CN114957405B - 一种抗蛋白酶降解的口服吸收蛋白及其制备方法以及应用 - Google Patents
一种抗蛋白酶降解的口服吸收蛋白及其制备方法以及应用 Download PDFInfo
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Abstract
本申请涉及生物技术的领域，尤其是涉及一种抗蛋白酶降解的口服吸收蛋白及其制备方法以及应用。抗蛋白酶降解的口服吸收蛋白，所述蛋白保留有FcRn亲和力且具有蛋白酶抗性，所述蛋白的氨基酸序列如SEQ ID NO：1所示，和/或SEQ ID NO：2，和/或SEQ ID NO：3，和/或SEQ ID NO：4，和/或SEQ ID NO：5。本申请制得的口服蛋白保留有FcRn亲和力且具有蛋白酶抗性，可以口服使用，也可以注射或吸入使用。
Description
技术领域
本申请涉及生物技术的领域，尤其是涉及一种抗蛋白酶降解的口服吸收蛋白及其制备方法以及应用。
背景技术
肽和蛋白质在糖尿病、心血管疾病、自身免疫和癌症等疾病中的治疗用途受到高度重视，具有大分子结构且亲水，保持蛋白质的空间组成或三级构型对于它们的药理活性十分必要。
肽和蛋白质的给药途径有多种，例如注射(皮下、静脉内和肌肉内)、口服、鼻腔、肺部、直肠和眼部途径。由于安全性和易用性，口服给药途径被患者更好地接受。然而，由于蛋白质的物理化学性质和大分子结构以及胃内酸性环境下胃肠酶的存在，口服给药通常会发生蛋白质和肽经历酶降解、三级结构改变和低吸收的现象。
口服给药是最广泛使用的处方药途径之一，因为它在大多数患者中具有更高的依从性和更少的副作用。尽管蛋白质药物的口服生物利用度有限，但蛋白质的口服给药仍然是传统肠外蛋白质给药的一种有趣且有吸引力的替代方案，因为它是一种无痛且更容易的给药途径。
全身口服剂量需要要求在胃肠道(GI)中溶解并且稳定，包括胃的酸性环境(禁食状态下的pH值1-2，进食状态下的pH值3-7)，接近中性环境的小肠的pH值4.4–6.6)。大多数化合物在小肠的第一部分(称为十二指肠)被专门的上皮细胞(肠上皮细胞)吸收。吸收可分为三类，细胞旁(肠细胞之间)、跨细胞(通过肠细胞的被动扩散)和主动转运(利用转运蛋白)。细胞旁吸收仅限于分子量(MW)<350Da和低亲脂性的小亲水化合物，可通过肠细胞之间紧密连接的限制。已知通过天然转运蛋白识别，少部分药物会发生主动摄取。
跨细胞转运是药物吸收最常见的途径，需要通过顶膜被动扩散，面向胃肠道，进入肠细胞，然后扩散穿过细胞并通过基底外侧膜进入血液。肠细胞具有许多外排转运蛋白，例如P-糖蛋白(Pgp)主动将化合物从血液转运到肠细胞进入胃肠道。此外，将药物靶向上皮细胞中表达的受体，例如FcRn，可能会因此增强通过粘膜屏障进行摄取和转运。
在整个吸收过程中，胃肠道、肠细胞、血液和淋巴中的酶可能将药物代谢为活性较低且渗透性较低的物质。因此，目前亟需开发一种抗蛋白酶降解的口服蛋白。
发明内容
为了提高口服蛋白的抗蛋白酶降解性，本申请提供一种抗蛋白酶降解的口服吸收蛋白及其制备方法以及应用。
第一方面，本申请提供一种抗蛋白酶降解的口服吸收蛋白，采用如下技术方案：一种抗蛋白酶降解的口服吸收蛋白，所述蛋白保留有FcRn亲和力且具有蛋白酶抗性。
可选的，所述蛋白的氨基酸序列如SEQ ID NO：1所示，和/或SEQ ID NO：2，和/或SEQ ID NO：3，和/或SEQ ID NO：4，和/或SEQ ID NO：5。
可选的，所述蛋白的氨基酸序列如SEQ ID NO：1所示。
可选的，所述蛋白的氨基酸序列如SEQ ID NO：2所示。
可选的，所述蛋白的氨基酸序列如SEQ ID NO：3所示。
可选的，所述蛋白的氨基酸序列如SEQ ID NO：4所示。
可选的，所述蛋白的氨基酸序列如SEQ ID NO：5所示。
第二方面，本申请提供编码上述抗蛋白酶降解的口服吸收蛋白的核酸。
第三方面，本申请提供上述抗蛋白酶降解的口服吸收蛋白的制备方法。
