CN116240175A - 一种嵌合抗hiv广谱中和抗体外泌体的制备方法以及在抗hiv感染中的应用 - Google Patents
一种嵌合抗hiv广谱中和抗体外泌体的制备方法以及在抗hiv感染中的应用 Download PDFInfo
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Abstract
本发明属于外泌体领域，具体涉及一种嵌合抗HIV广谱中和抗体外泌体的制备方法以及在抗HIV感染中的应用。抗HIV广谱中和抗体在293F细胞中表达并分泌的过程中，会同时将广谱中和抗体嵌合在外泌体膜外，再通过探究广谱中和抗体在外泌体上的展示方式证明它能与HIVgp120三聚体蛋白结合以此达到中和病毒的作用，最后利用体外C8166细胞的抗HIV感染的实验验证其功能性，这种抗HIV感染的嵌合抗HIV广谱中和抗体外泌体不仅可以提高抗体治疗中抗体运输到人体的效率，还可以将抗体运输到身体更远的部位，并产生更长久的治疗效果。
Description
技术领域
本发明属于外泌体领域，具体涉及一种嵌合抗HIV广谱中和抗体外泌体的制备方法以及在抗HIV感染中的应用。
背景技术
人类免疫缺陷病毒(HIV)是导致艾滋病的病原体，已发现的主要有HIV-Ⅰ和HIV-Ⅱ两种，而对于HIV的研究主要是针对HIV-Ⅰ。HIV是一种单链RNA病毒，属于反转录病毒，具有慢病毒种属特征。电子显微镜下病毒粒子直径为100-200nm，20面体对称结构，球形。HIV最外层为脂蛋白包膜，膜上有表面蛋白(gp120)和镶嵌蛋白(gp41)两种糖蛋白，包膜内面为基质蛋白，其内是衣壳蛋白包裹着RNA。HIV的基因组为单股正链RNA二倍体，每条RNA链长约9.8kb，其主要包括3个结构基因：Gag基因、Env基因、Pol基因，3个调节基因：Tat基因、Rev基因、Nef基因，以及4个辅助基因：Vif基因、Vpr基因、Vpu基因、Vpx基因。HIV主要感染的是人体的T淋巴细胞和巨噬细胞，通过病毒膜蛋白gp120与细胞表面的CD4受体蛋白相结合，使细胞感染HIV。
广谱中和抗体(bNAbs)存在于HIV-1感染几年后并且没有进行抗逆转录病毒治疗的情况下，能够长时间保持极低病毒载量的精英控制者(Elite Controllers,EC)体内，在这些精英控制者体内，通过噬菌体展示技术和单个B细胞培养及直接功能筛选或抗原特异性单个B细胞分选技术可以分离出一系列具有高中和活力及中和广度的抗体，也就是所谓的广谱中和抗体(bNAbs)。根据中和活性和分离时间，可将bNAbs大致分为两代。第一代bNAbs在20世纪90年代被分离，它的中和能力是有限的，尽管第一代bNAbs在动物模型上取得不错的结果，但临床试验显示这些bNAbs并不能有效抑制人体内的HIV-1病毒；自2009年以来，科研人员利用特异性单个B细胞分选技术、B细胞受体测序技术、及高通量中和抗体检测等新技术，新一代高效广谱bNAbs陆续被分离，且有更显著的中和宽度和中和活性，被称为第二代广谱中和抗体。bNAbs可以直接特异性地靶向HIV病毒膜上的Env三聚体，并与之结合，阻断Env-CD4受体结合或病毒与宿主细胞融合，从而降低宿主体内的病毒载量，达到治疗HIV的效果。
目前，感染HIV-1仍然是一个无法治愈的公共卫生问题，其主要疗法是鸡尾酒疗法，即高效抗逆转录病毒疗法(HAART)。HAART是通过三种或三种以上的抗病毒药物联合使用来治疗艾滋病，因为潜伏的前病毒会整合到CD4+T细胞的基因组中，从而形成稳定的病毒储存库，因此HAART虽然具有一定治疗效果，但需要终身服药。另一种治疗方法是使用抗HIV-1抗体的免疫疗法，这种治疗方法可以抑制病毒的感染并提高感染细胞的清除率。有临床研究表明，在有或没有HAART的情况下，HIV感染者在20周内接受7剂两种广谱中和抗体的组合试剂，其HIV-1储存库会被影响，但还需要更多更长时间的研究来确定抗体免疫疗法对HIV-1病毒储存库的确切影响。
