可溶性HLA-G1-肽復(fù)合物的體外折疊及其對混合淋巴細(xì)胞培養(yǎng)中T細(xì)胞增殖的影響
發(fā)布時(shí)間：2018-07-16 17:46
【摘要】： 人類白細(xì)胞抗原(humanleukocyteantigen,HLA)G基因是1987年由Geraghty等克隆并測序的一類非經(jīng)典HLA I類基因,可通過不同的剪切方式翻譯出七種不同結(jié)構(gòu)的蛋白,包括4種跨膜型和3種可溶性HLA-G(soluble HLA-G,sHLA-G)分子。對HLA-G的關(guān)注源于母胎耐受。胎兒基因組的一半來自父方,這些基因編碼的產(chǎn)物對母體而言是同種異體抗原,但正常情況下母體并不對胎兒的同種異體抗原產(chǎn)生免疫應(yīng)答而導(dǎo)致對胎兒的排斥。因此,母胎之間必然存在誘導(dǎo)并維持母胎耐受的機(jī)制。研究發(fā)現(xiàn),胎盤絨毛膜細(xì)胞不表達(dá)經(jīng)典的HLA I、II類分子,卻表達(dá)非經(jīng)典的HLA-G分子。母胎界面上HLA分子表達(dá)的特殊性是導(dǎo)致母胎耐受的重要機(jī)制之一。 對HLA-G的研究最初集中于跨模型HLA-G1分子。研究證實(shí),跨膜型HLA-G1分子可直接或間接與NK細(xì)胞、T細(xì)胞、樹突狀細(xì)胞、單核細(xì)胞及巨嗜細(xì)胞等免疫細(xì)胞表面的殺傷細(xì)胞抑制性受體(killer inhibitory receptor, KIR)及ILT(immunoglobulin-like transcripts/CD85)結(jié)合,激活其胞內(nèi)段的免疫受體酪氨酸抑制基序(Immunology receptor tyrosine-based inhibitory motif, ITIM),啟動(dòng)抑制性信號傳導(dǎo)通路,從而抑制免疫細(xì)胞的功能,誘導(dǎo)免疫耐受。 在妊娠早期,胎盤所有類型的滋養(yǎng)層細(xì)胞都分泌sHLA-G,母體血中sHLA-G含量與妊娠是否成功相關(guān):若母體血中sHLA-G含量高,則妊娠易成功;否則,妊娠不易成功。在某些腫瘤、感染、器官移植、自身免疫性疾病病人的血清及組織中檢測到sHLA-G。不表達(dá)膜型HLA-G1的HLA-G*0105N純合型健康個(gè)體能正常妊娠,這些現(xiàn)象提示sHLA-G同樣有著重要的功能。近年來對sHLA-G生物學(xué)功能研究成為HLA-G研究的新熱點(diǎn)。Contini P等研究證實(shí)sHLA-G能抑制NK細(xì)胞,CD4+、CD8+ T細(xì)胞的功能,但其具體機(jī)制沒有闡明。Fournel等研究表明,純化的HLA-G5可在0.25μg/ml的低濃度下與CD8分子結(jié)合,刺激CD8+細(xì)胞表達(dá)CD95配體,通過Fas/FasL的途徑誘導(dǎo)CD8+細(xì)胞凋亡。而有些研究者在用分泌HLA-G5的細(xì)胞與T細(xì)胞一起孵育時(shí)并沒有觀察到T細(xì)胞的凋亡增加。最近有研究表明HLA-G5可以抑制同種反應(yīng)T細(xì)胞的細(xì)胞周期但不促進(jìn)其凋亡。Le Rond等在體外用HLA-G5致敏初始T細(xì)胞18個(gè)小時(shí),發(fā)現(xiàn)這些處理過的初始T細(xì)胞喪失了對其后同種抗原刺激的反應(yīng)能力,并能抑制其他T細(xì)胞的反應(yīng)性。在混合淋巴細(xì)胞培養(yǎng)中,CD4+、CD8+ T細(xì)胞均能表達(dá)可溶性或膜型HLA-G,作為一種負(fù)反饋信號調(diào)節(jié)CD4+同種反應(yīng)性T細(xì)胞的增殖。 為闡明sHLA-G的生物學(xué)作用及其作用機(jī)制,有必要獲取大量有功能的sHLA-G分子,真核表達(dá)系統(tǒng)表達(dá)的可溶性蛋白雖然保持了蛋白原有的構(gòu)象,但不能大量表達(dá),且純化困難。