CN113136366B - 27位赖氨酸突变泛素分子增强树突状细胞交叉提呈的方法 - Google Patents
27位赖氨酸突变泛素分子增强树突状细胞交叉提呈的方法 Download PDFInfo
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Abstract
27位赖氨酸突变泛素分子增强树突状细胞交叉提呈的方法，涉及基因工程技术领域。从小鼠骨髓中分离得到树突状细胞；用27位赖氨酸突变泛素分子预处理骨髓来源树突状细胞：先进行siRNA干扰实验，再进行体外过继免疫，最后进行酶联免疫斑点实验和免疫印迹实验。以27位赖氨酸突变为精氨酸的泛素分子处理树突状细胞，实验证明，处理后的树突状细胞可明显增强树突状细胞交叉提呈能力，并促进DC介导的抗原特异性CTL诱生能力；27位赖氨酸突变为精氨酸的泛素分子可增加树突状细胞转输受体内淋巴细胞产生穿孔素、颗粒酶B能力。
Description
技术领域
本发明涉及基因工程技术领域，尤其是涉及一种27位赖氨酸突变泛素分子增强树突状细胞交叉提呈的方法。
背景技术
树突状细胞(Dendritic cells,DC)是机体介导适应性免疫的重要抗原提呈细胞，对于固有免疫与适应性免疫起着重要的桥梁作用。交叉提呈途径是指外源性抗原经DC作用后与MHC-I类分子形成复合体，提呈给CD8+T(cytotoxic T lymphocyte,CTL)细胞并使其增殖分化。CTL细胞对肿瘤、病毒感染的宿主细胞进行杀伤过程则涉及释放穿孔素(perforin)颗粒酶(granzyme B)所介导的效应有关。穿孔素是Ca2+依赖的疏水性分子，它能在靶细胞膜上融出通道，并进一步诱导颗粒酶进入靶细胞的细胞质中，促进靶细胞发生凋亡。由于外源性抗原的相对分子质量较大，限制其通过内源性途径对抗原进行处理，而通过交叉提呈途径提呈抗原，可起到杀伤靶细胞的作用，因此，DC交叉提呈所诱导的CTL对肿瘤及病毒的清除非常重要(Fu C,Jiang A.Dendritic cells and CD8 T cell immunity in tumormicroenvironment[J].Front Immunol,2018,9:3059.DOI:10.3389/fimmu.2018.03059)。
泛素有7个赖氨酸(Lysine,K)残基，因此，蛋白的泛素化也可根据其发生泛素化的赖氨酸位置而分为K6、K11、K27、K29、K33、K48和K63等方式的泛素化(Zinngrebe J,Montinaro A,Peltzer N,et al.Ubiquitin in the immune system[J].EMBO Rep,2014,15(1):28-45.DOI:10.1002/embr.201338025)。一个完整的泛素化过程需由泛素活化酶E1、结合酶E2及连接酶E3共同完成(Swatek KN,Komander D.Ubiquitin modification[J].Cell Res,2016,26(4):399-422.DOI:10.1038/cr.2016.39)。活化酶E1在ATP催化下，将泛素的羧基端与半胱氨酸形成硫酯键、在结合酶E2作用下活化的泛素与结合酶E2半胱氨酸残基结合、再由连接酶E3催化泛素分子羧基结合到靶蛋白赖氨酸侧链上(Park Y,Jin HS,AkiD,et al.The ubiquitin system in immune regulation[J].A dv Immunol,2014,124:17-66.DOI:10.1016/B978-0-12-800147-9.00002-9)。DC交叉提呈所涉及的内吞抗原移位进入细胞质的亚细胞器定位与泛素化密切相关，前期研究发现，K48泛素化使泛素化的蛋白靶向蛋白酶体，而K63泛素化的蛋白则靶向溶酶体，K48泛素化突变使得树突状细胞交叉提呈能力减弱(Zehner M,Chasan AI,Schuette V,et al.Mannose receptor poly-ubiquitination regulates endosomal recruitment of p97 and cytosolic antigentranslocation for cross-presentation[J].PNAS,2011,108:9933-9938.DOI:10.1073/pnas.1102397108)。
