可溶性HLA-G1-肽复合物的体外折叠及其对混合淋巴细胞培养中T细胞增殖的影响
发布时间:2018-07-16 17:46
【摘要】: 人类白细胞抗原(humanleukocyteantigen,HLA)G基因是1987年由Geraghty等克隆并测序的一类非经典HLA I类基因,可通过不同的剪切方式翻译出七种不同结构的蛋白,包括4种跨膜型和3种可溶性HLA-G(soluble HLA-G,sHLA-G)分子。对HLA-G的关注源于母胎耐受。胎儿基因组的一半来自父方,这些基因编码的产物对母体而言是同种异体抗原,但正常情况下母体并不对胎儿的同种异体抗原产生免疫应答而导致对胎儿的排斥。因此,母胎之间必然存在诱导并维持母胎耐受的机制。研究发现,胎盘绒毛膜细胞不表达经典的HLA I、II类分子,却表达非经典的HLA-G分子。母胎界面上HLA分子表达的特殊性是导致母胎耐受的重要机制之一。 对HLA-G的研究最初集中于跨模型HLA-G1分子。研究证实,跨膜型HLA-G1分子可直接或间接与NK细胞、T细胞、树突状细胞、单核细胞及巨嗜细胞等免疫细胞表面的杀伤细胞抑制性受体(killer inhibitory receptor, KIR)及ILT(immunoglobulin-like transcripts/CD85)结合,激活其胞内段的免疫受体酪氨酸抑制基序(Immunology receptor tyrosine-based inhibitory motif, ITIM),启动抑制性信号传导通路,从而抑制免疫细胞的功能,诱导免疫耐受。 在妊娠早期,胎盘所有类型的滋养层细胞都分泌sHLA-G,母体血中sHLA-G含量与妊娠是否成功相关:若母体血中sHLA-G含量高,则妊娠易成功;否则,妊娠不易成功。在某些肿瘤、感染、器官移植、自身免疫性疾病病人的血清及组织中检测到sHLA-G。不表达膜型HLA-G1的HLA-G*0105N纯合型健康个体能正常妊娠,这些现象提示sHLA-G同样有着重要的功能。近年来对sHLA-G生物学功能研究成为HLA-G研究的新热点。Contini P等研究证实sHLA-G能抑制NK细胞,CD4+、CD8+ T细胞的功能,但其具体机制没有阐明。Fournel等研究表明,纯化的HLA-G5可在0.25μg/ml的低浓度下与CD8分子结合,刺激CD8+细胞表达CD95配体,通过Fas/FasL的途径诱导CD8+细胞凋亡。而有些研究者在用分泌HLA-G5的细胞与T细胞一起孵育时并没有观察到T细胞的凋亡增加。最近有研究表明HLA-G5可以抑制同种反应T细胞的细胞周期但不促进其凋亡。Le Rond等在体外用HLA-G5致敏初始T细胞18个小时,发现这些处理过的初始T细胞丧失了对其后同种抗原刺激的反应能力,并能抑制其他T细胞的反应性。在混合淋巴细胞培养中,CD4+、CD8+ T细胞均能表达可溶性或膜型HLA-G,作为一种负反馈信号调节CD4+同种反应性T细胞的增殖。 为阐明sHLA-G的生物学作用及其作用机制,有必要获取大量有功能的sHLA-G分子,真核表达系统表达的可溶性蛋白虽然保持了蛋白原有的构象,但不能大量表达,且纯化困难。而原核表达系统表达的包涵体蛋白不能保持蛋白原有的构象,也不能进行糖基化,因此需在体外进行折叠复性和功能鉴定。本课题研究的目的是采用原核表达系统,制备大量有功能的sHLA-G1-肽复合物,并且利用同种异体T细胞混合培养的方法,观察体外折叠的sHLA-G1-肽复合物对T细胞增殖反应的影响,为进一步阐明其作用机制及临床应用奠定物质基础。我们采用HLA-G限制性的九肽KGPPAALTL与sHLA-G1重链及轻链在体外经稀释复性制备sHLA-G1-肽复合物,并建立同种异体混合淋巴细胞培养体系,验证其生物学功能。本课题的研究内容和结果如下: 1、sHLA-G1重链、β2m的高效表达和纯化 sHLA-G1分子的两个亚基sHLA-G1重链和β2m(轻链)均以包涵体的形式在细菌内高效表达,经IPTG诱导表达目的蛋白,提取包涵体并初步纯化后溶于8mol/L尿素中。sHLA-G1重链和β2m的蛋白产量分别为180mg/L菌液和150mg/L菌液,灰度扫描纯度分别为75.7%及65.8%,能够满足本研究制备sHLA-G1-抗原肽复合物的需要。 