而原核表達(dá)系統(tǒng)表達(dá)的包涵體蛋白不能保持蛋白原有的構(gòu)象,也不能進(jìn)行糖基化,因此需在體外進(jìn)行折疊復(fù)性和功能鑒定。本課題研究的目的是采用原核表達(dá)系統(tǒng),制備大量有功能的sHLA-G1-肽復(fù)合物,并且利用同種異體T細(xì)胞混合培養(yǎng)的方法,觀察體外折疊的sHLA-G1-肽復(fù)合物對T細(xì)胞增殖反應(yīng)的影響,為進(jìn)一步闡明其作用機(jī)制及臨床應(yīng)用奠定物質(zhì)基礎(chǔ)。我們采用HLA-G限制性的九肽KGPPAALTL與sHLA-G1重鏈及輕鏈在體外經(jīng)稀釋復(fù)性制備sHLA-G1-肽復(fù)合物,并建立同種異體混合淋巴細(xì)胞培養(yǎng)體系,驗(yàn)證其生物學(xué)功能。本課題的研究內(nèi)容和結(jié)果如下: 1、sHLA-G1重鏈、β2m的高效表達(dá)和純化 sHLA-G1分子的兩個(gè)亞基sHLA-G1重鏈和β2m(輕鏈)均以包涵體的形式在細(xì)菌內(nèi)高效表達(dá),經(jīng)IPTG誘導(dǎo)表達(dá)目的蛋白,提取包涵體并初步純化后溶于8mol/L尿素中。sHLA-G1重鏈和β2m的蛋白產(chǎn)量分別為180mg/L菌液和150mg/L菌液,灰度掃描純度分別為75.7%及65.8%,能夠滿足本研究制備sHLA-G1-抗原肽復(fù)合物的需要。 2、sHLA-G1-抗原肽復(fù)合物分子的稀釋折疊復(fù)性 在分子結(jié)構(gòu)上,HLA-G1與經(jīng)典的HLA I類分子一樣,均為包括重鏈、輕鏈和抗原肽的三分子復(fù)合物。在HLA-G1限制性的抗原肽存在的情況下,通過稀釋制備得到的β2m與sHLA-G1中的變性劑而實(shí)現(xiàn)體外復(fù)性折疊。來自于胎盤滋養(yǎng)層的九肽KGPPAALTL是從HLA-G上洗脫的三種內(nèi)源性自身肽的一種,它的存在可幫助共折疊形成穩(wěn)定的sHLA-G1-抗原肽復(fù)合物分子。用已知僅與天然構(gòu)象的HLA I類分子結(jié)合的單克隆抗體W6/32進(jìn)行Western-blot鑒定,結(jié)果顯示W(wǎng)6/32同樣能夠結(jié)合于折疊復(fù)合物中的成份,進(jìn)一步應(yīng)用W6/32與抗β2m抗體進(jìn)行雙抗夾心ELISA,證實(shí)體外折疊sHLA-G1-抗原肽復(fù)合物具有HLA I類分子的天然構(gòu)象,說明通過原核表達(dá)、體外折疊能夠成功地制備sHLA-G1單體。 3、sHLA-G1對混合淋巴細(xì)胞培養(yǎng)中T細(xì)胞增殖的影響 外周血淋巴細(xì)胞用羧基熒光素乙酰乙酸琥珀酰亞胺酯(carboxyfluorescein diacetate succinimidyl ester,CFSE)標(biāo)記后,用同種異體的EB病毒轉(zhuǎn)化的B淋巴母樣細(xì)胞(EBV-LCL)刺激,此為本實(shí)驗(yàn)的混合淋巴細(xì)胞培養(yǎng)體系,代表針對同種異體抗原的T細(xì)胞應(yīng)答。在96孔培養(yǎng)板中加入2×105熒光素標(biāo)記的反應(yīng)細(xì)胞和2×104刺激細(xì)胞,37℃,5%CO2培養(yǎng)箱中培養(yǎng)。在培養(yǎng)的第一天和第四天加入300μg/ml sHLA-G1-肽復(fù)合物及相同濃度的sHLA-A2-肽復(fù)合物、β2m作為對照,在第七天再次加入刺激細(xì)胞和蛋白,繼續(xù)培養(yǎng)三天后流式檢測T細(xì)胞的增殖情況,結(jié)果發(fā)現(xiàn):三個(gè)個(gè)體中,sHLA-G1-肽復(fù)合物實(shí)驗(yàn)組的增殖指數(shù)均明顯低于培養(yǎng)基對照組(個(gè)體一:5.5266±0.6124 vs 7.7350±0.4879;個(gè)體二:6.0566±0.4660 vs 8.9700±0.2353;個(gè)體三:5.8133±0.3470 vs 7.4600±0.5726; P0.05),而HLA-G的單克隆抗體G11E5+sHLA-G1組與培養(yǎng)基組比較沒有明顯差異(個(gè)體一:7.7050±0.0777 vs 7.7350±0.4879;個(gè)體二:9.5700±0.5091 vs 8.9700±0.2353;個(gè)體三:7.200±0.6763 vs 7.4600±0.5726; P0.05)。