发明内容
本发明的目的在于提供一种27位赖氨酸突变泛素分子增强树突状细胞交叉提呈的方法。
本发明包括以下步骤：
1)从小鼠骨髓中分离得到树突状细胞；
2)用27位赖氨酸突变泛素分子预处理骨髓来源树突状细胞：先进行siRNA干扰实验，再进行体外过继免疫，最后进行酶联免疫斑点实验和免疫印迹实验。
在步骤1)中，所述从小鼠骨髓中分离得到树突状细胞具体方法可为：首先脱臼处死小鼠，酒精消毒后，无菌分离股骨和胫骨，剔除多余肌肉组织，将骨髓冲出，直至骨头由红变灰白色，以无菌注射器底部研磨骨髓成单细胞悬液；离心弃上清后，加入红细胞裂解液裂解红细胞，待溶液由红变白后，加入等体积完全培养液终止；再次离心、弃上清，以培养液调整细胞浓度为1×106个/mL；加入细胞因子GM-CSF和IL-4，使其终浓度为10ng/mL和1ng/mL；置于培养箱中培养；在第4天时，弃去上清中悬浮细胞，以PBS冲洗皿底，得到的皿底贴壁的细胞继续培养2～4天，即为骨髓来源的树突状细胞。
在步骤2)中，所述siRNA干扰实验用于诱导骨髓来源树突状细胞，具体步骤可为：首先siRNA、transfection reagent、transfection medium置于常温避光待其恢复室温；其次配制ubiquitin siRNA稀释液后，以PBS清洗树突状细胞后，加入ubiquitin siRNA稀释液轻轻覆盖于细胞，作用5～7h；继而加入20％FBS的1640培养液，作用24h；最后换液，加入10％FBS的RMPI 1640培养基，继续培养48～72h待用；
所述配制ubiquitin siRNA稀释液的具体方法可为：先配制A液：加入ubiquitinsiRNA+transfection medium；再配制B液：加入transfection reagent+transfectionmedium；两者分别充分混匀后，将A液加入到B液中，轻柔混匀后避光孵育得到ubiquitinsiRNA稀释液。
所述体外过继免疫实验的具体步骤可为：DC在干扰ubiquitin后同时加入泛素化突变链K27R(27位赖氨酸突变泛素分子，10～30μM)及外源性抗原(ovalbumin,OVA 50～100μg/mL),作用4～6h后，收集细胞至离心管中，以PBS重悬细胞并离心去除多余突变链及鸡卵清白蛋白影响；再次以适量生理盐水重悬细胞并调整细胞浓度，经腹腔将相同数量的DC细胞转输到相同基因背景的C57BL/6雌性小鼠体内；5～7天后取其脾脏、淋巴结制成细胞悬液，进行后续实验。
所述酶联免疫斑点实验的具体步骤可为：首先制备小鼠脾脏悬液浓度，脱臼处死过继免疫实验小鼠并无菌分离脾脏，以红细胞裂解液裂解红细胞后，调整悬液浓度为1×107/mL；其次以完全培养液活化预包被IFN-γ抗体检测板,加入悬液，即1×106/well，加入SIINFEKL2～4μg/mL，培养16～20h；以预冷的双蒸水低渗裂解细胞后，相继加入生物素-IFN-γ和亲和素-HRP抗体孵育期间以washing buffer洗板；最后现配AEC显色液显色，并以清水冲洗终止实验，待检测板自然晾干后，用ELISPOT图像分析系统拍照并计数斑点的个数。
所述免疫印迹实验的具体步骤可为：
(1)蛋白的提取
首先蛋白磷酸酶抑制剂按1︰100加入蛋白裂解液(RIPA)，脱臼处死过继免疫实验小鼠，分离得到脾脏；接着加入蛋白裂解液裂解并超声数分钟；继而收集裂解液于eppendorf管，离心后取上清，加入1/4的5×loading buffer溶液(含有β巯基乙醇)，涡旋混匀，最后金属浴加热100℃，5～15min,并置于冰箱保存。
(2)SDS-PAGE
