抗人CD40人—鼠嵌合抗体的构建表达及功能的初步研究
发布时间：2018-10-24 13:57
【摘要】： 本研究在自行成功研制的鼠抗人CD40激发型单克隆抗体(5C11)和鼠抗人muCD40单克隆抗体(5H6)的基础上,采用嵌合抗体构建方法,在真核细胞293T中实现表达,通过计算机辅助设计方法提高嵌合抗体表达量,并对嵌合抗体生物学功能进行初步研究。 第一部分:CD40人-鼠嵌合抗体的构建、表达及功能初步研究 从5C11杂交瘤细胞中抽取总RNA,常规逆转录,应用简并引物进行PCR扩增,钓取重链Fd及轻链基因。根据重、轻链基因序列设计引物,应用PCR方法扩增VH和VL基因。双酶切装载有信号肽序列及人IgG1γ链的Fc、CH1基因的重链表达载体pCMV-VH以及装载有信号肽序列及κ链的Cκ基因的轻链表达载体pCMV-VL。将同样双酶切的VH和VL基因连接入表达载体,构建成嵌合抗体(ch5C11)的轻链真核表达载体5C11L-pCMV及重链真核表达载体5C11H-pCMV。将嵌合抗体的轻重链真核表达载体共转染入293T细胞,利用夹心ELISA法测定上清中c5C11的浓度,利用流式细胞术检测c5C11与膜抗原的特异性结合。选择天然高表达CD40分子的人B淋巴瘤细胞株Daudi(阳性表达率90%)与ch5C11共培养,镜下观察细胞生长形态, CCK-8分析ch5C11对Daudi细胞的生长与存活的影响。研究结果表明:成功构建了分别含嵌合重、轻链基因的真核表达载体5C11H-pCMV及5C11L-pCMV,并在293T细胞中得到瞬时表达。ch5C11能够有效识别Daudi细胞膜型CD40分子。ch5C11能有效抑制Daudi细胞体外增殖。提示CD40嵌合抗体具有良好的生物学活性。 通过计算机辅助设计方法对c5C11的氨基酸序列进行改造,分析并设计突变影响嵌合抗体表达量的氨基酸。将改造后的轻链表达载体5C11L-pCMV及重链表达载体5C11H(M)-pCMV共转染的293T细胞。利用夹心ELISA法测定上清中ch5C11的浓度,利用流式细胞术检测ch5C11与膜抗原的特异性结合。选择天然高表达CD40分子的人B淋巴瘤细胞株Daudi(阳性表达率90%)与ch5C11(M)共培养,镜下观察细胞生长形态, CCK-8分析ch5C11(M)对Daudi细胞的生长与存活的影响。结果表明:ch5C11(M)表达量有较显著的提高。ch5C11(M)能够有效识别Daudi细胞膜型CD40分子。ch5C11能有效抑制Daudi细胞体外增殖。提示CD40嵌合抗体具有良好的生物学活性。 第二部分:muCD40人-鼠嵌合抗体的构建、表达及功能初步研究 从5H6杂交瘤细胞中抽取总RNA,常规逆转录,应用简并引物进行PCR扩增,钓取重链Fd及轻链基因。根据重、轻链基因序列设计引物,应用PCR方法扩增VH和VL基因。双酶切装载有信号肽序列及人IgG1γ链的Fc、CH1基因的重链表达载体pCMV-VH以及装载有信号肽序列及κ链的Cκ基因的轻链表达载体pCMV-VL。将同样双酶切的VH和VL基因连接入表达载体,构建成嵌合抗体(ch5H6)的轻链真核表达载体5H6L-pCMV及重链真核表达载体5H6H-pCMV。将嵌合抗体的轻重链真核表达载体共转染入293T细胞,利用夹心ELISA法测定上清中ch5H6的浓度,利用流式细胞术检测ch5H6与膜抗原的特异性结合。 综上所述:1、成功构建了抗人CD40人-鼠嵌合抗体(ch5C11)。2、CD40嵌合抗体可有效抑制天然高表达CD40分子的人B淋巴瘤细胞株Daudi的体外增殖。3、通过计算机辅助设计方法对ch5C11的氨基酸序列进行改造后,瞬时表达量明显提高。该抗体在某些肿瘤的免疫治疗及移植抗排异中具有潜在的应用价值。4、成功构建了抗人muCD40人-鼠嵌合抗体(ch5H6)。 这两株工程化抗人CD40抗体的研制,为CD40抗体在肿瘤治疗及诊断中的应用研究,奠定了厚实的基础。
[Abstract]:On the basis of the mouse anti-human CD40 stimulating monoclonal antibody (5C11) and the mouse anti-human muCD40 monoclonal antibody (5H6), the mouse anti-human CD40 stimulating type monoclonal antibody (5C11) and the mouse anti-human muCD40 monoclonal antibody (5H6) are successfully developed, the expression is realized in the eukaryotic cell 293T, the expression amount of the chimeric antibody is improved by the computer aided design method, The biological function of chimeric antibody was studied. Part one: Construction, expression and function of CD40 human-mouse chimeric antibody In the preliminary study, total RNA was extracted from 5C11 hybridoma cells, conventional reverse transcription was performed, degenerate primers were applied for PCR amplification, and heavy chain was fished. Fd and light chain genes are designed according to heavy and light chain gene sequences and amplified by PCR method. VH and VL genes. The double enzyme is loaded with a signal peptide sequence and an Fc, CH1 gene of the human IgG1 replicon chain, a heavy chain expression vector pCMV-VH of the CH1 gene, and a light chain expression vector loaded with a signal peptide sequence and a C-jun gene of the human IgG1 chain. pCMV-VL. The VH and VL genes of the same bisenzyme cleavage were ligated into the expression vector, and the light chain true nuclear expression vector 5C11L-pCMV and the heavy chain true nuclear expression vector 5C1 of the chimeric antibody (ch5C11) were constructed. 1H-pCMV was transfected into 293T cells. The concentration of c5C11 in supernatant was determined by sandwich ELISA, and c5C11 and membrane were detected by flow cytometry. Human B lymphoma cell line Daudi (positive expression rate 90%) selected from natural high-expression CD40 molecule was co-cultured with ch5C11, the cell growth pattern was observed under the mirror, and CCK-8 was used to analyze the growth of Daudi cell. The results showed that the eukaryotic expression vector 5C11H-pCMV and 5C11L-pCMV containing chimeric heavy and light chain genes were successfully constructed, and 293T cells were constructed successfully. Transient expression was obtained. Ch5C11 was able to identify Daudi cell membrane effectively. type CD40 molecule. ch5C11 can effectively inhibit Daud In vitro proliferation of i-cells showed that the CD40 chimeric antibody was good. The amino acid sequence of c5C11 was transformed, analyzed and designed by computer aided design method. Chimeric antibody expression vector 5C11L-pCMV and heavy chain expression vector 5C11H (M)-pCM The concentration of ch5C11 in supernatant was determined by sandwich ELISA. Ch5C1 was detected by flow cytometry. 