而sHLA-A2對照組在個(gè)體一、個(gè)體二中增殖指數(shù)都明顯低于培養(yǎng)基對照組(個(gè)體一:5.5633±0.3523 vs 7.7350±0.4879;個(gè)體二:7.0100±0.3111 vs 8.9700±0.2353;P0.05),而在個(gè)體三中卻高于培養(yǎng)基對照組(9.0933±0.3859 vs 7.4600±0.5726; P0.05)。這些結(jié)果顯示原核表達(dá)、體外折疊的sHLA-G1能夠抑制T細(xì)胞針對同種抗原的增殖反應(yīng)。并且這種抑制作用能被針對HLA-Gα1區(qū)的單克隆抗體G11E5封閉。 可溶性HLA-G是近年來HLA-G研究的一個(gè)新的熱點(diǎn),在體內(nèi)可能發(fā)揮比膜型HLA-G更為重要的作用。本研究利用原核表達(dá)體系,在體外大量制備正確構(gòu)像的sHLA-G1-肽復(fù)合物,并首次采用CFSE標(biāo)記反應(yīng)細(xì)胞,利用流式細(xì)胞儀準(zhǔn)確快速的檢測sHLA-G1-肽復(fù)合物對混合淋巴細(xì)胞反應(yīng)中T細(xì)胞增殖的影響。成功制備出有功能的sHLA-G1-肽復(fù)合物,為進(jìn)一步闡明其作用機(jī)制及臨床應(yīng)用奠定物質(zhì)基礎(chǔ)。
[Abstract]:Human leukocyte antigen (humanleukocyteantigen, HLA) G gene is a class of non classical HLA I gene cloned and sequenced by Geraghty in 1987. It can translate seven different structural proteins through different shear ways, including 4 transmembrane and 3 soluble HLA-G (soluble HLA-G, sHLA-G) molecules. Half of the fetal genome comes from the parent, and the genes encoded by these genes are allogenic antigens to the mother body, but in normal cases the mother does not produce an immune response to the fetal alloantigen and causes rejection of the fetus. Therefore, the mother fetus is bound to have a mechanism to induce and maintain maternal tolerance. The hair membrane cells do not express the classic HLA I, II class, but express non classical HLA-G molecules. The specific expression of HLA molecules on the maternal fetal interface is one of the important mechanisms that lead to maternal tolerance.