首先根据目的蛋白的相对分子质量制备分离胶后，再制备上层的浓缩胶，待其凝固后，蛋白样品上样，并用1×loading buffer补齐至相同体积，加入合适的电泳缓冲液进行电泳，浓缩胶范围内，电泳电压为60～80V,待其跑到分离胶后，加大电压，分离胶电泳电压为100～120V,100～150min；其次弃去浓缩胶，把分离胶含有目的条带所在位置的凝胶切下，以甲醇活化PVDF膜并按照滤纸-凝胶-PVDF膜-滤纸放置的顺序制备“转膜夹心”，倒入预冷的转膜液，加入冰袋，转膜时间为80～100V,1～1.5h；接着用丽春红染色后,转移至甲醇溶液中脱色，并置于PBS溶液漂洗；继而把膜置于提前以PBST溶解的5％脱脂牛奶室温孵育1.5～2h，置于配制的抗体稀释液中，4℃，孵育过夜，以PBST漂洗数次后，膜条带置于偶联HRP的二抗稀释液，常温孵育1.5～2h；最后在避光环境下，以ECL发光液孵育膜条带，置于暗盒中，以胶片记录条带发光情况。
与现有技术相比，本发明具有以下突出优点：
本发明以27位赖氨酸突变为精氨酸的泛素分子处理树突状细胞，实验证明，处理后的树突状细胞可明显增强树突状细胞交叉提呈能力，并促进DC介导的抗原特异性CTL诱生能力；27位赖氨酸突变为精氨酸的泛素分子可增加树突状细胞转输受体内淋巴细胞产生穿孔素、颗粒酶B能力。
附图说明
图1为27位赖氨酸突变为精氨酸的泛素分子增强转输受体脾脏抗原特异性CTL图。
图2为27位赖氨酸突变为精氨酸的泛素分子增强转输受体淋巴结抗原特异性CTL图。
图3为27位赖氨酸突变为精氨酸的泛素分子增强转输受体脾细胞穿孔素产生图。
图4为27位赖氨酸突变为精氨酸的泛素分子增强转输受体脾细胞颗粒酶B产生图。
具体实施方式
以下实施例将结合附图对本发明作进一步的说明。
本发明实施例以27位赖氨酸突变为精氨酸的泛素分子处理树突状细胞，以酶联免疫斑点实验(Elispot)、western blot技术检测树突状细胞介导抗原特异性CTL的诱生能力。
本发明实施例包括以下步骤：
1)骨髓来源树突状细胞诱导
首先脱臼处死小鼠，酒精消毒后，以眼科剪和眼科镊无菌分离股骨和胫骨，剔除多余肌肉组织；其次以RMPI 1640完全培养液将骨髓冲出，直至骨头由红变灰白色，以无菌注射器底部研磨骨髓成单细胞悬液；离心弃上清后，加入红细胞裂解液裂解红细胞，待溶液由红变白后，加入等体积完全培养液终止；再次离心、弃上清，以培养液调整细胞浓度为1×106个/mL；加入细胞因子GM-CSF和IL-4，使其终浓度为10ng/mL和1ng/mL；置于培养箱中培养；最后在第4天时，弃去上清中悬浮细胞，以PBS冲洗皿底，得到的皿底贴壁的细胞继续培养2～4天，即为骨髓来源的树突状细胞。
2)骨髓来源树突状细胞的预处理
(1)siRNA干扰实验
诱导骨髓来源树突状细胞。首先siRNA、transfection reagent、transfectionmedium置于常温避光待其恢复室温；其次配制ubiquitin siRNA稀释液后，以PBS清洗树突状细胞后，加入ubiquitin siRNA稀释液轻轻覆盖于细胞，作用5～7h；继而加入20％FBS的1640培养液，作用24h；最后换液，加入10％FBS的RMPI 1640培养基，继续培养48-72h待用。
在上述步骤中，所述配制ubiquitin siRNA稀释液的具体方法可为：先配制A液：加入ubiquitin siRNA+transfection medium；再配制B液：加入transfection reagent+transfection medium；两者分别充分混匀后，将A液加入到B液中，轻柔混匀后避光孵育，得ubiquitin siRNA稀释液。
(2)体外过继免疫实验
DC在干扰ubiquitin后同时加入泛素化突变链K27R(27位赖氨酸突变泛素分子，10-30μM)及外源性抗原(ovalbumin,OVA 50-100μg/mL),作用4～6h后，收集细胞至离心管中。以PBS重悬细胞并离心去除多余突变链及鸡卵清白蛋白影响。再次以适量生理盐水重悬细胞并调整细胞浓度，经腹腔将相同数量的DC细胞转输到相同基因背景的C57BL/6雌性小鼠体内。5～7天后取其脾脏、淋巴结制成细胞悬液，进行后续实验。
(3)酶联免疫斑点实验(Enzyme-linked immunospot assay,Elispot)