发明内容
本发明提供了一种嵌合抗HIV广谱中和抗体外泌体的制备方法以及在抗HIV感染中的应用并验证了效果。具体发明内容如下：
一种嵌合外泌体，通过利用293F表达抗HIV-1的广谱中和抗体VRC01,在产生并分泌抗体的时候，同时产生嵌合抗HIV广谱中和抗体外泌体，收集分离纯化外泌体获得。
嵌合外泌体制备方法如下：
(1)收集转染了VRC01抗体表达质粒的293F细胞上清：收集至少200mL
(2)超高速离心分离外泌体：
a将上述收集的细胞培养上清，4℃，300×g离心5min，去掉沉淀；
b将a中的上清在4℃，3000×g离心30min，去掉沉淀；
c将b中的上清在4℃，10000×g离心60min，去掉沉淀；
d将c中的上清在4℃，100000×g离心90min，留沉淀；
e用预冷的无菌PBS重悬沉淀，将外泌体洗涤1遍，100000×g离心90min，留沉淀；
f用预冷的无菌适量PBS重悬沉淀，即得到外泌体悬液，-80℃保存。本发明具有以下有益效果：
1.探究了广谱中和抗体在分泌过程中会将其展现在外泌体膜外的现象，为治疗艾滋病相关治疗方法提供了新的理论基础。
2.本发明有望优化抗体治疗中的不足和限制，利用外泌体的高生物相容性和高传递效率等特点使抗体治疗具有更长的作用时间并可以透过血脑屏障到达大脑，在基于广谱中和抗体抗HIV感染研究相关研究中与外泌体结合具有一定的方法创新。
附图说明
图1抗HIV-1的广谱中和抗体VRC01其轻链和重链表达质粒简构图谱
图2抗HIV-1的广谱中和抗体VRC01其轻链和重链表达质粒酶切琼脂糖凝胶跑胶图
图3来源于293F的嵌合抗HIV广谱中和抗体外泌体分离纯化示意图
图4 293F抗体表达细胞上的抗体各结构部分流式检测
图5考马斯亮蓝染色和蛋白免疫印记检测目标抗体蛋白表达
图6嵌合抗HIV广谱中和抗体外泌体的示意图
图7电镜、NTA检测分离纯化的外泌体实验结果显示
图8蛋白免疫印记法检测外泌体标记蛋白和抗体
图9流式细胞术检测嵌合抗HIV广谱中和抗体外泌体上抗体的展示方式
图10 BLI(生物膜层干涉技术)实验检测嵌合抗HIV广谱中和抗体外泌体的中和活力
图11体外中和实验检测嵌合抗HIV广谱中和抗体外泌体的抗HIV感染效果
具体实施方式
实施例1、针对HIV的广谱中和抗体VRC01表达质粒图谱及跑胶验证以NCBI中的数据为模板，首先得到VRC01的轻链和重链序列，设计相应的抗体表达质粒，使其可以在293F细胞中表达，图1是VRC01轻链和重链质粒简构图谱。
图2是通过对构建的质粒用EcoRⅠ限制性内切酶进行单酶切，然后在1％的琼脂糖凝胶中电泳，在紫外光下成像即得到VRC01轻链和重链质粒酶切琼脂糖凝胶跑胶图，通过Snapgene模拟酶切对比片段大小，图2结果表明质粒大小正确。
实施例2、利用293F蛋白表达系统产生嵌合外泌体的广谱中和抗体
2.1293F培养
1.细胞复苏
取出冻存于﹣80℃或液氮内的293F细胞，迅速将细胞冻存管移至37℃恒温水浴中，轻轻摇晃使其快速融化，尽量将时间控制在1min内。用75％酒精擦拭冻存管，后迅速在超净工作台内加入1mL37℃预热F培养基，轻轻混匀，300g离心5min，于超净工作台内吸去上清，用移液枪吸取1mL37℃预热的F培养基加入冻存管内，轻轻吹打制备细胞悬液,避免剪切力过大损伤细胞。将细胞悬液转移至125mL细胞培养瓶中，并补充24mL新鲜预热的F培养基，轻轻旋转摇晃培养瓶，使细胞在培养瓶中均匀分布。将培养瓶移至37℃细胞培养箱中，每天观察细胞状态。