The study of HLA-G was initially focused on cross model HLA-G1 molecules. Studies have shown that transmembrane HLA-G1 molecules can be directly or indirectly associated with NK cells, T cells, dendritic cells, monocytes and giant cells and other immune cells, such as killer inhibitory receptor, KIR, and ILT (immunoglobulin-like transcripts/CD85). It activates the immune receptor tyrosine inhibition (Immunology receptor tyrosine-based inhibitory motif, ITIM) in its intracellular segment and activates the inhibitory signal transduction pathway, thus inhibiting the function of immune cells and inducing immune tolerance.
In the early pregnancy, all types of trophoblast cells in the placenta secrete sHLA-G, and the content of sHLA-G in the maternal blood is related to the success of pregnancy: if the content of sHLA-G in the maternal blood is high, the pregnancy is easy to succeed; otherwise, the pregnancy is not easy to succeed. In some tumors, infections, organ transplantation, and in the serum and tissue of patients with autoimmune diseases, sHLA-G. is not detected. The HLA-G*0105N homozygous healthy individuals expressing the membrane type HLA-G1 can be pregnant with normal pregnancy. These phenomena suggest that sHLA-G also has important functions. In recent years, the study of the biological function of sHLA-G has become a new hot spot in HLA-G research,.Contini P and other studies have confirmed that sHLA-G can inhibit the function of NK cells, CD4+, CD8+ T cells, but the specific mechanism of sHLA-G has not been clarified. Rnel and other studies show that purified HLA-G5 can bind to CD8 molecules at a low concentration of 0.25 u g/ml, stimulate CD8+ cells to express CD95 ligands and induce apoptosis in CD8+ cells via Fas/FasL pathway. Some researchers have not observed the increase of apoptosis in T cells when incubated with HLA-G5 cells with T cells. -G5 can inhibit the cell cycle of the homologous T cell but does not promote its apoptosis.Le Rond and so on to sensitized the initial T cells with HLA-G5 for 18 hours in vitro. It is found that these treated initial T cells lose their response to the subsequent alloantigen stimulation and inhibit the reverse response of other T cells. In mixed lymphocyte culture, CD4+, CD8+ T. Cells can express soluble or membranous HLA-G as a negative feedback signal to regulate the proliferation of CD4+ allo T cells.
In order to clarify the biological function and mechanism of sHLA-G, it is necessary to obtain a large number of functional sHLA-G molecules. The soluble protein expressed in eukaryotic expression system, although it maintains the original conformation of the protein, can not be expressed in large quantities and is difficult to purify, and the inclusion body protein expressed in the prokaryotic expression system can not maintain the original conformation of the protein, The purpose of this study is to use the prokaryotic expression system to prepare a large number of functional sHLA-G1- peptide complexes, and to observe the effect of the sHLA-G1- peptide complex folded in vitro on the proliferation of T cells by using the method of mixed culture of allogeneic T cells. In order to further clarify its mechanism of action and its clinical application, we used the HLA-G limited nine peptide KGPPAALTL and sHLA-G1 heavy chain and light chain to prepare sHLA-G1- peptide complex in vitro, and establish a allogenic mixed lymphocyte culture system to verify its biological function. As follows:
1, high expression and purification of sHLA-G1 heavy chain and beta 2M
The two subunits of sHLA-G1 molecule sHLA-G1 heavy chain and beta 2m (light chain) are highly expressed in the form of inclusion body in the form of inclusion body. The target protein is induced by IPTG, the inclusion body is extracted, and the protein yield of.SHLA-G1 heavy chain and beta 2m in 8mol/L urea, which is dissolved in 8mol/L urea, is 180mg/L and 150mg/L, respectively. The purity of the gray scale scanning is 75.7%, respectively. And 65.8%, it can meet the needs of preparing sHLA-G1- antigen peptide complexes in this study.
2, dilution and folding renaturation of sHLA-G1- antigen peptide complexes.