首先制备小鼠脾脏悬液浓度，脱臼处死过继免疫实验小鼠并无菌分离脾脏，以红细胞裂解液裂解红细胞后，调整悬液浓度为1×107/mL；其次以完全培养液活化预包被IFN-γ抗体检测板20min,加入悬液100μL,即1×106/well,加入SIINFEKL(2～4μg/mL)，培养16-20h；接着以预冷的双蒸水低渗裂解细胞后，相继加入生物素-IFN-γ和亲和素-HRP抗体孵育期间以washing buffer洗板；最后现配AEC显色液显色，并以清水冲洗终止实验，待检测板自然晾干后，用ELISPOT图像分析系统拍照并计数斑点的个数。
(4)免疫印迹实验
(4.1)蛋白的提取
首先蛋白磷酸酶抑制剂按(1︰100)加入蛋白裂解液(RIPA)，脱臼处死过继免疫实验小鼠，分离得到脾脏；接着加入蛋白裂解液裂解并超声数分钟；继而收集裂解液于eppendorf管，离心后取上清，加入1/4的5×loading buffer溶液(含有β巯基乙醇)，涡旋混匀，最后金属浴加热100℃，5～15min,并置于-20℃冰箱保存。
(4.2)SDS-PAGE
首先根据目的蛋白的相对分子质量制备分离胶后，再制备上层的浓缩胶，待其凝固后，蛋白样品上样，并用1×loading buffer补齐至相同体积，加入合适的电泳缓冲液进行电泳，浓缩胶范围内，电泳电压为60～80V,待其跑到分离胶后，加大电压，分离胶电泳电压为100～120V,100～150min；其次弃去浓缩胶，把分离胶含有目的条带所在位置的凝胶切下，以甲醇活化PVDF膜并按照滤纸-凝胶-PVDF膜-滤纸放置的顺序制备“转膜夹心”，倒入预冷的转膜液，加入冰袋，并在槽外周围覆盖冰块，转膜时间为80～100V,1～1.5h；接着用丽春红染色数分钟后,转移至甲醇溶液中脱色，并置于PBS溶液漂洗数分钟；继而把膜置于提前以PBST溶解的5％脱脂牛奶室温孵育1.5～2h，置于配制的抗体稀释液中，4℃，孵育过夜，以PBST漂洗数次后，膜条带置于配制的对应的偶联HRP的二抗稀释液，常温孵育1.5～2h；最后在避光的环境下，以ECL发光液孵育膜条带，置于暗盒中，以胶片记录条带的发光情况。
统计方法如下：
各组计量资料以均数(Mean)±标准差(Sd)表示。应用GraphPad Prism 5进行统计分析，差异采用student t检验法(Student's t test)。P<0.05为差异有统计学意义。
27位赖氨酸突变为精氨酸的泛素分子增强转输受体脾脏抗原特异性CTL图见图1，对GM-CSF和IL-4诱导的DC，ubi干扰处理，同时予以27位赖氨酸突变为精氨酸的泛素分子K27R(10-30μM)和外源性OVA(50-100μg/mL)，经腹腔转输等数量DC至同基因C57BL/6小鼠体内。DC转输小鼠体内5～7天后，取小鼠脾细胞(spleen,SP)进行Elispot实验。数据以均数+-标准差显示，组间采用student t检验法(Student's t test)，*P<0.05，**P<0.01,***P<0.001,n＝3。OVA：ovalbumin；Ubi:ubiquitin。
实验结果：Elispot实验结果显示，27位赖氨酸突变为精氨酸的泛素分子处理后的树突状细胞负载外源性抗原转输小鼠体内，诱导脾脏细胞特异性CTL产生能力提高了200％
结论：27位赖氨酸突变为精氨酸的泛素分子联合OVA抗原处理后的DC交叉提呈能力增加，并进一步诱导脾脏抗原特异性CTL产生能力增强。
27位赖氨酸突变为精氨酸的泛素分子增强转输受体淋巴结抗原特异性CTL图见图2，对GM-CSF和IL-4诱导的DC，ubi干扰后，同时予以27位赖氨酸突变为精氨酸的泛素分子K27R(10～30μM)，和外源性OVA(50～100μg/mL)，经腹腔注射等数量DC至同基因C57BL/6小鼠体内。DC转输小鼠体内5～7天后，取小鼠淋巴结(lymph node,LN)进行Elispot实验。数据以均数+-标准差显示，组间采用student t检验法(Student's t test)，*P<0.05，**P<0.01,***P<0.001,n＝3。OVA：ovalbumin；Ubi:ubiquitin。