抗蛋白酶降解的口服吸收蛋白的制备方法，包括如下步骤：
FcRn亲和蛋白G8通过易错PCR突变并随机筛选，构建重组表达体；
构建所述口服吸收蛋白表达的基因工程菌；
纯化所述口服吸收蛋白。
可选的，构建所述口服吸收蛋白表达的基因工程菌的方法包括如下步骤：
所述口服蛋白的重组载体转化感受态细胞；
培养菌液；
进行降温并诱导、离心收集菌体。
可选的，所述口服吸收蛋白的纯化，包括如下步骤：
将基因工程菌进行重悬、菌体破碎、离心后收集上清液，对所述上清液进行纯化、洗脱后得到目标蛋白。
可选的，所述步骤还包括二次纯化。
可选的，采用随机突变试剂盒获得目的基因。
第四方面，本申请提供上述抗蛋白酶降解的口服吸收蛋白在制备糖尿病、心血管疾病、自身免疫和癌症等疾病中的口服药物上的应用。
综上所述，本申请包括以下至少一种有益技术效果：
1.本申请提供的抗蛋白酶降解的口服吸收蛋白，保留有FcRn亲和力且具有蛋白酶抗性。
2.本申请提供的抗蛋白酶降解的口服吸收蛋白不仅可应用于口服药物的开发，同样也可以应用于注射药物或吸入药物的开发。
3.本申请提供的口服吸收蛋白应用于口服药物，经胃肠道、肠细胞、血液和淋巴中的酶代谢后，仍能保持较高的活性以及渗透性。
附图说明
图1是本申请随机突变的流程示意图。
图2是本申请蛋白纯化图。
图3是本申请蛋白凝胶电泳图，其中，泳道1，流穿；泳道2，菌液；泳道3-8，分别为不同浓度咪唑梯度洗脱液；泳道9-11，第二次洗脱的目的条带。
图4是本申请氨基酸序列比对图。
图5是本申请氨基酸保守性分析图。
图6是BLI原理图。
图7是蛋白Gator结合图。
图8是本申请突变型蛋白在不同条件下的结合图。
图9是本申请突变型蛋白在不同pH条件下的结合。
图10是本申请突变型蛋白凝胶电泳胃蛋白酶消化图。
具体实施方式
以下结合附图1-10对本申请作进一步详细说明，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例
本申请实施例公开了一种抗蛋白酶降解的口服吸收蛋白，具体的，参照图1，本申请在FcRn亲和蛋白G8的基础上通过易错PCR突变并随机筛选，得到保留FcRn亲和力且有胃蛋白酶抗性的蛋白。具体的，本申请突变质粒由金斯瑞合成。基于FcRn亲和蛋白G8，通过随机突变试剂盒得到目的基因，并对目的基因扩增，通过易错PCR获得PCR产物后重组合成突变质粒。
抗蛋白酶降解的口服吸收蛋白的氨基酸序列如SEQ ID NO：1、SEQ ID NO：2、SEQID NO：3、SEQ ID NO：4、SEQ ID NO：5所示。其中，
SEQ ID NO：1对应c4蛋白氨基酸序列；
SEQ ID NO：2对应b10蛋白氨基酸序列；
SEQ ID NO：3对应d11蛋白氨基酸序列；
SEQ ID NO：4对应d13蛋白氨基酸序列；
SEQ ID NO：5对应d14蛋白氨基酸序列。
本申请还公开了一种编码上述蛋白的核酸，本领域人员公知的是，核酸通常是RNA或DNA。核酸可以是单链或双链的。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。当其连入载体时优选采用DNA核酸。核酸可以经过密码子优化以在期望的宿主细胞中获得更为高效的表达。
本申请实施例提供的抗蛋白酶降解的口服吸收蛋白的制备方法：
1、诱导表达蛋白
1.1将金斯瑞合成的突变质粒进行转化BL21(DE3)，并培养菌液，以1：500的比例扩增到500ml上述液体LB培养基中，37℃，250rpm，扩增至OD600为0.5左右，降温降速并加入0.03mMIPTG，18℃，250rpm，过夜培养16小时；
1.2在6000g，4℃的条件下，离心30分钟收集细菌沉淀。
2、裂解细胞
2.1称量菌块的质量，每0.5L菌液约有2.5mg湿菌块，按照每毫克菌重加10ml裂解液，1mg/ml溶菌酶，100μl100mMPMSF，10μl1MDTT，600UBenz-Neburas，4℃混合均匀，冰上缓慢搅拌孵育30min；
