抗人CD40人—鼠嵌合抗體的構建表達及功能的初步研究
發(fā)布時間：2018-10-24 13:57
【摘要】： 本研究在自行成功研制的鼠抗人CD40激發(fā)型單克隆抗體(5C11)和鼠抗人muCD40單克隆抗體(5H6)的基礎上,采用嵌合抗體構建方法,在真核細胞293T中實現(xiàn)表達,通過計算機輔助設計方法提高嵌合抗體表達量,并對嵌合抗體生物學功能進行初步研究。 第一部分:CD40人-鼠嵌合抗體的構建、表達及功能初步研究 從5C11雜交瘤細胞中抽取總RNA,常規(guī)逆轉錄,應用簡并引物進行PCR擴增,釣取重鏈Fd及輕鏈基因。根據(jù)重、輕鏈基因序列設計引物,應用PCR方法擴增VH和VL基因。雙酶切裝載有信號肽序列及人IgG1γ鏈的Fc、CH1基因的重鏈表達載體pCMV-VH以及裝載有信號肽序列及κ鏈的Cκ基因的輕鏈表達載體pCMV-VL。將同樣雙酶切的VH和VL基因連接入表達載體,構建成嵌合抗體(ch5C11)的輕鏈真核表達載體5C11L-pCMV及重鏈真核表達載體5C11H-pCMV。將嵌合抗體的輕重鏈真核表達載體共轉染入293T細胞,利用夾心ELISA法測定上清中c5C11的濃度,利用流式細胞術檢測c5C11與膜抗原的特異性結合。選擇天然高表達CD40分子的人B淋巴瘤細胞株Daudi(陽性表達率90%)與ch5C11共培養(yǎng),鏡下觀察細胞生長形態(tài), CCK-8分析ch5C11對Daudi細胞的生長與存活的影響。研究結果表明:成功構建了分別含嵌合重、輕鏈基因的真核表達載體5C11H-pCMV及5C11L-pCMV,并在293T細胞中得到瞬時表達。ch5C11能夠有效識別Daudi細胞膜型CD40分子。ch5C11能有效抑制Daudi細胞體外增殖。提示CD40嵌合抗體具有良好的生物學活性。 通過計算機輔助設計方法對c5C11的氨基酸序列進行改造,分析并設計突變影響嵌合抗體表達量的氨基酸。將改造后的輕鏈表達載體5C11L-pCMV及重鏈表達載體5C11H(M)-pCMV共轉染的293T細胞。利用夾心ELISA法測定上清中ch5C11的濃度,利用流式細胞術檢測ch5C11與膜抗原的特異性結合。選擇天然高表達CD40分子的人B淋巴瘤細胞株Daudi(陽性表達率90%)與ch5C11(M)共培養(yǎng),鏡下觀察細胞生長形態(tài), CCK-8分析ch5C11(M)對Daudi細胞的生長與存活的影響。結果表明:ch5C11(M)表達量有較顯著的提高。ch5C11(M)能夠有效識別Daudi細胞膜型CD40分子。ch5C11能有效抑制Daudi細胞體外增殖。提示CD40嵌合抗體具有良好的生物學活性。 第二部分:muCD40人-鼠嵌合抗體的構建、表達及功能初步研究 從5H6雜交瘤細胞中抽取總RNA,常規(guī)逆轉錄,應用簡并引物進行PCR擴增,釣取重鏈Fd及輕鏈基因。根據(jù)重、輕鏈基因序列設計引物,應用PCR方法擴增VH和VL基因。雙酶切裝載有信號肽序列及人IgG1γ鏈的Fc、CH1基因的重鏈表達載體pCMV-VH以及裝載有信號肽序列及κ鏈的Cκ基因的輕鏈表達載體pCMV-VL。將同樣雙酶切的VH和VL基因連接入表達載體,構建成嵌合抗體(ch5H6)的輕鏈真核表達載體5H6L-pCMV及重鏈真核表達載體5H6H-pCMV。將嵌合抗體的輕重鏈真核表達載體共轉染入293T細胞,利用夾心ELISA法測定上清中ch5H6的濃度,利用流式細胞術檢測ch5H6與膜抗原的特異性結合。 綜上所述:1、成功構建了抗人CD40人-鼠嵌合抗體(ch5C11)。2、CD40嵌合抗體可有效抑制天然高表達CD40分子的人B淋巴瘤細胞株Daudi的體外增殖。3、通過計算機輔助設計方法對ch5C11的氨基酸序列進行改造后,瞬時表達量明顯提高。該抗體在某些腫瘤的免疫治療及移植抗排異中具有潛在的應用價值。4、成功構建了抗人muCD40人-鼠嵌合抗體(ch5H6)。 這兩株工程化抗人CD40抗體的研制,為CD40抗體在腫瘤治療及診斷中的應用研究,奠定了厚實的基礎。
[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
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
本文編號：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
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
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