本文關(guān)鍵詞：“PQRSPT”基序在人B7-H3基因生物學(xué)功能中的分子機(jī)制研究　出處：《蘇州大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： “PQRSPT” 2IgB7-H3-Add 4IgB7-H3-Del sB7-H3 生物學(xué)功能

【摘要】：T細(xì)胞的有效活化需要雙信號(hào),其中共刺激分子在免疫調(diào)節(jié)中發(fā)揮著重要的作用,B7-H3是共刺激分子B7家族成員之一。人B7-H3有兩種不同形式的剪切體:2IgB7-H3和4IgB7-H3。2IgB7-H3胞外段由IgV-IgC兩個(gè)免疫球蛋白結(jié)構(gòu)域組成,而4IgB7-H3胞外段由IgV1-IgC1-IgV2-IgC2四個(gè)免疫球蛋白結(jié)構(gòu)域組成。研究報(bào)道發(fā)現(xiàn)4IgB7-H3是人各組織細(xì)胞中B7-H3的主要表達(dá)形式,目前仍缺乏對(duì)兩種剪切體的差異研究。 許多共刺激分子分別以細(xì)胞膜型和可溶型兩種形式存在。本實(shí)驗(yàn)室在健康人外周血和組織液中發(fā)現(xiàn)存在天然形式的sB7-H3,且發(fā)現(xiàn)sB7-H3僅源于2IgB7-H3�；蛐蛄醒芯堪l(fā)現(xiàn)4IgB7-H3胞外段4個(gè)結(jié)構(gòu)域是由編碼2IgB7-H3 IgV和IgC外顯子復(fù)制的結(jié)果,2IgB7-H3和4IgB7-H3兩種剪切體的同源性超過(guò)95%,本研究通過(guò)氨基酸序列比對(duì)和分析,發(fā)現(xiàn)在人4IgB7-H3分子第一個(gè)C樣免疫球蛋白結(jié)構(gòu)域碳端末端存在一段獨(dú)特的氨基酸序列為“PQRSPT”,其在2IgB7-H3分子序列中缺失。本研究以此為出發(fā)點(diǎn)獲得了增加“PQRSPT”基序的2IgB7-H3-Add基因和缺失“PQRSPT”基序的4IgB7-H3-Del基因,并構(gòu)建相應(yīng)的基因轉(zhuǎn)染細(xì)胞株,以此來(lái)探討“PQRSPT”基序在人B7-H3基因生物學(xué)功能及可溶性B7-H3產(chǎn)生過(guò)程中的分子機(jī)制。 一、2IgB7-H3-Add與4IgB7-H3-Del基因轉(zhuǎn)染細(xì)胞株的建立 RT-PCR法分別擴(kuò)增2IgB7-H3與4IgB7-H3編碼區(qū)全長(zhǎng)基因,并通過(guò)拼接PCR的方法獲得增加“PQRSPT”基序的2IgB7-H3-Add基因和缺失“PQRSPT”基序的4IgB7-H3-Del基因。將兩個(gè)目的基因片段雙酶切后與pIRES2-EGFP真核表達(dá)載體連接,構(gòu)建重組子pIRES2-EGFP/2IgB7-H3-Add和pIRES2-EGFP/4IgB7-H3- Del,經(jīng)測(cè)序鑒定正確后通過(guò)脂質(zhì)體轉(zhuǎn)染法將兩個(gè)重組載體分別導(dǎo)入小鼠L929細(xì)胞。RT-PCR結(jié)果表明表明導(dǎo)入的外源性2IgB7-H3-Add和4IgB7-H3-Del基因已分別成功整合到L929細(xì)胞基因組中并能成功地進(jìn)行轉(zhuǎn)錄;通過(guò)流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)GFP和B7-H3蛋白在兩株細(xì)胞表面均穩(wěn)定高表達(dá);Western Blot分析結(jié)果顯示L929/2IgB7-H3-Add和L929/4IgB7-H3-Del細(xì)胞均能成功表達(dá)B7-H3蛋白。 二、“PQRSPT”基序在人B7-H3基因生物學(xué)功能中的分子機(jī)制研究 收集上述基因轉(zhuǎn)染細(xì)胞的培養(yǎng)上清進(jìn)行ELISA和Western Blot分析,結(jié)果顯示在2IgB7-H3和4IgB7-H3-Del基因轉(zhuǎn)染細(xì)胞培養(yǎng)上清中存在可溶性B7-H3蛋白,而4IgB7-H3和2IgB7-H3-Add基因轉(zhuǎn)染細(xì)胞培養(yǎng)上清與對(duì)照組一樣,都未檢測(cè)出可溶性形式的存在。該研究結(jié)果表明保守性氨基酸“PQRSPT”可能是人4IgB7-H3不能形成可溶性形式的一個(gè)重要基序。同時(shí),基因轉(zhuǎn)染細(xì)胞與外周血T細(xì)胞共培養(yǎng)的淋巴細(xì)胞增殖實(shí)驗(yàn)及分泌細(xì)胞因子的檢測(cè)實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),2IgB7-H3基因轉(zhuǎn)染細(xì)胞能顯著地促進(jìn)T細(xì)胞體外增殖與細(xì)胞因子IL-2和IFN-γ的分泌,相比之下,增加“PQRSPT”基序的2IgB7-H3-Add基因轉(zhuǎn)染細(xì)胞則無(wú)此效應(yīng);4IgB7-H3基因轉(zhuǎn)染細(xì)胞能顯著抑制T細(xì)胞的增殖與細(xì)胞因子IL-2和IFN-γ的分泌,而缺失“PQRSPT”基序的4IgB7-H3-Del基因轉(zhuǎn)染細(xì)胞則無(wú)明顯作用。以上研究結(jié)果均證實(shí)“PQRSPT”基序可能是4IgB7-H3分子中的功能結(jié)構(gòu)域,且對(duì)B7-H3生物學(xué)功能的改變具有重要作用。綜上所述,本實(shí)驗(yàn)成功構(gòu)建了L929/2IgB7-H3-Add和L929/ 4IgB7-H3-Del兩株轉(zhuǎn)基因細(xì)胞,對(duì)基因轉(zhuǎn)染細(xì)胞株培養(yǎng)上清中sB7-H3的檢測(cè)結(jié)果表明了“PQRSPT”基序與人4IgB7-H3分子不能分泌可溶性形式密切相關(guān)。T細(xì)胞體外增殖實(shí)驗(yàn)顯示,兩株基因轉(zhuǎn)染細(xì)胞均對(duì)T細(xì)胞的增殖和細(xì)胞因子的分泌無(wú)效應(yīng),表明“PQRSPT”基序可能是4IgB7-H3分子發(fā)揮抑制作用的重要基序。上述結(jié)果為進(jìn)一步闡明sB7-H3的來(lái)源機(jī)制提供新的思路,以及為研究人B7-H3兩種剪切體的差別性奠定了分子基礎(chǔ)。