On the molecular structure, HLA-G1, like the classical HLA I class, consists of three molecular complexes including heavy chains, light chains and antigenic peptides. In the presence of HLA-G1 restrictive peptides, a denaturant in beta 2M and sHLA-G1, prepared by dilution, is refolding in vitro. The nine peptide KGPPAALTL from the placental trophoblastic layer is from H One of the three endogenous endogenous peptides eluted from LA-G, and its existence helps to fold up a stable sHLA-G1- peptide complex molecule. Western-blot identification with a monoclonal antibody W6/32 that is known only to be combined with the natural conformation of HLA I molecules. The results show that W6/32 can also be combined in the composition of the folded complex, and the same can be found. W6/32 and anti beta 2m antibody were used to carry out double anti sandwich ELISA, which proved that the complex of sHLA-G1- antigen peptide complex in vitro has the natural conformation of HLA I molecules, indicating that the sHLA-G1 monomer can be successfully prepared by the prokaryotic expression.
3, the effect of sHLA-G1 on the proliferation of T cells in mixed lymphocyte culture.
The peripheral blood lymphocytes were labeled with carboxyl fluorescein acetoacetate succinimide (carboxyfluorescein diacetate succinimidyl ester, CFSE) and stimulated by B lymphoblastic cells (EBV-LCL) transformed by allogeneic EB virus. This was a mixed lymphocyte culture system in this experiment, representing the T cell response to allogenic antigen. In the 96 hole culture plate, 2 x 105 fluorescein labeled reactive cells and 2 x 104 stimulation cells were cultured in the incubator at 37 C, 5%CO2 culture box. 300 mu sHLA-G1- peptide complex and the same concentration of sHLA-A2- peptide complex were added to the first and fourth days of culture, and beta 2m was used as the control, and the cells and proteins were added again on seventh days, and continued to be cultured. The proliferation of T cells was detected after three days. The results showed that among the three individuals, the proliferation index of the sHLA-G1- peptide complex was significantly lower than that of the culture group (individual 1: 5.5266 + 0.6124 vs 7.7350 + 0.4879; individuals two: 6.0566 + 0.4660 vs 8.9700 + three; individual three: 5.8133 + vs, P0.05), and HLA-G There was no significant difference between the G11E5+sHLA-G1 group and the medium group (individual: 7.7050 + 0.0777 vs 7.7350 + 0.4879; individuals two: 9.5700 + 0.5091 vs 8.9700 + 0.2353; individuals three: 7.200 + 0.6763 vs 7.4600 + 7.7350 +; P0.05). And sHLA-A2 control group was significantly lower than that of the medium control group in the individual one. The individual one: 5.5633 + 0.3523 vs 7.7350 + 0.4879; individuals two: 7.0100 + 0.3111 vs 8.9700 + 0.2353; P0.05), but higher in the individual 3 than the medium control group (9.0933 + 0.3859 vs 7.4600 + 0.5726; P0.05). These results showed that the prokaryotic expression, the in vitro folded sHLA-G1 could inhibit the proliferation reaction of the T cells to the same antigen. The inhibitory effect can be blocked by monoclonal antibody G11E5 targeting HLA-G alpha 1 region.
Soluble HLA-G is a new hot spot in HLA-G research in recent years, and it may play a more important role than membrane type HLA-G in the body. This study uses the prokaryotic expression system to prepare sHLA-G1- peptide complexes with correct structure in vitro, and first uses CFSE labeled reactive cells, and uses flow cytometry to detect sHLA-G1- peptide accurately and quickly. The effect of complex on the proliferation of T cells in mixed lymphocyte reaction. The functional sHLA-G1- peptide complex was successfully prepared, which laid the material basis for further elucidating its mechanism and clinical application.
【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
本文編號：2127145
[Abstract]:Human leukocyte antigen (humanleukocyteantigen, HLA) G gene is a class of non classical HLA I gene cloned and sequenced by Geraghty in 1987. It can translate seven different structural proteins through different shear ways, including 4 transmembrane and 3 soluble HLA-G (soluble HLA-G, sHLA-G) molecules. Half of the fetal genome comes from the parent, and the genes encoded by these genes are allogenic antigens to the mother body, but in normal cases the mother does not produce an immune response to the fetal alloantigen and causes rejection of the fetus. Therefore, the mother fetus is bound to have a mechanism to induce and maintain maternal tolerance. The hair membrane cells do not express the classic HLA I, II class, but express non classical HLA-G molecules. The specific expression of HLA molecules on the maternal fetal interface is one of the important mechanisms that lead to maternal tolerance.