2.2置于冰上超声波细胞粉碎机以3son，7soff的频率超声20min；
2.3在10000rpm、4℃的条件下离心15min，得到细胞沉淀。
3、包涵体蛋白的变性
3.1将细胞沉淀完全重悬在10ml变性缓冲液E中；
3.2将裂解物在室温下孵育30分钟，翻转；
3.3在室温下以转速为10000g离心裂解物30分钟；
3.4收集上清液，并梯度透析复性。
4、融合蛋白质的纯化
4.1稳定AKATA仪器后，将HP预装柱连接到AKATA仪器，使用1ml/min的流速，用结合缓冲液洗涤纯化柱10个柱体积；
4.2将20ml上清以1ml/min流速流过纯化柱；
4.3以1ml/min流速，用结合缓冲液洗涤纯化柱5个柱体积或至流穿物中没有可见的蛋白杂质峰；
4.4洗脱液梯度洗脱，收集洗脱峰，SDS-PAGE电泳鉴定；
4.5以上步骤A液平衡，B液洗脱，收集到的蛋白再C液D液循环一次；
4.6 SDS-PAGE电泳鉴定，测蛋白浓度，冻存于-80℃备用。
检测结果分析
一、纯化结果分析
参见图2和图3，结果显示，经过第一次纯化仍有许多杂质，还需第二次纯化进行蛋白富集。其回收量较少。得到的第二次纯化胶图杂蛋白明显比第一次少很多。目的蛋白分子量为31kDa。
参照图4，将筛选出的十五个蛋白氨基酸序列通过SnapGene软件分析得到以下突变点数据，分析并对比结果。结果显示其氨基酸突变点随机分布在各CDR和FR区。其圈出的部位为CDR区域，未圈出的部分则为FR区，且(G4S)3为连接链(Linker)。
参照图5，通过SnapGene软件评估每个氨基酸的保守性。结果显示，无论是在CDR区还是在FR区，其突变点都是均匀分布的，没有特殊的偏好性。也进一步验证了突变的随机性结果。
二、蛋白的结合活性分析
参照图6，本申请使用的GATOR仪器基于BLI原理，主要描述了一种使用基于链霉亲和素的BLI分析突变型蛋白质与FcRn相互作用的有效方法，同时也使用Fc探针测定了突变型蛋白与GLP-1r的结合能力，从该仪器上可以直接读取结合亲和力、结合率和解离率。
1、实验器材与材料
表1.1实验器材
| 名称 | 厂家 |
| Gator非标记生物分析仪 | Gatorbio，美国 |
| IMS-100制冰机 | 常熟市雪科电器有限公司，中国 |
| S10-3型数显恒温磁力搅拌器 | 上海司乐仪器有限公司，中国 |
| 优普ΜLUP超纯水器 | 四川超纯优普科技有限公司，中国 |
| BS2000S电子天平 | Sartorius，德国 |
| ORION3-Star精密台式pH计 | Thermo Fisher Scientific，美国 |
表1.2实验材料
2.实验方法与步骤
2.1溶液配制
(1)Q Buffer配制：
量取25mL20xPBS于干净的500ml容量瓶，用超纯水定容至刻度，随后转移到一个干净的500ml瓶子，加入称取的1gBSA，1ml 10％(w/v)Tween-20，磁力搅拌器充分搅拌均匀，过0.22μm滤膜，放于4℃冰箱内备用。
(2)K Buffer配制：
量取50mlQBuffer和25ml20xPBS于干净的0.5L容量瓶，用超纯水定容至刻度，随后转移到一个干净的0.5L瓶子，搅拌均匀，过0.22μm滤膜，放于4℃冰箱内备用。
(3)Regeneration Buffer配制：
称取600mg甘氨酸和8.77g氯化钠于干净的1L烧杯中，加入800ml超纯水，搅拌溶解，并用盐酸调节pH为2，随后转移至干净的1L容量瓶中，用超纯水定容至刻度，再过0.22μm滤膜，放于4℃冰箱内备用。
以上溶液均为一月一换，且实验温度应为室温下进行。
2.2抗原抗体结合的初步表征
将生物素化的FcRn抗原稀释到Q buffer溶液中，使终浓度为5μg/ml；
i.统一配制蛋白浓度为20μg/ml，250μl备用；