[Abstract]:Effective activation of the T cell requires two signals. The costimulatory molecules play an important role in immune regulation, B7-H3 is one of the costimulatory molecules of B7 family members. B7-H3 there are two different forms of shear: 2IgB7-H3 and 4IgB7-H3.2IgB7-H3 extracellular domain is composed of IgV-IgC two immunoglobulin domains, 4IgB7-H3 cell the outer segment consists of IgV1-IgC1-IgV2-IgC2 four immunoglobulin domains. Studies suggest that 4IgB7-H3 is the main form of expression of B7-H3 in various tissues, there is still a lack of study on the differences of the two variants.
Many costimulatory molecules exist respectively in cell membrane and soluble forms. There are two natural forms of sB7-H3 were found in our laboratory in healthy human peripheral blood and tissue fluid, and found that sB7-H3 only from the sequence of 2IgB7-H3. gene showed that 4IgB7-H3 extracellular domain 4 is copied by the code 2IgB7-H3 IgV and IgC, 2IgB7-H3 and 4IgB7-H3 two variants homology of more than 95%, the amino acid sequence alignment and analysis, found in human 4IgB7-H3 molecules first C like immunoglobulindomains carbon terminal has a unique amino acid sequence "PQRSPT", and its deletion in 2IgB7-H3 molecules sequence. This study as a starting point for the "PQRSPT" motif of the 2IgB7-H3-Add gene and the deletion of "PQRSPT" motif of 4IgB7-H3-Del gene, and construct the corresponding gene transfected cell line, in order to explore the The "PQRSPT" motif is a molecular mechanism in the biological function of human B7-H3 gene and the production of soluble B7-H3.
The establishment of 2IgB7-H3-Add and 4IgB7-H3-Del gene transfected cell lines
Full length 2IgB7-H3 were amplified with 4IgB7-H3 gene encoding RT-PCR and PCR method, by splicing method to obtain "PQRSPT" motif of the 2IgB7-H3-Add gene and the deletion of "PQRSPT" motif of the 4IgB7-H3-Del gene. The two gene fragments were digested with pIRES2-EGFP eukaryotic expression vector, recombinant pIRES2-EGFP/2IgB7-H3-Add and pIRES2-EGFP/4IgB7-H3- Del, after identification by sequencing by Lipofectamine two recombinant plasmids were respectively transfected into.RT-PCR L929 cells in mice results showed that exogenous 2IgB7-H3-Add and 4IgB7-H3-Del gene were successfully integrated into the genome of L929 cells and successfully through transcription; flow cytometry showed that GFP and B7-H3 protein in two strains the cell surface was stable and high expression; Western Blot analysis showed that L929/2IgB7-H3-Add and L929/4IgB7-H3-Del cells B7-H3 protein can be successfully expressed.
