CD86人—鼠嵌合抗體的構(gòu)建、表達(dá)及其生物學(xué)活性的初步研究
發(fā)布時(shí)間：2018-10-21 10:29
【摘要】： B7分子(包括B7-1和B7-2,又稱CD80和CD86)是表達(dá)在APC表面的共刺激分子,其與表達(dá)于T細(xì)胞表面的相應(yīng)受體CD28/CTLA-4結(jié)合產(chǎn)生共刺激信號,對T細(xì)胞的活化和耐受起關(guān)鍵性調(diào)節(jié)作用。CD86為一個(gè)由1120bp的基因所編碼,其基因序列與B7-1有26%的同源性,整個(gè)蛋白由306個(gè)氨基酸組成,屬于I型跨膜糖蛋白。本研究在已獲得穩(wěn)定分泌鼠抗人CD86單克隆抗體雜交瘤細(xì)胞株的基礎(chǔ)上,采用基因重組技術(shù)及細(xì)胞與分子生物學(xué)方法等進(jìn)行了以下研究:1.CD86人-鼠嵌合抗體表達(dá)載體的構(gòu)建及在CHO細(xì)胞中的表達(dá) 首先采用RT-PCR從特異性分泌抗人CD86單克隆抗體的雜交瘤細(xì)胞株1D1中提取總RNA,常規(guī)逆轉(zhuǎn)錄,應(yīng)用簡并引物進(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)增含信號肽序列的V_H和V_L基因,進(jìn)行測序鑒定。在本科室構(gòu)建的表達(dá)質(zhì)粒PIRES/ch5C11基礎(chǔ)上,通過PCR獲得人IgG1γ鏈、κ鏈恒定區(qū)基因。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(jìn)行拼接獲得嵌合重鏈,將含信號肽序列的V_L序列和人的κ鏈恒定區(qū)序列進(jìn)行拼接獲得嵌合輕鏈,拼接產(chǎn)物測序鑒定,構(gòu)建共表達(dá)嵌合重、輕鏈的重組質(zhì)粒pIRES/ch1D1。采用脂質(zhì)體法將重組質(zhì)粒轉(zhuǎn)染293T細(xì)胞。以CD86-L929基因轉(zhuǎn)染細(xì)胞株作為檢測細(xì)胞,經(jīng)流式細(xì)胞術(shù)對293T細(xì)胞培養(yǎng)上清鑒定后轉(zhuǎn)染CHO細(xì)胞,再行鑒定后大量收集無血清培養(yǎng)上清,經(jīng)Protein G親和層析純化及Lowry法定量。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CD86人-鼠嵌合抗體真核表達(dá)載體pIRES/ch1D1。選擇中國倉鼠卵巢細(xì)胞(CHO)作為表達(dá)宿主,經(jīng)試劑盒抽提的pIRES/ch1D1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,經(jīng)G418加壓篩選后進(jìn)行亞克隆,獲得持續(xù)穩(wěn)定分泌抗人CD86人-鼠嵌合抗體(ch1D1)的基因轉(zhuǎn)染細(xì)胞株(ch1D1-CHO)。RT-PCR鑒定結(jié)果表明ch1D1表達(dá)基因成功整合在ch1D1-CHO細(xì)胞中;FCM結(jié)果表明,轉(zhuǎn)染pIRES/ch1D1質(zhì)粒的CHO培養(yǎng)上清中的嵌合抗體與L929-CD86基因轉(zhuǎn)染細(xì)胞膜型CD86分子的陽性結(jié)合率為98.5%。從CHO細(xì)胞培養(yǎng)上清中獲取嵌合抗體純化品的量約為3.0mg/L。 2.CD86人—鼠嵌合抗體的生物學(xué)活性的初步研究 采用間接免疫熒光及流式細(xì)胞術(shù)方法,對多株腫瘤細(xì)胞株Daudi、Sub-T、U937、Jurkat、HO8910、H1299及WERI-RB1細(xì)胞膜性CD86分子進(jìn)行分析。在此基礎(chǔ)上,選取天然高表達(dá)CD86及CD80分子的Daudi細(xì)胞為靶細(xì)胞,觀察和分析了抗CD86嵌合抗體單獨(dú)及聯(lián)合抗CD80嵌合抗體對細(xì)胞生長和增殖的影響作用。將嵌合抗體(終濃度為5μg/mL)加入到Daudi細(xì)胞的培養(yǎng)體系中,經(jīng)顯微鏡觀察及MTT法分析,嵌合抗體對Daudi細(xì)胞的生長具有抑制作用(p＜0.05),且CD86與CD80嵌合抗體具有協(xié)同抑制作用。將兩個(gè)健康人外周血單個(gè)核細(xì)胞(PBMC)加入96孔板中,分別加入CD86嵌合抗體及聯(lián)合CD80嵌合抗體(終濃度為5μg/mL),于培養(yǎng)72小時(shí)采用MTT法檢測增殖情況。結(jié)果顯示在雙向混合淋巴細(xì)胞反應(yīng)中,CD86嵌合抗體能抑制混合淋巴細(xì)胞的增殖效應(yīng),且CD86與CD80嵌合抗體具有協(xié)同抑制作用。 綜上所述,本實(shí)驗(yàn)成功構(gòu)建了CD86人-鼠嵌合抗體(ch1D1)真核表達(dá)質(zhì)粒,并在CHO細(xì)胞中實(shí)現(xiàn)了穩(wěn)定表達(dá),表達(dá)的嵌合抗體對B淋巴瘤細(xì)胞株Daudi細(xì)胞的生長增殖具有抑制作用,并且在混合淋巴細(xì)胞反應(yīng)中,能抑制混合淋巴細(xì)胞的增殖效應(yīng)。
[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
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392
本文編號：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
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 聶靜苑;劉煜;;人源單克隆抗體藥物質(zhì)量控制與分析[J];中國生化藥物雜志;2012年02期
，本文編號：2284820
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