原核表达，体外折叠的HLA-G5促进人PBMC产生TNF

发布时间：2018-04-01 10:35

本文选题：HLA-G5-抗原肽复合物　切入点：稀释复性　出处：《华中科技大学》2006年硕士论文

【摘要】： 背景:MHC编码的产物在启动和调节免疫应答的过程中发挥重要作用。HLA-G基因座位位于人类MHC中,属于非经典HLA I类基因(Non-Classical class I gene),即HLA Ib。HLA-G基因编码的产物即HLA-G分子,由于mRNA的不同拼接方式,HLA-G分子有多种同种型,其中最为常见的是完整的HLA-G1(表达在细胞膜上)和缺乏跨膜区的HLA-G5(可溶性,又称sHLA-G1)。已有的研究显示HLA-G分子在免疫应答的调节中起着重要作用。含一半父源基因的胎儿在母体内不被排斥,原因之一是母胎界面有大量HLA-G分子的表达,HLA-G被认为参与诱导母胎界面的免疫耐受、被认为是妊娠成功的必要条件。近年来国内外学者对HLA-G都给予极大的关注,但真核表达难以达到需要的量,阻碍了对其功能的认识,而原核表达可弥补此不足。本研究在已有工作基础上,通过原核表达、体外折叠的方法制备具有天然构象的可溶性HLA-G,并应用于探讨其对天然免疫细胞的影响。我们选择外周血单核细胞在LPS作用下产生TNF作为模型,观察原核表达、体外折叠的HLA-G对该模型的影响,从而进一步了解HLA-G的生物学功能。目的:通过原核表达、体外折叠的方法制备具有天然构象的可溶性HLA-G,并探讨其对人PBMC产生TNF的影响。方法:通过原核表达获得的HLA-G5重链和轻链(β2m),经初步纯化后,利用稀释法与人工合成的九肽(KGPPAALTL)一起进行体外复性折叠,形成HLA-G5-抗原肽复合物;利用与天然HLA I类分子构象表位结合的单抗W6/32,经非变性电泳(Native-PAGE)/Western blot和双抗夹心ELISA法来鉴定折叠产物的构象。利用单核细胞受LPS刺激后产生TNF的特点,观察原核表达,体外折叠的HLA-G5对单核细胞分泌TNF的影响,TNF分泌量采用TNF敏感的L929细胞进行生物法测定。结果:原核表达体外折叠的HLA-G5经非变性电泳后,除了见到HC、LC位置的条带外,还可见到一新增条带;用Western blot检测该条带能够与HLAⅠ类分子的单抗W6/32结合;用W6/32和β2M抗体进行夹心ELISA鉴定折叠HLA-G5(3个复孔吸光度值的均值,OD490nm)为2.6168,而折叠HC为0.3716、折叠LC为0.6046、HLA-A*2402为1.8122、PBS为0.3784,折叠HLA-G5组的吸光度值均值与其他各组相比较,差异具有显著性(P0.01)。从人外周血分离得到的PBMC分别加入HLA-G5+LPS、HC+LPS、LC+LPS及单独LPS(空白对照组)培养12h后获得无色的含TNF的上清,另设只加DMEM的阴性对照组,培养至24 h时,镜下可见到阴性对照组长势一般但无细胞碎屑出现,HLA-G实验组呈现满视野细胞碎片,其它组细胞碎片也有所增加但还有少许存活细胞。以结晶紫染色后用酶标仪测吸光度值(OD570nm),各个组均设三个复孔,取50%杀伤处吸光度值均值,结果(杀伤率)如下:折叠HLA-G5为45.85%、折叠HC为18.37%、折叠LC为16.55%、空白对照组为7.09%,折叠HLA-G5组的吸光度值均值与其他各组相比较,差异具有显著性(P0.05)。结论:原核表达可以获得足够量的HLA-G5的轻链及重链,经过体外折叠复性的HLA-G5具有HLAⅠ类分子的天然构象,体外细胞功能实验显示折叠的HLA-G5能够促进人单核细胞分泌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..

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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