Two, study on the molecular mechanism of "PQRSPT" motif in human B7-H3 gene biological function
Culture supernatants were collected the transfected cells was analyzed by ELISA and Western Blot, showed the presence of soluble B7-H3 protein in the supernatant of cultured in 2IgB7-H3 and 4IgB7-H3-Del transfected cells, while 4IgB7-H3 and 2IgB7-H3-Add gene transfected cell culture supernatant as control group, were not detected in soluble form. The results of the study indicate that conservative amino acid "PQRSPT" may be an important motif of 4IgB7-H3 cannot form a soluble form. At the same time, lymphocytes were cultured transfected cells and peripheral blood T cell proliferation and cytokine secretion in experimental detection experimental results showed that the 2IgB7-H3 gene transfected cells could significantly promote cell proliferation and secretion of T cytokines IL-2 and IFN- gamma in contrast, the increase of "PQRSPT" motif of 2IgB7-H3-Add gene transfected cells had no such effect; 4IgB7-H3 gene transfection Secretory cells can significantly inhibit the proliferation of T cells and cytokines IL-2 and IFN- gamma, but the lack of a "PQRSPT" motif of 4IgB7-H3-Del gene transfected cells has no obvious effect. The above results demonstrated that the "PQRSPT" motif may be the functional domain of 4IgB7-H3 molecules, and the biological function of B7-H3 has great change. In summary, we successfully constructed L929/2IgB7-H3-Add and L929/ 4IgB7-H3-Del two transgenic cells. The detection results of culture supernatant of sB7-H3 transfected cells showed a "PQRSPT" motif and 4IgB7-H3 molecules cannot display the secreted soluble forms of.T is closely related to cell proliferation in vitro, two strains were transfected cells on T cells the proliferation and cytokine secretion had no effect, that the "PQRSPT" motif may be an important motif in 4IgB7-H3 molecules play inhibition. The above results It provides a new idea for further clarifying the source mechanism of sB7-H3 and laying a molecular basis for the study of the difference between the two kinds of human B7-H3 shear bodies.

【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】：2011
【分類號(hào)】：R392.1

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相關(guān)期刊論文 前1條

1 張光波,陳永井,姜智,於葛華,楊明峰,施勤,王勤,李文香,張學(xué)光;人B7-H3基因轉(zhuǎn)染及其生物學(xué)功能的研究[J];現(xiàn)代免疫學(xué);2004年06期

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本文編號(hào)：1407343