抗人CD40人—鼠嵌合抗體的構建、表達及其生物學活性的初步研究
發(fā)布時間：2018-07-29 17:11
【摘要】：本研究在本科室自行研制成功的國內首株激發(fā)型抗人CD40單克隆抗體(5C11)的基礎上，采用經典的嵌合抗體構建方法，對其進行基因工程改造，并在CHO細胞中實現(xiàn)真核表達，進而對其生物學活性進行初步的研究，以期獲得能持續(xù)有效分泌特異性的抗人CD40人-鼠嵌合抗體的穩(wěn)定細胞株，為靶向CD40分子的腫瘤免疫治療提供必要的物質基礎，也期在治療性單抗的研發(fā)領域中取得突破。 第一部分首先采用RT-PCR從特異性分泌抗人CD40單克隆抗體的雜交瘤細胞株5C11中提取總RNA，常規(guī)逆轉錄，應用簡并引物進行PCR擴增，并根據重、輕鏈基因序列分析結果設計引物，應用SMART-PCR方法擴增含信號肽序列的V_H和V_L基因，進行測序鑒定。同時采用RT-PCR從人脾臟細胞中抽提總RNA，分別設計特異性引物擴增人IgG1_(gamma)鏈huFc、huCH1基因和κappa鏈恒定區(qū)基因huCκ，再利用TP-PCR方法將huFc和huCH1進行拼接得到人IgG1_(gamma)(CH1-CH3)鏈恒定區(qū)基因，測序鑒定。將含信號肽序列的V_H序列和人的IgG1重鏈恒定區(qū)序列進行拼接獲得嵌合重鏈，將含信號肽序列的V_L序列和人的κappa鏈恒定區(qū)序列進行拼接獲得嵌合輕鏈，拼接產物測序鑒定，構建共表達嵌合重、輕鏈的重組質粒pIRES／hu5C11。pIRES／hu5C11重組質粒用脂質體法轉染293T細胞，利用CD40-L929和mock-L929細胞株，通過流式細胞術對293T細胞瞬時表達上清中抗人CD40人-鼠嵌合抗體進行鑒定。結果顯示，本研究成功構建了含人-鼠嵌合重鏈和嵌合輕鏈基因的抗人CD40人-鼠嵌合抗體真核表達載體pIRES／huSC11。 第二部分選擇中國倉鼠卵巢細胞(CHO)作為表達宿主，經試劑盒抽提的pIRES／hu5C11嵌合抗體重組表達質粒用脂質體法轉染CHO細胞，獲得持續(xù)穩(wěn)定分泌表達抗人CD40人-鼠嵌合抗體(ch-SC11)的基因轉染細胞株(hu5C11-CHO)。RT-PCR鑒定結果表明ch-SC11表達基因成功整合在huSC11-CHO細胞中；FCM和Western blot結果表明，轉染pIRES／hu5C11質粒的CHO培養(yǎng)上清中含有抗人CD40人-鼠嵌合抗體，，其不僅保留了特異識別CD40分子活性，并且含人免疫球蛋白Fc
[Abstract]:On the basis of the first domestic activated anti-human CD40 monoclonal antibody (5C11) developed by our department, the classical chimeric antibody construction method was used to carry out genetic engineering modification and eukaryotic expression in CHO cells. In order to obtain the stable cell line which can continuously secrete specific anti-human CD40 human-mouse chimeric antibody, and provide the necessary material basis for tumor immunotherapy targeting CD40 molecule, the biological activity of the cell line was studied preliminarily in order to obtain the stable cell line which can effectively secrete the specific anti-human CD40 human-mouse chimeric antibody. There is also a breakthrough in the research and development of therapeutic McAbs. In the first part, RT-PCR was used to extract total RNAs from hybridoma cell line 5C11 which specifically secreted monoclonal antibody against human CD40. Routine reverse transcription was used to amplify PCR with degenerate primers, and primers were designed according to the results of sequence analysis of heavy and light chain genes. The V _ (th) and V _ (th) L genes containing signal peptide sequences were amplified by SMART-PCR and sequenced. At the same time, RT-PCR was used to extract total RNAs from human spleen cells. Specific primers were designed to amplify the gene of huFchuCH1 and huC 魏 of human IgG1 _ (gamma) chain huFchuCH1 and 魏 appa chain. Then huFc and huCH1 were spliced to obtain the constant region gene of human IgG1 _ (gamma) (CH1-CH3 chain by TP-PCR and sequenced. The chimeric heavy chain was obtained by splicing the VSP sequence containing the signal peptide sequence and the constant region sequence of the human IgG1 heavy chain. The chimeric light chain was obtained by splicing the VSP sequence containing the signal peptide sequence and the constant region sequence of the human 魏 appa chain, and the splicing product was sequenced. The recombinant plasmid pIRES/hu5C11.pIRES/hu5C11 was constructed and transfected into 293T cells by liposome method. CD40-L929 and mock-L929 cell lines were used to transfect 293T cells. Human-mouse chimeric antibody against human CD40 in the supernatant of 293T cells was identified by flow cytometry. The results showed that the eukaryotic expression vector pIRES- rhuSC11 containing human mouse chimeric heavy chain and chimeric light chain gene against human CD40 human-mouse chimeric antibody was successfully constructed in this study. In the second part, Chinese hamster ovarian cell (CHO) was selected as the expression host. The recombinant expression plasmid of pIRES/hu5C11 chimeric antibody extracted by kit was transfected into CHO cells by liposome method. Gene transfection cell lines (hu5C11-CHO) expressing human chimeric antibody (ch-SC11) against human CD40 were obtained and identified by RT-PCR. The results of RT-PCR showed that ch-SC11 gene was successfully integrated into huSC11-CHO cells by FCM and Western blot. The supernatant of CHO transfected with pIRES/hu5C11 plasmid contains anti-human CD40 human-mouse chimeric antibody, which not only retains the activity of specifically recognizing CD40 molecule, but also contains human immunoglobulin FC.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392
