【摘要】：本研究在本科室自行研制成功的國(guó)內(nèi)首株激發(fā)型抗人CD40單克隆抗體(5C11)的基礎(chǔ)上，采用經(jīng)典的嵌合抗體構(gòu)建方法，對(duì)其進(jìn)行基因工程改造，并在CHO細(xì)胞中實(shí)現(xiàn)真核表達(dá)，進(jìn)而對(duì)其生物學(xué)活性進(jìn)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(xì)胞株，為靶向CD40分子的腫瘤免疫治療提供必要的物質(zhì)基礎(chǔ)，也期在治療性單抗的研發(fā)領(lǐng)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(xì)胞株5C11中提取總RNA，常規(guī)逆轉(zhuǎn)錄，應(yīng)用簡(jiǎn)并引物進(jìn)行PCR擴(kuò)增，并根據(jù)重、輕鏈基因序列分析結(jié)果設(shè)計(jì)引物，應(yīng)用SMART-PCR方法擴(kuò)增含信號(hào)肽序列的V_H和V_L基因，進(jìn)行測(cè)序鑒定。同時(shí)采用RT-PCR從人脾臟細(xì)胞中抽提總RNA，分別設(shè)計(jì)特異性引物擴(kuò)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(jìn)行拼接得到人IgG1_(gamma)(CH1-CH3)鏈恒定區(qū)基因，測(cè)序鑒定。將含信號(hào)肽序列的V_H序列和人的IgG1重鏈恒定區(qū)序列進(jìn)行拼接獲得嵌合重鏈，將含信號(hào)肽序列的V_L序列和人的κappa鏈恒定區(qū)序列進(jìn)行拼接獲得嵌合輕鏈，拼接產(chǎn)物測(cè)序鑒定，構(gòu)建共表達(dá)嵌合重、輕鏈的重組質(zhì)粒pIRES／hu5C11。pIRES／hu5C11重組質(zhì)粒用脂質(zhì)體法轉(zhuǎn)染293T細(xì)胞，利用CD40-L929和mock-L929細(xì)胞株，通過流式細(xì)胞術(shù)對(duì)293T細(xì)胞瞬時(shí)表達(dá)上清中抗人CD40人-鼠嵌合抗體進(jìn)行鑒定。結(jié)果顯示，本研究成功構(gòu)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(dá)載體pIRES／huSC11。 第二部分選擇中國(guó)倉(cāng)鼠卵巢細(xì)胞(CHO)作為表達(dá)宿主，經(jīng)試劑盒抽提的pIRES／hu5C11嵌合抗體重組表達(dá)質(zhì)粒用脂質(zhì)體法轉(zhuǎn)染CHO細(xì)胞，獲得持續(xù)穩(wěn)定分泌表達(dá)抗人CD40人-鼠嵌合抗體(ch-SC11)的基因轉(zhuǎn)染細(xì)胞株(hu5C11-CHO)。RT-PCR鑒定結(jié)果表明ch-SC11表達(dá)基因成功整合在huSC11-CHO細(xì)胞中；FCM和Western blot結(jié)果表明，轉(zhuǎn)染pIRES／hu5C11質(zhì)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(shí)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(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)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(nèi)皮生長(zhǎng)因子嵌合抗體在真核細(xì)胞中的高效表達(dá)[J];癌癥;2001年03期

2 沈倍奮;抗體藥物研究進(jìn)展[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2002年10期

3 羅萍;治療性單克隆抗體研究進(jìn)展及臨床應(yīng)用現(xiàn)狀[J];第三軍醫(yī)大學(xué)學(xué)報(bào);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(guó)外醫(yī)學(xué).預(yù)防.診斷.治療用生物制品分冊(cè);2002年01期

5 陳凌,廖曉龍;CD40配體在腫瘤免疫治療中的應(yīng)用[J];國(guó)外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));2004年08期

6 冉宇靚,孔健,趙澤國(guó),孫立新,遇瓏,劉軍,楊治華;抗CEA嵌合抗體在CHO細(xì)胞中的高效表達(dá)[J];免疫學(xué)雜志;2000年06期

7 古濤,李敏,陳成,朱一蓓,周時(shí)勇,周桓,陳永井,於葛華,張學(xué)光;PD-L1和PD-L2在樹突狀細(xì)胞上的表達(dá)及其生物學(xué)意義[J];現(xiàn)代免疫學(xué);2004年01期

8 劉國(guó)奇,王海濤;外源蛋白在中國(guó)倉(cāng)鼠卵巢細(xì)胞中高效表達(dá)的策略[J];生物化學(xué)與生物物理進(jìn)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(xì)胞中的高效表達(dá)及其活性分析[J];生物化學(xué)與生物物理學(xué)報(bào);2001年01期

10 林蕓,閻錫蘊(yùn);人源化抗體研究歷程及發(fā)展趨勢(shì)[J];生物工程學(xué)報(bào);2004年01期

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