实验结果：树突状细胞在加入27位赖氨酸突变为精氨酸的泛素分子及负载外源性抗原后，转输小鼠体内，诱导淋巴结产生针对抗原的特异性CTL增加了150％。
结论：27位赖氨酸突变为精氨酸的泛素分子联合OVA抗原处理后的DC诱导淋巴结产生抗原特异性CTL产生能力增强。
27位赖氨酸突变为精氨酸的泛素分子增强转输受体脾细胞穿孔素表达图见图3，对GM-CSF和IL-4诱导的DC，ubi干扰后，再经过27位赖氨酸突变为精氨酸的泛素分子K27R(10～30μM)和外源性OVA(50～100μg/mL)处理，经腹腔转移等数量DC至同基因C57BL/6小鼠体内。DC转输小鼠体内5～7天后，取小鼠脾脏提蛋白，并通过western blot检测穿孔素(perforin)的表达情况，western blot结果以Image J分析得到蛋白条带的密度(RelativeIntegrate density,RID)表示。OVA：ovalbumin；Ubi:ubiquitin。
结果：western blot结果显示，对照组的RID值设为1，经过K27R处理后DC介导CTL产生穿孔素增加了60％。
结论：27位赖氨酸突变为精氨酸的泛素分子联合OVA抗原处理后的DC介导脾脏CTL分泌的穿孔素增加。
27位赖氨酸突变为精氨酸的泛素分子增强转输受体脾细胞颗粒酶B表达图见图4。GM-CSF和IL-4诱导得到的DC，ubi干扰后，经过27位赖氨酸突变为精氨酸的泛素分子K27R(10～30μM)，和外源性OVA(50～100μg/mL)处理，经腹腔转移等数量DC至同基因C57BL/6小鼠体内。5～7天后，取小鼠脾脏提蛋白后，并通过western blot检测颗粒酶B(granzyme B)的表达情况，以Image J分析得到蛋白条带的密度(Relative Integrate density,RID)表示。OVA：ovalbumin；Ubi:ubiquitin。
实验结果：western blot结果显示，若对照组RID值设定为1，经过K27R处理后DC介导CTL产生穿孔素增加了50％。
结论：27位赖氨酸突变为精氨酸的泛素分子负载OVA抗原处理后的DC介导CTL分泌的颗粒酶B增加。
Claims (3)
1.增强树突状细胞交叉提呈的方法，其特征在于包括以下步骤：
1）从小鼠骨髓中分离得到树突状细胞；
2）进行ubiquitin的siRNA干扰，树突状细胞在干扰ubiquitin后同时加入泛素化突变链K27R 10～30μM 及外源性抗原OVA 50～100μg/mL，作用4～6 h后，收集树突状细胞至离心管中，以PBS重悬树突状细胞并离心去除多余泛素化突变链K27R及外源性抗原OVA影响。
2.如权利要求1所述增强树突状细胞交叉提呈的方法，其特征在于在步骤1）中，所述从小鼠骨髓中分离得到树突状细胞具体方法为：首先脱臼处死小鼠，酒精消毒后，无菌分离股骨和胫骨，剔除多余肌肉组织，将骨髓冲出，直至骨头由红变灰白色，以无菌注射器底部研磨骨髓成单细胞悬液；离心弃上清后，加入红细胞裂解液裂解红细胞，待溶液由红变白后，加入等体积完全培养液终止；再次离心、弃上清，以培养液调整细胞浓度为1×106个/mL；加入细胞因子GM-CSF和IL-4，使其终浓度为10 ng/mL和1 ng/mL；置于培养箱中培养；在第4天时，弃去上清中悬浮细胞，以 PBS 冲洗皿底，得到的皿底贴壁的细胞继续培养2～4天，即为从小鼠骨髓中分离得到树突状细胞。
3.如权利要求1所述增强树突状细胞交叉提呈的方法，其特征在于在步骤2）中，所述ubiquitin的siRNA干扰，具体步骤为：首先siRNA、transfection reagent、transfectionmedium置于常温避光待其恢复室温；其次配制ubiquitin siRNA稀释液后，以PBS清洗树突状细胞后，加入ubiquitin siRNA稀释液轻轻覆盖于细胞，作用5～7h；继而加入20% FBS的1640培养液，作用24h；最后换液，加入10 % FBS 的RMPI 1640培养基，继续培养48～72 h待用。
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