2.细胞计数
从37℃细胞培养箱中取出125mL培养瓶，置于超净工作台中，用移液管轻轻吹打均匀后取出100μL细胞悬液，然后加350μLPBS，再用50μL台盼蓝进行染色(单细胞悬液：台盼蓝＝9:1)混匀后计数，染色时间3-10min。取10μL染色后的悬液加入血球计数板，10倍镜下分别计算出左上、右上、右下、左下4个大格子中的总细胞数和染上蓝色的细胞数，并计算出细胞活率和所得总细胞数(取样细胞密度＝计算总细胞数/4×稀释倍数(5倍稀释)×104，细胞活率＝(计算总细胞数-染上蓝色的总细胞数)/计算总细胞数×100％，所得总细胞＝取样细胞密度×细胞悬液体积)。
3.293F细胞传代
当细胞密度达到2×106cells/mL及以上时，细胞需要进行传代，传代后的细胞密度应保持在3-5×105cells/mL。根据具体细胞计数的结果，计算传代所需的细胞悬液量，将细胞摇晃均匀后，用移液器吸取125mL培养瓶中相应量的细胞悬液到15mL无菌离心管中，300g离心5min，在超净工作台内吸去上清，加入1mL37℃预热的新鲜培养基，轻轻吹打成细胞悬液，将细胞悬液转移至新的500mL细胞培养瓶中，并补充99mL新鲜预热的F完全培养基，此时细胞密度应为3-5×105cells/mL。将瓶内的细胞轻轻摇晃均匀后，移至37℃细胞培养箱中，摇床上摇晃培养，次日观察。
4.细胞转染
通过将聚乙烯亚胺PEI(1mg/mL)和表达质粒瞬时转染到293F细胞中来表达VRC01抗体。
转染前三天按照3×105cells/mL的接种量往500mL摇瓶中的100mL培养基中接种细胞，37℃，125rpm，5％CO2浓度的摇床培养箱中孵育72h。转染当天的细胞密度预期应达到2×106cells/mL(细胞约24h能增殖一倍)。如果计数时发现密度不足2×106cells/mL(约1.0-2.0×106cells/mL)，若细胞状态良好，活率达到90％也能用于转染。
转染当天从培养箱内摇床上取出细胞培养瓶，使用电动移液枪，并用10mL移液管轻柔地吹打细胞悬液约20次(不要用力吹打细胞，极易导致细胞受到损伤)，防止细胞结团导致计数不准确。吹打混匀后获得单细胞悬液，取出少量单细胞悬液至1.5mL EP管中用于细胞密度计数，根据细胞密度计数结果，计算细胞活率及总细胞数。吸取100μg的质粒(pFUSEss-VRC01-VH：pFUSE2ss-VRC01-VL＝2：3，即pFUSEss-VRC01-VH 40μg，pFUSE2ss-VRC01-VL 60μg)加到1mL的F培养基中，吸取300μL已过滤除菌的PEI溶液(1mg/mL)加到1mL的F培养基中，使用移液枪充分混匀，静置5min。左手拿电枪吹打质粒/F培养基混合液，右手用移液枪逐渐滴加PEI/F培养基混合液到质粒/F培养基混合液中，室温静置30min。将质粒/PEI混合液逐滴滴加到细胞里。转染后，于37℃，125rpm，5％CO2浓度的摇床培养箱中孵育72h。
转染24h后需要添加OPM-CHO PFF06无蛋白补料培养基(添加体积为转染体系的1/20)和L-Glutamine(添加体积为转染体系1/50)。
2.2细胞培养上清处理及分离纯化外泌体
1.收集细胞培养上清：收集至少200mL
2.超高速离心法分离外泌体：
(1)将上述收集的细胞培养上清，4℃，300×g离心5min，去掉沉淀；
(2)将(1)中的上清在4℃，3000×g离心30min，去掉沉淀；
(3)将(2)中的上清在4℃，10000×g离心60min，去掉沉淀；
(4)将(3)中的上清在4℃，100000×g离心90min，留沉淀；
(5)用预冷的无菌PBS重悬沉淀，将外泌体洗涤1遍，100000×g离心90min，留沉淀；