ii.96孔板中加样200μl，每列按照Q buffer,Loading buffer,Q buffer,sample,Q buffer的顺序进行加样；
iii.SA探针置于MAX板中，MAX板需要提前加入250μl Q buffer预湿；
iv.将两块板分别安置在仪器的正确位置上，并进行参数设置；反应温度37℃，转速为1000rpm；采用以下步骤对数据采集软件进行编程：
a)基线：120s
b)加载：120s(设置loading高度为1)
c)基线：120s
d)结合：300s
e)解离：300s
f)再生：15s(SA探针不可再生，此步骤应用于Fc探针)
g)中和：30s
v.反应结束后，回收样品。并进行动力学参数结果计算。
3.实验结果
经典的BLI分析步骤包括五个:1、平衡；2、配体固定；3、再平衡；4、分析物结合；5、解离。结果参照图7。
3.1亲和力分析
表3.1统计了蛋白分别在pH 7.4和pH 6.0条件下的KD值。从结合数据中筛选出亲和性较高的蛋白进行胃蛋白酶消化实验以及建模分析验证。
选取pH7.4的结合强度低于pH6.0条件并且亲和力高于G8蛋白的六个突变型蛋白：c4，b2，b10，d11，d13，d14。
表3.1蛋白在pH7.4和pH6.0条件下的KD值
| Protein | pH 6.0 | pH 7.4 |
| G8 | 69.7 | NA |
| c1 | 102.00 | 12.40 |
| c2 | NA | 2.94 |
| c3 | NA | 8.55 |
| c4 | 58.30 | NA |
| c5 | 165.00 | 9.81 |
| c6 | 128.00 | NA |
| b2 | 28.10 | 221.00 |
| b10 | 32.3 | 551.00 |
| d9 | NA | NA |
| d10 | NA | 48.70 |
| d11 | 25 | 474 |
| d12 | NA | 243.00 |
| d13 | 88.80 | 1210.00 |
| d14 | 29.20 | 52.70 |
| d21 | NA | NA |
| d23 | NA | 55.50 |
测序结果显示，b2大部分氨基酸被替换且缺失，不适用于下一步研究。表3.2统计了以上五个突变型蛋白(c4，b10，d11，d13，d14)分别在轻链和重链位置的突变位点。
表3.2五个蛋白突变点分析
所筛选出的scFv蛋白与FcRn在pH 6.0条件下高强度结合，在pH 7.4条件下也有结合，但是结合高度比pH 6.0低。基于BLI原理的Gator实验仪器拟合数据的特殊性，尽管在结合高度并且有趋势的情况下也能进行数据分析，并拟合出具体的亲和力数值。如下表所示：
表3.3 pH 6.0条件下的结合
| Protein | koff(1/s) | kon(1/Ms) | KD(nM) |
| G8 | 0.0051 | 7.32E+04 | 69.7 |
| c4 | 0.00485 | 3.46E+04 | 140 |
| b10 | 0.00969 | 3.00E+05 | 32.3 |
| d11 | 0.00121 | 4.76E+04 | 25 |
| d13 | 0.00486 | 3.91E+04 | 124 |
| d14 | 0.00456 | 4.68E+04 | 97.6 |
表3.4 pH 7.4条件下的结合
| Protein | koff(1/s) | kon(1/Ms) | KD(nM) |
| G8 | 0.0786 | NA | NA |
| c4 | NA | NA | NA |
| b10 | 0.000648 | 2.28E+04 | 28.4 |
| d11 | 0.00842 | 1.78E+04 | 474 |
| d13 | 0.00571 | NA | NA |
| d14 | 0.000831 | 1.58E+04 | 52.7 |
表4.7与GLP-1r的结合