The study of HLA-G was initially focused on cross model HLA-G1 molecules. Studies have shown that transmembrane HLA-G1 molecules can be directly or indirectly associated with NK cells, T cells, dendritic cells, monocytes and giant cells and other immune cells, such as killer inhibitory receptor, KIR, and ILT (immunoglobulin-like transcripts/CD85). It activates the immune receptor tyrosine inhibition (Immunology receptor tyrosine-based inhibitory motif, ITIM) in its intracellular segment and activates the inhibitory signal transduction pathway, thus inhibiting the function of immune cells and inducing immune tolerance.
In the early pregnancy, all types of trophoblast cells in the placenta secrete sHLA-G, and the content of sHLA-G in the maternal blood is related to the success of pregnancy: if the content of sHLA-G in the maternal blood is high, the pregnancy is easy to succeed; otherwise, the pregnancy is not easy to succeed. In some tumors, infections, organ transplantation, and in the serum and tissue of patients with autoimmune diseases, sHLA-G. is not detected. The HLA-G*0105N homozygous healthy individuals expressing the membrane type HLA-G1 can be pregnant with normal pregnancy. These phenomena suggest that sHLA-G also has important functions. In recent years, the study of the biological function of sHLA-G has become a new hot spot in HLA-G research,.Contini P and other studies have confirmed that sHLA-G can inhibit the function of NK cells, CD4+, CD8+ T cells, but the specific mechanism of sHLA-G has not been clarified. Rnel and other studies show that purified HLA-G5 can bind to CD8 molecules at a low concentration of 0.25 u g/ml, stimulate CD8+ cells to express CD95 ligands and induce apoptosis in CD8+ cells via Fas/FasL pathway. Some researchers have not observed the increase of apoptosis in T cells when incubated with HLA-G5 cells with T cells. -G5 can inhibit the cell cycle of the homologous T cell but does not promote its apoptosis.Le Rond and so on to sensitized the initial T cells with HLA-G5 for 18 hours in vitro. It is found that these treated initial T cells lose their response to the subsequent alloantigen stimulation and inhibit the reverse response of other T cells. In mixed lymphocyte culture, CD4+, CD8+ T. Cells can express soluble or membranous HLA-G as a negative feedback signal to regulate the proliferation of CD4+ allo T cells.
In order to clarify the biological function and mechanism of sHLA-G, it is necessary to obtain a large number of functional sHLA-G molecules. The soluble protein expressed in eukaryotic expression system, although it maintains the original conformation of the protein, can not be expressed in large quantities and is difficult to purify, and the inclusion body protein expressed in the prokaryotic expression system can not maintain the original conformation of the protein, The purpose of this study is to use the prokaryotic expression system to prepare a large number of functional sHLA-G1- peptide complexes, and to observe the effect of the sHLA-G1- peptide complex folded in vitro on the proliferation of T cells by using the method of mixed culture of allogeneic T cells. In order to further clarify its mechanism of action and its clinical application, we used the HLA-G limited nine peptide KGPPAALTL and sHLA-G1 heavy chain and light chain to prepare sHLA-G1- peptide complex in vitro, and establish a allogenic mixed lymphocyte culture system to verify its biological function. As follows:
1, high expression and purification of sHLA-G1 heavy chain and beta 2M
The two subunits of sHLA-G1 molecule sHLA-G1 heavy chain and beta 2m (light chain) are highly expressed in the form of inclusion body in the form of inclusion body. The target protein is induced by IPTG, the inclusion body is extracted, and the protein yield of.SHLA-G1 heavy chain and beta 2m in 8mol/L urea, which is dissolved in 8mol/L urea, is 180mg/L and 150mg/L, respectively. The purity of the gray scale scanning is 75.7%, respectively. And 65.8%, it can meet the needs of preparing sHLA-G1- antigen peptide complexes in this study.
2, dilution and folding renaturation of sHLA-G1- antigen peptide complexes.