(6)用适量预冷的无菌PBS重悬沉淀，即得到外泌体悬液，-80℃保存。
2.3转染了质粒的293F细胞流式检测
1.取转染质粒后的293F细胞，用枪吹打混匀之后进行计数，每管取1×106个细胞；
2.每管加入2mL孵育缓冲液，400g离心5min漂洗，除去上清；
3.重复漂洗一次；
4.将细胞沉淀重悬在100μL孵育缓冲液中；；
5.分别加入荧光染料抗体Fab-PE，Fd-APC，Fc-BV421各5μL，室温避光孵育30min；
6.再次往每管加入2mL孵育缓冲液，400g离心5min漂洗3次，去除多余的抗体；
7.最后一次用500μL PBS重悬，得到细胞悬液样品，4℃放置等待上机检测。
注：孵育缓冲液：含2％FBS的PBS，存储在4℃。
图4在293F转染VRC01质粒后，通过流式细胞术检测抗体各部分的存在，图4表明在转染了VRC01抗体质粒的293F细胞中，有10％以上的293F细胞存在抗体。
2.4转染后的293F抗体蛋白表达验证
1.考马斯亮蓝染色
对转染后的293F细胞上清样品进行SDS-PAGE跑胶。10％SDS-PAGE蛋白电泳配方如表1、表2所示：
表1 10％分离胶配方
表2浓缩胶配方
(1)将239F上清与蛋白上样缓冲液5×loading buffer按比例混合，来制备蛋白样品；
(2)将制备好的蛋白样品进行电泳上样操作，上样完成后连接电源，初始设置成80V恒压，待样品条带压至浓缩胶与分离胶交界线后将电压更换为120V恒压；
(3)电泳结束后将SDS-PAGE胶取下，放在去离子水中煮沸，倒掉更换新的去离子水，再煮沸1次；
(4)将煮好的SDS-PAGE胶放置在考马斯亮蓝染色液中，室温下摇晃染色约30min；
(5)弃去染色液，更换脱色液，室温下用摇床摇晃对胶进行脱色。
2.蛋白免疫印记(Western Blot)
(1)取15μL蛋白样品，依次加入到SDS－PAGE凝胶上样孔中，80V恒压电泳；
(2)待蛋白跑至浓缩胶和分离胶界面以下时，将电压调至120V恒压，继续电泳直至蛋白完全分离，关闭电源；
(3)将PVF膜在甲醇中浸泡2min；
(4)从电泳板上取下凝胶，按照负极-海绵-滤纸-凝胶-PVF膜－滤纸－海绵－正极的顺序放置在三明治转膜装置中，调节电流为100mA恒流，转膜时间为60min，(在转膜过程中需要将转膜装置放在冰水中进行降温)；
(5)转膜结束后，取出PVF膜放进洗膜盒中，加入5mL TBST缓冲液，在摇床上摇晃洗膜3次，每次5min；
(6)洗膜结束后，去除TBST缓冲液，加入5％脱脂奶粉稀释液，室温下封闭90min，封闭结束后，再次用TBST缓冲液洗膜3次，每次5min；
(7)将PVF膜放入按比例稀释好的抗人IgG抗体稀释液中，室温下孵育60min，抗体孵育结束后，用TBST缓冲液洗膜3次，每次5min；
(8)将化学发光底物A液与B液按1:1等体积预混，避光反应1-2min后均匀滴加在PVF膜上，于凝胶成像仪上曝光。
图5表明在293F转染VRC01抗体质粒后，成功表达了VRC01抗体，并将抗体分泌到了细胞上清中。
实施例3、检测鉴定嵌合抗HIV广谱中和抗体外泌体
图6是嵌合抗HIV广谱中和抗体外泌体的简易示意图，包括外泌体的各个组分及所携带的抗体分子。然后通过透射电子显微镜及NTA(粒子示踪技术)来对分离纯化到的外泌体进行表型鉴定。再利用Western Blot检测外泌体标记蛋白和抗体蛋白，最后通过外泌体流式试剂盒检测外泌体上抗体的存在。
图7是通过透射电镜和NTA分析外泌体，其形态和直径均呈现外泌体经典形态特征和直径大小；图8结果显示所获得的外泌体存在标记性蛋白CD63、CD81、CD9、TSG101，此外这些外泌体上还带有VRC01抗体蛋白；图9结果表明外泌体中有5％左右的外泌体携带VRC01抗体蛋白。
