (ch1D1) was obtained. The results of RT-PCR showed that the ch1D1 expression gene was successfully integrated in ch1D1-CHO cells; FCM junctions The results showed that the positive binding rate of chimeric antibody in CHO culture supernatant transfected with pIRES/ ch1D1 plasmid and L929-CD86 gene into cell membrane type CD86 was 98.. 5%. The amount of purified product purified from CHO cell culture was about 3.0m Bioassay of Chimeric Antibody in g/ L. 2.CD86 Human A preliminary study on the activity of the study was carried out by indirect immunofluorescence and flow cytometry. The cell membranes of Daudi, Sub-T, U937, HO8910, HO8910, H1299 and WERI-RB1 were studied by indirect immunofluorescence and flow cytometry. On this basis, Daudi cells with high expression of CD86 and CD86 were selected as target cells, and anti-CD86 chimeric antibody alone and anti-CD86 chimeric antibody were observed and analyzed. In the culture system of Daudi cells, the chimeric antibody (final concentration of 5. mu.g/ mL) was added to the culture system of Daudi cells, and the inhibition of the chimeric antibody on the growth of Daudi cells was observed by microscopic observation and MTT assay (p <0.05), and CD86 and CD86 were embedded in the culture system of Daudi cells. Two healthy human peripheral blood mononuclear cells (PBMC) were added to 96-well plates, CD86 chimeric antibody and combined anti-CD86 chimeric antibody (final concentration of 5. mu.g/ mL) were added to the 96-well plates. The results showed that CD86 chimeric antibody could inhibit the proliferation effect of mixed lymphocyte in the two-way mixed lymphocyte reaction, and CD86 and CD86 were embedded in the mixed lymphocyte. In conclusion, the expression plasmid of CD86 human-mouse chimeric antibody (ch1D1) was successfully constructed in this experiment, and stable expression was realized in CHO cells. The expression of chimeric antibody has inhibitory effect on the growth and proliferation of Daudi cells of B lymphoma cell line. Effect and in mixed lymphocyte reaction
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
【引证文献】
相关期刊论文 前1条
1 聂静苑;刘煜;;人源单克隆抗体药物质量控制与分析[J];中国生化药物杂志;2012年02期
，本文编号：2284820
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