1. Specific binding to membrane antigen. Daudi (positive expression rate 90%) of human B lymphoma cell line selected by natural high expression CD40 molecule was co-cultured with ch5C11 (M), the cell growth pattern was observed under the microscope, and CCK-8 analysis ch5C11 (M) on Daudi The results showed that ch5C11 (M) Ch5C11 (M) was able to identify Daud effectively. i cell membrane type CD40 molecule. ch5C11 can be effectively inhibited. In vitro proliferation of Daudi cells. The body has good biological activity. The second part: muCD40 human-mouse block Preliminary study on the construction, expression and function of antibody to extract total RNA from 5H6 hybridoma cells, conventional reverse transcription, and application of degenerate primers carrying out PCR amplification, fishing a heavy chain Fd and a light chain gene, designing a heavy chain Fd and a light chain gene, Primers are used to amplify VH and VL genes by PCR method. Two enzymes are loaded with signal peptide sequence and Fc, CH1 gene of human IgG1 chain chain, heavy chain expression vector pCMV-VH of CH1 gene, A light chain expression vector pCMV-VL of the C-type gene is ligated into an expression vector, a light chain true nuclear expression vector 5H6L-pCM is constructed as a chimeric antibody (ch5H6), V and heavy chain eukaryon expression vector 5H6H-pCMV. The expression vector of the heavy chain of chimeric antibody was transfected into 293T cells, and the concentration of ch5H6 in supernatant was determined by sandwich ELISA. The specific binding of ch5H6 to membrane antigen was detected by cell cytometry. In conclusion: 1, anti-human CD40 human-mouse chimeric antibody (ch5C11) was successfully constructed. CD40 chimeric antibody could effectively inhibit the proliferation of human B lymphoma cell line Daudi in vitro. Method of co-design to ch5C When the amino acid sequence of 11 is modified, the transient expression level is obviously improved. The antibody has potential application value in the immunotherapy and transplantation of certain tumors. 4. Successful construction Anti-human muCD40 human-mouse chimeric antibody (ch5H6) was developed. The two engineered anti-human CD40 antibodies were developed as CD
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2291606
[Abstract]:On the basis of the mouse anti-human CD40 stimulating monoclonal antibody (5C11) and the mouse anti-human muCD40 monoclonal antibody (5H6), the mouse anti-human CD40 stimulating type monoclonal antibody (5C11) and the mouse anti-human muCD40 monoclonal antibody (5H6) are successfully developed, the expression is realized in the eukaryotic cell 293T, the expression amount of the chimeric antibody is improved by the computer aided design method, The biological function of chimeric antibody was studied. Part one: Construction, expression and function of CD40 human-mouse chimeric antibody In the preliminary study, total RNA was extracted from 5C11 hybridoma cells, conventional reverse transcription was performed, degenerate primers were applied for PCR amplification, and heavy chain was fished. Fd and light chain genes are designed according to heavy and light chain gene sequences and amplified by PCR method. VH and VL genes. The double enzyme is loaded with a signal peptide sequence and an Fc, CH1 gene of the human IgG1 replicon chain, a heavy chain expression vector pCMV-VH of the CH1 gene, and a light chain expression vector loaded with a signal peptide sequence and a C-jun gene of the human IgG1 chain. pCMV-VL. The VH and VL genes of the same bisenzyme cleavage were ligated into the expression vector, and the light chain true nuclear expression vector 5C11L-pCMV and the heavy chain true nuclear expression vector 5C1 of the chimeric antibody (ch5C11) were constructed. 