本文選題：CD200 + 克隆��； 參考：《吉林大學(xué)》2005年碩士論文

【摘要】：CD200 分子是近年新發(fā)現(xiàn)的免疫超家族新成員,2000 年,在HLDA7(7th Workshop And Conference On Human Leukocyte Differentiation Antigens)會(huì)議上被正式命名。CD200(以前被稱為MOX2、MRC OX-2抗原)是一種具有免疫調(diào)節(jié)功能的分子,目前動(dòng)物實(shí)驗(yàn)已經(jīng)證實(shí)CD200 分子有減輕自身免疫性疾病癥狀、延長(zhǎng)移植物存活時(shí)間、抑制前炎癥因子引起的流產(chǎn)、打破腫瘤免疫耐受等重要作用,因其具有治療潛能而備受青睞,它獨(dú)特的表達(dá)方式和效應(yīng)功能有著廣泛的應(yīng)用前景,目前CD200 分子已成為一個(gè)新的研究熱點(diǎn)。 本研究從經(jīng)Con A 刺激后的正常人外周血白細(xì)胞中獲得總RNA,逆轉(zhuǎn)錄PCR 反應(yīng)獲得大量人CD200 基因,將其插入pcDNA3 表達(dá)載體,構(gòu)建了真核表達(dá)質(zhì)粒pcDNA3-CD200。用電穿孔法將重組質(zhì)粒轉(zhuǎn)染入U(xiǎn)937 細(xì)胞中,經(jīng)G418 篩選并連續(xù)傳代培養(yǎng)30 代以上,采用流式細(xì)胞術(shù)技術(shù)測(cè)定其表達(dá)情況。 本研究結(jié)果表明:成功的克隆了人CD200 基因并構(gòu)建了真核表達(dá)質(zhì)粒pcDNA3-CD200;用電穿孔的方法轉(zhuǎn)染了U937 細(xì)胞,經(jīng)G418 篩選后,空白對(duì)照組細(xì)胞全部死亡,轉(zhuǎn)染目的基因組及空質(zhì)粒組的細(xì)胞存活,并連續(xù)傳代培養(yǎng)30 代以上,用FITC 標(biāo)記的鼠抗人CD200 單克隆抗體采用流式細(xì)胞術(shù)技術(shù)監(jiān)測(cè)該重組質(zhì)粒的表達(dá)活性�？召|(zhì)粒組CD200 表達(dá)陽性細(xì)胞的百分率與對(duì)照組計(jì)數(shù)結(jié)果相比無統(tǒng)計(jì)學(xué)差異,而轉(zhuǎn)染CD200 基因組表達(dá)陽性細(xì)胞的百分率與空質(zhì)粒組及對(duì)照組結(jié)果相比有顯著差異(p㩳0.01),前者陽性細(xì)胞百分率明顯高于后兩者。說明CD200 重組質(zhì)粒轉(zhuǎn)染成功并具有穩(wěn)定的表達(dá)功能,穩(wěn)定高效表達(dá)人CD200 基因的U937 細(xì)胞株已建立。
[Abstract]:CD200 molecule is a newly discovered member of the immune superfamily in recent years. It was officially named. CD200 (formerly known as MOX2MRC OX-2 antigen) at the HLDA7(7th Workshop And Conference on Human Leukocyte Differentiation antigens is a kind of immunomodulatory molecule.At present, animal experiments have proved that CD200 molecules have the important effects of relieving autoimmune disease symptoms, prolonging graft survival time, inhibiting abortion caused by preinflammatory factors, breaking tumor immune tolerance and so on.Its unique expression mode and effect function have a wide application prospect, CD200 molecule has become a new research hotspot at present.In this study, total RNAs were obtained from peripheral blood leukocytes stimulated by Con A, a large number of human CD200 genes were obtained by reverse transcription PCR reaction, and inserted into pcDNA3 expression vector, and the eukaryotic expression plasmid pcDNA3-CD200 was constructed.The recombinant plasmid was transfected into U937 cells by electroporation. The recombinant plasmid was screened by G418 and cultured for more than 30 generations. The expression of recombinant plasmid was determined by flow cytometry.The results showed that human CD200 gene was cloned successfully and eukaryotic expression plasmid pcDNA3-CD200 was constructed, and U937 cells were transfected by electroporation.The cells transfected with the genome and empty plasmid group survived and were cultured for more than 30 generations. The expression activity of the recombinant plasmid was detected by flow cytometry with FITC labeled mouse monoclonal antibody against human CD200.There was no significant difference in the percentage of CD200 positive cells between the empty plasmid group and the control group.The percentage of positive cells transfected with CD200 genome was significantly different from that of blank plasmid group and control group. The percentage of positive cells in the former group was significantly higher than that in the latter two groups.The results showed that the CD200 recombinant plasmid was successfully transfected and had stable expression function. The U937 cell line expressing human CD200 gene stably and efficiently was established.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 蘇曉健;豬細(xì)小病毒NS1與VP2基因的研究[D];河北農(nóng)業(yè)大學(xué);2007年

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本文編號(hào)：1760528