本文選題：HLA-G5-抗原肽復(fù)合物　切入點(diǎn)：稀釋復(fù)性　出處：《華中科技大學(xué)》2006年碩士論文

【摘要】： 背景:MHC編碼的產(chǎn)物在啟動和調(diào)節(jié)免疫應(yīng)答的過程中發(fā)揮重要作用。HLA-G基因座位位于人類MHC中,屬于非經(jīng)典HLA I類基因(Non-Classical class I gene),即HLA Ib。HLA-G基因編碼的產(chǎn)物即HLA-G分子,由于mRNA的不同拼接方式,HLA-G分子有多種同種型,其中最為常見的是完整的HLA-G1(表達(dá)在細(xì)胞膜上)和缺乏跨膜區(qū)的HLA-G5(可溶性,又稱sHLA-G1)。已有的研究顯示HLA-G分子在免疫應(yīng)答的調(diào)節(jié)中起著重要作用。含一半父源基因的胎兒在母體內(nèi)不被排斥,原因之一是母胎界面有大量HLA-G分子的表達(dá),HLA-G被認(rèn)為參與誘導(dǎo)母胎界面的免疫耐受、被認(rèn)為是妊娠成功的必要條件。近年來國內(nèi)外學(xué)者對HLA-G都給予極大的關(guān)注,但真核表達(dá)難以達(dá)到需要的量,阻礙了對其功能的認(rèn)識,而原核表達(dá)可彌補(bǔ)此不足。本研究在已有工作基礎(chǔ)上,通過原核表達(dá)、體外折疊的方法制備具有天然構(gòu)象的可溶性HLA-G,并應(yīng)用于探討其對天然免疫細(xì)胞的影響。我們選擇外周血單核細(xì)胞在LPS作用下產(chǎn)生TNF作為模型,觀察原核表達(dá)、體外折疊的HLA-G對該模型的影響,從而進(jìn)一步了解HLA-G的生物學(xué)功能。 目的:通過原核表達(dá)、體外折疊的方法制備具有天然構(gòu)象的可溶性HLA-G,并探討其對人PBMC產(chǎn)生TNF的影響。 方法:通過原核表達(dá)獲得的HLA-G5重鏈和輕鏈(β2m),經(jīng)初步純化后,利用稀釋法與人工合成的九肽(KGPPAALTL)一起進(jìn)行體外復(fù)性折疊,形成HLA-G5-抗原肽復(fù)合物;利用與天然HLA I類分子構(gòu)象表位結(jié)合的單抗W6/32,經(jīng)非變性電泳(Native-PAGE)/Western blot和雙抗夾心ELISA法來鑒定折疊產(chǎn)物的構(gòu)象。利用單核細(xì)胞受LPS刺激后產(chǎn)生TNF的特點(diǎn),觀察原核表達(dá),體外折疊的HLA-G5對單核細(xì)胞分泌TNF的影響,TNF分泌量采用TNF敏感的L929細(xì)胞進(jìn)行生物法測定。 結(jié)果:原核表達(dá)體外折疊的HLA-G5經(jīng)非變性電泳后,除了見到HC、LC位置的條帶外,還可見到一新增條帶;用Western blot檢測該條帶能夠與HLAⅠ類分子的單抗W6/32結(jié)合;用W6/32和β2M抗體進(jìn)行夾心ELISA鑒定折疊HLA-G5(3個復(fù)孔吸光度值的均值,OD490nm)為2.6168,而折疊HC為0.3716、折疊LC為0.6046、HLA-A*2402為1.8122、PBS為0.3784,折疊HLA-G5組的吸光度值均值與其他各組相比較,差異具有顯著性(P0.01)。從人外周血分離得到的PBMC分別加入HLA-G5+LPS、HC+LPS、LC+LPS及單獨(dú)LPS(空白對照組)培養(yǎng)12h后獲得無色的含TNF的上清,另設(shè)只加DMEM的陰性對照組,培養(yǎng)至24 h時(shí),鏡下可見到陰性對照組長勢一般但無細(xì)胞碎屑出現(xiàn),HLA-G實(shí)驗(yàn)組呈現(xiàn)滿視野細(xì)胞碎片,其它組細(xì)胞碎片也有所增加但還有少許存活細(xì)胞。以結(jié)晶紫染色后用酶標(biāo)儀測吸光度值(OD570nm),各個組均設(shè)三個復(fù)孔,取50%殺傷處吸光度值均值,結(jié)果(殺傷率)如下:折疊HLA-G5為45.85%、折疊HC為18.37%、折疊LC為16.55%、空白對照組為7.09%,折疊HLA-G5組的吸光度值均值與其他各組相比較,差異具有顯著性(P0.05)。 結(jié)論:原核表達(dá)可以獲得足夠量的HLA-G5的輕鏈及重鏈,經(jīng)過體外折疊復(fù)性的HLA-G5具有HLAⅠ類分子的天然構(gòu)象,體外細(xì)胞功能實(shí)驗(yàn)顯示折疊的HLA-G5能夠促進(jìn)人單核細(xì)胞分泌TNF。
[Abstract]:Background: MHC encoding products play an important role in the.HLA-G gene locus in human MHC in the process of initiating and regulating immune responses, belonging to the non classical HLA class I gene (Non-Classical class I gene), the product of HLA Ib.HLA-G gene encoding the HLA-G molecules, due to different splicing mRNA, HLA-G molecules with multiple isotypes among them, the most common is the complete HLA-G1 (expressed in the cell membrane) and the lack of transmembrane region (soluble HLA-G5, also known as sHLA-G1). Previous study showed that HLA-G molecules in regulating the immune response plays an important role. With half the paternal gene of fetal rejection in the womb. One of the reasons is the maternal fetal interface and abundant expression of HLA-G molecules, HLA-G is believed to participate in the induction of immune tolerance to maternal fetal interface, is considered essential to successful pregnancy. In recent years, domestic and foreign scholars on the HLA-G are given a great deal of attention, But the eukaryotic expression is difficult to achieve the required amount, hindering the understanding of its function, and prokaryotic expression can solve this problem. The research work on the basis of the prokaryotic expression of soluble HLA-G method for preparation of folding in vitro with the native conformation, and applied to investigate its effect on immune cells we choose the peripheral blood mononuclear cells to produce TNF in the presence of LPS as a model, to observe the prokaryotic expression, refolding in vitro effect of HLA-G on the model, in order to further understand the biological function of HLA-G.