本文編號：2153401
[Abstract]:On the basis of the first domestic activated anti-human CD40 monoclonal antibody (5C11) developed by our department, the classical chimeric antibody construction method was used to carry out genetic engineering modification and eukaryotic expression in CHO cells. In order to obtain the stable cell line which can continuously secrete specific anti-human CD40 human-mouse chimeric antibody, and provide the necessary material basis for tumor immunotherapy targeting CD40 molecule, the biological activity of the cell line was studied preliminarily in order to obtain the stable cell line which can effectively secrete the specific anti-human CD40 human-mouse chimeric antibody. There is also a breakthrough in the research and development of therapeutic McAbs. In the first part, RT-PCR was used to extract total RNAs from hybridoma cell line 5C11 which specifically secreted monoclonal antibody against human CD40. Routine reverse transcription was used to amplify PCR with degenerate primers, and primers were designed according to the results of sequence analysis of heavy and light chain genes. The V _ (th) and V _ (th) L genes containing signal peptide sequences were amplified by SMART-PCR and sequenced. At the same time, RT-PCR was used to extract total RNAs from human spleen cells. Specific primers were designed to amplify the gene of huFchuCH1 and huC 魏 of human IgG1 _ (gamma) chain huFchuCH1 and 魏 appa chain. Then huFc and huCH1 were spliced to obtain the constant region gene of human IgG1 _ (gamma) (CH1-CH3 chain by TP-PCR and sequenced. The chimeric heavy chain was obtained by splicing the VSP sequence containing the signal peptide sequence and the constant region sequence of the human IgG1 heavy chain. The chimeric light chain was obtained by splicing the VSP sequence containing the signal peptide sequence and the constant region sequence of the human 魏 appa chain, and the splicing product was sequenced. The recombinant plasmid pIRES/hu5C11.pIRES/hu5C11 was constructed and transfected into 293T cells by liposome method. CD40-L929 and mock-L929 cell lines were used to transfect 293T cells. Human-mouse chimeric antibody against human CD40 in the supernatant of 293T cells was identified by flow cytometry. The results showed that the eukaryotic expression vector pIRES- rhuSC11 containing human mouse chimeric heavy chain and chimeric light chain gene against human CD40 human-mouse chimeric antibody was successfully constructed in this study. In the second part, Chinese hamster ovarian cell (CHO) was selected as the expression host. The recombinant expression plasmid of pIRES/hu5C11 chimeric antibody extracted by kit was transfected into CHO cells by liposome method. Gene transfection cell lines (hu5C11-CHO) expressing human chimeric antibody (ch-SC11) against human CD40 were obtained and identified by RT-PCR. The results of RT-PCR showed that ch-SC11 gene was successfully integrated into huSC11-CHO cells by FCM and Western blot. The supernatant of CHO transfected with pIRES/hu5C11 plasmid contains anti-human CD40 human-mouse chimeric antibody, which not only retains the activity of specifically recognizing CD40 molecule, but also contains human immunoglobulin FC.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392
【參考文獻】
相關期刊論文 前10條
1 冉宇靚,楊治華,孫立新,遇瓏,劉軍,董志偉;抗人血管內皮生長因子嵌合抗體在真核細胞中的高效表達[J];癌癥;2001年03期
2 沈倍奮;抗體藥物研究進展[J];第二軍醫(yī)大學學報;2002年10期
3 羅萍;治療性單克隆抗體研究進展及臨床應用現(xiàn)狀[J];第三軍醫(yī)大學學報;2004年05期
4 史久華;全人單克隆抗體與人源化單克隆抗體[J];國外醫(yī)學.預防.診斷.治療用生物制品分冊;2002年01期
5 陳凌,廖曉龍;CD40配體在腫瘤免疫治療中的應用[J];國外醫(yī)學(腫瘤學分冊);2004年08期
6 冉宇靚,孔健,趙澤國,孫立新,遇瓏,劉軍,楊治華;抗CEA嵌合抗體在CHO細胞中的高效表達[J];免疫學雜志;2000年06期
7 古濤,李敏,陳成,朱一蓓,周時勇,周桓,陳永井,於葛華,張學光;PD-L1和PD-L2在樹突狀細胞上的表達及其生物學意義[J];現(xiàn)代免疫學;2004年01期
8 劉國奇,王海濤;外源蛋白在中國倉鼠卵巢細胞中高效表達的策略[J];生物化學與生物物理進展;2000年05期
9 楊光,冉宇靚,孫立新,劉軍,遇龍,楊治華;抗人P185~(erbB2)嵌合抗體在CHO細胞中的高效表達及其活性分析[J];生物化學與生物物理學報;2001年01期
10 林蕓,閻錫蘊;人源化抗體研究歷程及發(fā)展趨勢[J];生物工程學報;2004年01期
本文編號：2153401
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2153401.html
最近更新
教材專著