实施例4、生物层干涉技术(BLI)和中和实验检测嵌合抗HIV广谱中和抗体外泌体的中和活力
4.1生物层干涉技术(BLI)测定嵌合抗HIV广谱中和抗体外泌体的中和活力
为了测试嵌合抗HIV广谱中和抗体外泌体与HIVgp120结合的亲和力，我们通过生物层干涉技术(BLI)，利用Octet QK2相互作用仪进行了动力学实验。将嵌合抗HIV广谱中和抗体外泌体固定在生物传感器上，选定800nM浓度的07BCgp120蛋白稀释液，分别与固定在传感器上的嵌合抗HIV广谱中和抗体外泌体进行结合和解离。结合常数(ka)与解离常数(kd)的比率决定了此处报告的KD值。结果表明，VRC01 Exo与VRC01抗体一样，对HIV-1具有结合亲和力，KD值都小于10-8M(100nM)。
图10的结果表明嵌合抗HIV广谱中和抗体外泌体可以07BC-gp120结合，也就表明嵌合抗HIV广谱中和抗体外泌体对于HIV具有中和活力。
4.2外泌体体外中和实验测定嵌合抗HIV广谱中和抗体外泌体的中和活力
(1)-80℃冰箱保存的假病毒上清取出置于4℃备用，配制10％FBS的DMEM；配制10％FBS的1640生长培养基；
(2)用DMEM(10％FBS)将NL-4TAA(150000TCID50)稀释到800TCID50，每孔加入20μL到96孔板中；
(3)将VRC01抗体，VRC01 Exo及空白Exo分别加入到96孔板，置于细胞培养箱中(37℃，5％CO2)与NL-4TAA孵育1h；
(4)孵育1h后，向96孔板中每孔加100uL 10％FBS的1640生长培养基(含C8166细胞数量为4×104个)；
(5)将96孔板放在细胞培养箱中(37℃，5％CO2)培养48h；
(6)直接在荧光倒置显微镜下观察NL-4TAA感染C8166的荧光，拍细胞和荧光2个视野。
图11是通过嵌合抗HIV广谱中和抗体外泌体与NL-4TAA进行体外中和实验，利用病毒感染细胞表达的EGFP荧光蛋白表达情况评判嵌合抗HIV广谱中和抗体外泌体对HIV的抗感染作用，根据结果显示，嵌合抗HIV广谱中和抗体外泌体具有抗HIV感染的作用。
Claims (4)
1.一种嵌合外泌体，其特征在于，通过利用293F表达抗HIV-1广谱中和抗体VRC01,在产生并分泌抗体的时候，同时产生嵌合抗HIV广谱中和抗体外泌体，收集分离纯化外泌体获得。
2.根据权利要求1所述的利用293F表达抗HIV-1广谱中和抗体VRC01并生产嵌合抗HIV广谱中和抗体外泌体的方法，其特征在于来源细胞293F的构建方法，包括以下步骤：
(1)细胞复苏；
(2)细胞计数；
(3)293F细胞传代；
(4)细胞转染：通过将聚乙烯亚胺PEI(1mg/mL)和表达质粒瞬时转染到293F细胞中来表达VRC01抗体并生产嵌合抗HIV广谱中和抗体外泌体。
3.根据权利要求1所述的收集分离纯化外泌体方法，其特征在于，具体包括以下步骤：
(1)收集293F细胞培养基上清：收集至少200mL
(2)超高速离心分离外泌体：
a将上述收集的细胞培养上清，4℃，300×g离心5min，去掉沉淀；
b将a中的上清在4℃，3000×g离心30min，去掉沉淀；
c将b中的上清在4℃，10000×g离心60min，去掉沉淀；
d将c中的上清在4℃，100000×g离心90min，留沉淀；
e用预冷的无菌PBS重悬沉淀，将外泌体洗涤1遍，100000×g离心90min，留沉淀；
f用预冷的无菌适量PBS重悬沉淀，即得到外泌体悬液，-80℃保存。
4.一种如权利要求1所述的嵌合抗HIV广谱中和抗体外泌体在抗HIV感染中的应用。
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