Objective: to prepare a soluble HLA-G with natural conformation by the method of prokaryotic expression and in vitro folding, and to explore its effect on the production of TNF by human PBMC.
Methods: the prokaryotic expression of the HLA-G5 heavy chain and light chain (beta 2m), after preliminary purification, using nine dilution method and synthetic peptide (KGPPAALTL) in vitro refolding folded together, forming the HLA-G5- peptide complexes; the use of natural and HLA class I molecules conformational epitopes with monoclonal antibody W6/32 by non denaturing gel electrophoresis, /Western blot (Native-PAGE) conformation and double antibody ELISA method to identify the folding products. The characteristics of TNF produced by LPS stimulated monocytes, observe the prokaryotic expression, in vitro folding HLA-G5 secretion of TNF on mononuclear cells, secretion of TNF was determined by TNF sensitive biological method the L929 cells.
Results: the prokaryotic expression of in vitro folding HLA-G5 by non denaturing gel electrophoresis, in addition to see HC, LC position of the band, you can still see a new strip; using Western detection of blot of the strip and HLA class I molecules are combined with monoclonal antibody W6/32; sandwich ELISA identified by W6/32 and 2M beta folding HLA-G5 antibody (mean, 3 wells in the absorbance of OD490nm) was 2.6168, HC was 0.3716 fold and 0.6046 fold, LC, HLA-A*2402 1.8122, PBS was 0.3784, compared with the other groups the mean absorbance folding values for the HLA-G5 group, the difference was significant (P0.01). The PBMC isolated from human the peripheral blood were added to HLA-G5+LPS, HC+LPS, LC+LPS and LPS alone (control group) after 12h culture was colorless TNF containing supernatant, a negative DMEM control group, cultured for 24 h, observed under the light microscope and the negative control group growth in general but no cell debris appeared, the experimental group was HLA-G The full view of cell debris, other cell debris also increased cell survival. But there is little to crystal violet staining with a microplate absorbance (OD570nm), each group has three holes, 50% killer mean absorbance value, the results are as follows: (killing rate) of HLA-G5 was 45.85% fold folding, HC is 18.37%, LC is 16.55% fold, the control group was 7.09%, compared with the other groups the mean absorbance folding values for the HLA-G5 group, the difference was significant (P0.05).
Conclusion: the light chain and heavy chain of HLA-G5 can be obtained by prokaryotic expression. After folding in vitro, HLA-G5 has the natural conformation of HLA class I molecules. In vitro cell function experiments show that folded HLA-G5 can promote human monocyte to secrete TNF..

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

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相關(guān)期刊論文 前5條

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