1H-pCMV was transfected into 293T cells. The concentration of c5C11 in supernatant was determined by sandwich ELISA, and c5C11 and membrane were detected by flow cytometry. Human B lymphoma cell line Daudi (positive expression rate 90%) selected from natural high-expression CD40 molecule was co-cultured with ch5C11, the cell growth pattern was observed under the mirror, and CCK-8 was used to analyze the growth of Daudi cell. The results showed that the eukaryotic expression vector 5C11H-pCMV and 5C11L-pCMV containing chimeric heavy and light chain genes were successfully constructed, and 293T cells were constructed successfully. Transient expression was obtained. Ch5C11 was able to identify Daudi cell membrane effectively. type CD40 molecule. ch5C11 can effectively inhibit Daud In vitro proliferation of i-cells showed that the CD40 chimeric antibody was good. The amino acid sequence of c5C11 was transformed, analyzed and designed by computer aided design method. Chimeric antibody expression vector 5C11L-pCMV and heavy chain expression vector 5C11H (M)-pCM The concentration of ch5C11 in supernatant was determined by sandwich ELISA. Ch5C1 was detected by flow cytometry. 1. Specific binding to membrane antigen. Daudi (positive expression rate 90%) of human B lymphoma cell line selected by natural high expression CD40 molecule was co-cultured with ch5C11 (M), the cell growth pattern was observed under the microscope, and CCK-8 analysis ch5C11 (M) on Daudi The results showed that ch5C11 (M) Ch5C11 (M) was able to identify Daud effectively. i cell membrane type CD40 molecule. ch5C11 can be effectively inhibited. In vitro proliferation of Daudi cells. The body has good biological activity. The second part: muCD40 human-mouse block Preliminary study on the construction, expression and function of antibody to extract total RNA from 5H6 hybridoma cells, conventional reverse transcription, and application of degenerate primers carrying out PCR amplification, fishing a heavy chain Fd and a light chain gene, designing a heavy chain Fd and a light chain gene, Primers are used to amplify VH and VL genes by PCR method. Two enzymes are loaded with signal peptide sequence and Fc, CH1 gene of human IgG1 chain chain, heavy chain expression vector pCMV-VH of CH1 gene, A light chain expression vector pCMV-VL of the C-type gene is ligated into an expression vector, a light chain true nuclear expression vector 5H6L-pCM is constructed as a chimeric antibody (ch5H6), V and heavy chain eukaryon expression vector 5H6H-pCMV. The expression vector of the heavy chain of chimeric antibody was transfected into 293T cells, and the concentration of ch5H6 in supernatant was determined by sandwich ELISA. The specific binding of ch5H6 to membrane antigen was detected by cell cytometry. In conclusion: 1, anti-human CD40 human-mouse chimeric antibody (ch5C11) was successfully constructed. CD40 chimeric antibody could effectively inhibit the proliferation of human B lymphoma cell line Daudi in vitro. Method of co-design to ch5C When the amino acid sequence of 11 is modified, the transient expression level is obviously improved. The antibody has potential application value in the immunotherapy and transplantation of certain tumors. 4. Successful construction Anti-human muCD40 human-mouse chimeric antibody (ch5H6) was developed. The two engineered anti-human CD40 antibodies were developed as CD
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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