| Protein | koff(1/s) | kon(1/Ms) | KD(nM) |
| G8 | 0.00819 | 1.60E+05 | 51.1 |
| c4 | 0.0494 | 6.33E+04 | 781 |
| b10 | 0.0182 | 2.01E+04 | 905 |
| d11 | 0.00209 | 2.95E+04 | 70.9 |
| d13 | 0.0129 | 3.90E+04 | 330 |
| d14 | 0.00343 | 8.03E+04 | 42.7 |
需要说明的是，本申请所有数据均由GraphPad Prism 8进行分析。GraphPadPrism 8软件描绘了最佳拟合线，参照图8，可更直观的观察到同一浓度下，不同突变蛋白在相同pH条件下的结合差别。其中各蛋白与GLP-1r的结合无明显差异。
参照图9，每个蛋白在不同pH溶液中的结合，可以观察到，在同一浓度下，pH6的结合高度如预期一样均比pH7.4条件的高。
三、蛋白酶降解实验
1.1胃蛋白酶耐受实验
反应条件：pH 2-3，每1μg蛋白分别用32U的3，6，9倍pepsin(比活力：10000U/mg；浓度：2.5μg/ml)降解，分别在0.5h，1h两个时间点取出并将pH调至中性以终止反应，蛋白凝胶电泳观察条带分解状况。统一上样量为5μg，即第一泳道为对照组(未经胃蛋白酶处理)。
1.2胃蛋白酶抗性
参照图10，胃蛋白酶(pepsin)处理后，五个蛋白表现出比原来的蛋白更具耐受性。G8突变前胃蛋白酶的耐受性实验可明显看出其被迅速分解，并且在1h在目标条带处已经看不到明显条带，可以分析在最后一个浓度条件下几乎被完全降解。而筛选剩下的几个蛋白均有保留，并未完全被降解。
然而突变型蛋白d14在做后续pepsin耐受实验时出现蛋白聚集现象，从而无法进行分析。原因可能是因为长时间放置导致蛋白自身的不稳定性导致其产生二聚体甚至是多聚体等。
综合以上的主要实验研究，初步拟定突变型蛋白d11为筛选得到的最终蛋白。G8蛋白的突变蛋白G8(d11)结构稳定，与靶点具有更高的结合活性，并具有对胃蛋白酶的耐受能力，可以作为G8蛋白口服给药的候选分子进行进一步研究。
应用例
本申请实施例提供的抗蛋白酶降解的口服吸收蛋白在制备糖尿病、心血管疾病、自身免疫和癌症等疾病中的口服药物上的应用。当然，本申请提供的抗蛋白酶降解的口服吸收蛋白不仅可应用于口服药物的开发，同样也可以应用于注射药物或吸入药物的开发。
以上均为本申请的较佳实施例，并非依此限制本申请的保护范围，故：凡依本申请的结构、形状、原理所做的等效变化，均应涵盖于本申请的保护范围之内。
序列表
<110> 杭州高田生物医药有限公司
<120> 一种抗蛋白酶降解的口服吸收蛋白及其制备方法以及应用
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Claims (4)
1.一种抗蛋白酶降解的口服吸收蛋白，其特征在于：所述蛋白保留有FcRn亲和力且具有蛋白酶抗性；所述蛋白的氨基酸序列如SEQ ID NO：3所示。
2.编码权利要求1所述的抗蛋白酶降解的口服吸收蛋白的核酸。
3.权利要求1所述的抗蛋白酶降解的口服吸收蛋白的制备方法，包括如下步骤：构建重组表达体，构建所述口服吸收蛋白表达的基因工程菌，纯化所述口服吸收蛋白。
4.权利要求1所述的抗蛋白酶降解的口服吸收蛋白在制备糖尿病、心血管疾病、自身免疫和癌症等疾病中的口服药物上的应用。
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| CN111484558A (zh) * | 2019-01-28 | 2020-08-04 | 上海交通大学 | 信号调节蛋白α片段-抗FcRn单链抗体融合蛋白及其制备与应用 |
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