On the molecular structure, HLA-G1, like the classical HLA I class, consists of three molecular complexes including heavy chains, light chains and antigenic peptides. In the presence of HLA-G1 restrictive peptides, a denaturant in beta 2M and sHLA-G1, prepared by dilution, is refolding in vitro. The nine peptide KGPPAALTL from the placental trophoblastic layer is from H One of the three endogenous endogenous peptides eluted from LA-G, and its existence helps to fold up a stable sHLA-G1- peptide complex molecule. Western-blot identification with a monoclonal antibody W6/32 that is known only to be combined with the natural conformation of HLA I molecules. The results show that W6/32 can also be combined in the composition of the folded complex, and the same can be found. W6/32 and anti beta 2m antibody were used to carry out double anti sandwich ELISA, which proved that the complex of sHLA-G1- antigen peptide complex in vitro has the natural conformation of HLA I molecules, indicating that the sHLA-G1 monomer can be successfully prepared by the prokaryotic expression.
3, the effect of sHLA-G1 on the proliferation of T cells in mixed lymphocyte culture.
The peripheral blood lymphocytes were labeled with carboxyl fluorescein acetoacetate succinimide (carboxyfluorescein diacetate succinimidyl ester, CFSE) and stimulated by B lymphoblastic cells (EBV-LCL) transformed by allogeneic EB virus. This was a mixed lymphocyte culture system in this experiment, representing the T cell response to allogenic antigen. In the 96 hole culture plate, 2 x 105 fluorescein labeled reactive cells and 2 x 104 stimulation cells were cultured in the incubator at 37 C, 5%CO2 culture box. 300 mu sHLA-G1- peptide complex and the same concentration of sHLA-A2- peptide complex were added to the first and fourth days of culture, and beta 2m was used as the control, and the cells and proteins were added again on seventh days, and continued to be cultured. The proliferation of T cells was detected after three days. The results showed that among the three individuals, the proliferation index of the sHLA-G1- peptide complex was significantly lower than that of the culture group (individual 1: 5.5266 + 0.6124 vs 7.7350 + 0.4879; individuals two: 6.0566 + 0.4660 vs 8.9700 + three; individual three: 5.8133 + vs, P0.05), and HLA-G There was no significant difference between the G11E5+sHLA-G1 group and the medium group (individual: 7.7050 + 0.0777 vs 7.7350 + 0.4879; individuals two: 9.5700 + 0.5091 vs 8.9700 + 0.2353; individuals three: 7.200 + 0.6763 vs 7.4600 + 7.7350 +; P0.05). And sHLA-A2 control group was significantly lower than that of the medium control group in the individual one. The individual one: 5.5633 + 0.3523 vs 7.7350 + 0.4879; individuals two: 7.0100 + 0.3111 vs 8.9700 + 0.2353; P0.05), but higher in the individual 3 than the medium control group (9.0933 + 0.3859 vs 7.4600 + 0.5726; P0.05). These results showed that the prokaryotic expression, the in vitro folded sHLA-G1 could inhibit the proliferation reaction of the T cells to the same antigen. The inhibitory effect can be blocked by monoclonal antibody G11E5 targeting HLA-G alpha 1 region.
Soluble HLA-G is a new hot spot in HLA-G research in recent years, and it may play a more important role than membrane type HLA-G in the body. This study uses the prokaryotic expression system to prepare sHLA-G1- peptide complexes with correct structure in vitro, and first uses CFSE labeled reactive cells, and uses flow cytometry to detect sHLA-G1- peptide accurately and quickly. The effect of complex on the proliferation of T cells in mixed lymphocyte reaction. The functional sHLA-G1- peptide complex was successfully prepared, which laid the material basis for further elucidating its mechanism and clinical application.
【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
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 楊敬寧,吳雄文,梁智輝,陸盛軍,尹姝晗,蔡蕾,韓軍艷,黃亞非,龔非力;可溶性HLA-G1重鏈分子的構(gòu)建、表達(dá)及純化[J];中國免疫學(xué)雜志;2005年04期
，本文編號：2127145
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