2、sHLA-G1-抗原肽复合物分子的稀释折叠复性 在分子结构上,HLA-G1与经典的HLA I类分子一样,均为包括重链、轻链和抗原肽的三分子复合物。在HLA-G1限制性的抗原肽存在的情况下,通过稀释制备得到的β2m与sHLA-G1中的变性剂而实现体外复性折叠。来自于胎盘滋养层的九肽KGPPAALTL是从HLA-G上洗脱的三种内源性自身肽的一种,它的存在可帮助共折叠形成稳定的sHLA-G1-抗原肽复合物分子。用已知仅与天然构象的HLA I类分子结合的单克隆抗体W6/32进行Western-blot鉴定,结果显示W6/32同样能够结合于折叠复合物中的成份,进一步应用W6/32与抗β2m抗体进行双抗夹心ELISA,证实体外折叠sHLA-G1-抗原肽复合物具有HLA I类分子的天然构象,说明通过原核表达、体外折叠能够成功地制备sHLA-G1单体。 3、sHLA-G1对混合淋巴细胞培养中T细胞增殖的影响 外周血淋巴细胞用羧基荧光素乙酰乙酸琥珀酰亚胺酯(carboxyfluorescein diacetate succinimidyl ester,CFSE)标记后,用同种异体的EB病毒转化的B淋巴母样细胞(EBV-LCL)刺激,此为本实验的混合淋巴细胞培养体系,代表针对同种异体抗原的T细胞应答。在96孔培养板中加入2×105荧光素标记的反应细胞和2×104刺激细胞,37℃,5%CO2培养箱中培养。在培养的第一天和第四天加入300μg/ml sHLA-G1-肽复合物及相同浓度的sHLA-A2-肽复合物、β2m作为对照,在第七天再次加入刺激细胞和蛋白,继续培养三天后流式检测T细胞的增殖情况,结果发现:三个个体中,sHLA-G1-肽复合物实验组的增殖指数均明显低于培养基对照组(个体一:5.5266±0.6124 vs 7.7350±0.4879;个体二:6.0566±0.4660 vs 8.9700±0.2353;个体三:5.8133±0.3470 vs 7.4600±0.5726; P0.05),而HLA-G的单克隆抗体G11E5+sHLA-G1组与培养基组比较没有明显差异(个体一:7.7050±0.0777 vs 7.7350±0.4879;个体二:9.5700±0.5091 vs 8.9700±0.2353;个体三:7.200±0.6763 vs 7.4600±0.5726; P0.05)。而sHLA-A2对照组在个体一、个体二中增殖指数都明显低于培养基对照组(个体一:5.5633±0.3523 vs 7.7350±0.4879;个体二:7.0100±0.3111 vs 8.9700±0.2353;P0.05),而在个体三中却高于培养基对照组(9.0933±0.3859 vs 7.4600±0.5726; P0.05)。这些结果显示原核表达、体外折叠的sHLA-G1能够抑制T细胞针对同种抗原的增殖反应。并且这种抑制作用能被针对HLA-Gα1区的单克隆抗体G11E5封闭。 可溶性HLA-G是近年来HLA-G研究的一个新的热点,在体内可能发挥比膜型HLA-G更为重要的作用。本研究利用原核表达体系,在体外大量制备正确构像的sHLA-G1-肽复合物,并首次采用CFSE标记反应细胞,利用流式细胞仪准确快速的检测sHLA-G1-肽复合物对混合淋巴细胞反应中T细胞增殖的影响。成功制备出有功能的sHLA-G1-肽复合物,为进一步阐明其作用机制及临床应用奠定物质基础。
[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.
【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
本文编号: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.
【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
【参考文献】
相关期刊论文 前1条
1 杨敬宁,吴雄文,梁智辉,陆盛军,尹姝晗,蔡蕾,韩军艳,黄亚非,龚非力;可溶性HLA-G1重链分子的构建、表达及纯化[J];中国免疫学杂志;2005年04期
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