CN116640227B - 一种新型、长效且高活性的促卵泡激素融合蛋白 - Google Patents
一种新型、长效且高活性的促卵泡激素融合蛋白 Download PDFInfo
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Abstract
本发明公开一种新型、长效且高活性的促卵泡激素融合蛋白，其结构式为Y‑L‑α:β和α:β‑L‑Y，式中，“:”为非共价键，表示α亚基与β亚基之间通过非共价键相互作用；“‑”为肽键；“α”为人促卵泡激素α亚基(FSHα)，其氨基酸包括SEQ ID NO:1或其至少一部分；“β”为人促卵泡激素β亚基(FSHβ)，其氨基酸包括SEQ ID NO:2或其至少一部分；“L”为连接肽；“Y”为单链Fc(sFc)，其氨基酸序列包括在SEQ ID NO:3中或上述任一序列的至少一部分；“β‑L‑Y”表示单链Fc通过连接肽共价链接到FSHβ氨基酸序列的N端或C端；“Y‑L‑α”表示单链Fc通过连接肽共价链接到FSHα氨基酸序列的N端；本发明同现有技术相比，不但保留了其FcRn结合能力，还可以降低融合蛋白分子量，增加组织渗透能力，维持被融合蛋白活性。
Description
技术领域
本发明涉及分子生物学及药物技术领域，具体来说是一种新型、长效且高活性的促卵泡激素融合蛋白。
背景技术
促卵泡激素(follicle stimulating hormone,FSH)是一种促进女性卵泡发育和成熟、男性精子正常发生的重要糖蛋白激素，常用FSH药物半衰期相对较短，体内代谢和消除迅速，患者一般需要每天1次或2次皮下注射才能维持FSH血药浓度在卵巢刺激的阈值水平以上，长效FSH的开发有利于增强生物活性和患者的依从性，减缓患者的精神压力和用药不当。
目前用于长效FSH分子设计的技术手段包括在FSH亚基末端融合绒毛膜促性腺激素β亚基的羧端肽(CTP)(B.C.J.M.Fauser等人，Human Reproduction Update,Vol.15,No.3pp.309–321,2009),或引进新的糖基化位点序列(Gross AW等人，J Biol Chem,2006,281:2024-2032)，或创建由CTP或糖基化连接序列连接而成的FSHβ和α亚基单链融合蛋白(Klein J等人，Fertil Steril,2002,77:1248-1255)，或创建FSH与IgG1的Fc片段的融合蛋白等(S.C.Low等人，Human Reproduction Vol.20,No.7pp.1805–1813,2005；Yin-Li Zhang等人，Human Reproduction,Vol.31,No.1pp.169–182,2016)。
调研发现，长效促卵泡激素目前主要有以下几种专利：CTP融合蛋白形式(US5338835A，US5585345A，CN109942717A)，Fc融合蛋白形式(CN1926237A，US2005186662A1，CN103509121A，US2016060321A1，CN106496331A)，CTP与Fc双重融合蛋白形式(CN103539860 B)等。
已有研究发展了使用免疫球蛋白Fc制备融合蛋白，以增加融合蛋白的半衰期(Cheol Ryong Ku等人，Eur J Endocrinol.2018Sep；179(3):169-179；Wolfgang Glaesner等人，Diabetes Metab Res Rev.2010May；26(4):287-96.)。其中上述Fc融合蛋白形式所用Fc均为双链形式，一方面该形式会明显增加被融合蛋白药物的分子量；另一方面二聚体形式的融合蛋白可能会互相干扰影响药效。由于促卵泡激素生物学活性的发挥需要一定的空间效应，因此融合蛋白中Fc的连接不当会造成空间位阻效应。
本发明创新性的采用sFc(单链Fc)融合蛋白形式(WO2019/080858A1)，sFc为针对IgG1恒定区改造后的新型Fc单体，通过抗体工程技术对人抗体IgG1恒定区Fc进行改造，使其二聚体变为Fc单体，且维持FcRn结合功能，具有极低的无关蛋白非特异性结合特性。利用此单体与hGH融合，一方面可以使延长hGH半衰期，另一方面可以采用Protein A进行纯化。
常规同源二聚形式的Fc其本身分子量约有60kDa，且被融合蛋白只能是二聚体形式，这使得融合蛋白的分子量往往会增加数倍，同时二聚形式的药物分子之间可能会互相干扰而影响药效。本发明相较于常规同源二聚形式的Fc不但保留了其FcRn结合能力，还可以降低融合蛋白分子量，增加组织渗透能力，维持被融合蛋白活性。同时本发明在增强FSH的体内半衰期的同时，将α亚基游离或使其C端暴露出来，最大程度的保留FSH的活性。
发明内容
本发明的目的在于解决现有技术的不足，提供一种新型、长效且高活性的促卵泡激素融合蛋白。
为了实现上述目的，设计一种新型、长效且高活性的促卵泡激素融合蛋白，其结构式为：Y-L-α:β和α:β-L-Y；
式中，“:”为非共价键，表示α亚基与β亚基之间通过非共价键相互作用；“-”为肽键；
“α”为人促卵泡激素α亚基(FSHα)，其氨基酸包括SEQ ID NO:1或其至少一部分；
“β”为人促卵泡激素β亚基(FSHβ)，其氨基酸包括SEQ ID NO:2或其至少一部分；
“L”为连接肽；
“Y”为单链Fc(sFc)，其氨基酸序列包括在SEQ ID NO:3中或上述任一序列的至少一部分；
“β-L-Y”表示单链Fc通过连接肽共价链接到FSHβ氨基酸序列的N端或C端；
“Y-L-α”表示单链Fc通过连接肽共价链接到FSHα氨基酸序列的N端。
本发明还包括如下优选的技术方案：
进一步，所述连接肽含有1个或更多个选自甘氨酸、丝氨酸、丙氨酸和苏氨酸的氨基酸。
进一步，所述连接肽氨基酸序列包括在SEQ ID NO:4、SEQ ID NO:5、SEQ IDNO:6、SEQ ID NO:7、SEQ ID NO:8中或上述任一序列的至少一部分。
进一步，Y-L-α:β和α:β-L-Y组合形式氨基酸序列包括在SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14中或上述任一序列的至少一部分。
进一步，DNA分子选自以下序列：SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28或其密码子优化序列。
进一步，所述表达载体含有所述的DNA分子。
进一步，所述宿主细胞含有所述的表达载体，或者所述的宿主细胞的基因组中整合有所述的DNA分子。
进一步，所述宿主细胞包括：原核细胞或真核细胞。
进一步，所述宿主细胞选自：大肠杆菌、酵母、CHO细胞、BHK、HEK293。
进一步，所述融合蛋白的生产方法如下:
S1.培养所述的宿主细胞，获得含融合蛋白的培养液；
S2.从培养液中分离出所述融合蛋白。
进一步，所述药物制剂含有所述的融合蛋白和制剂。
进一步，所述制剂为注射剂、冻干粉针剂或预充针注射剂。
本发明同现有技术相比，其优点在于：创新性的采用sFc(单链Fc)融合蛋白形式(WO2019/080858A1)，不但保留了其FcRn结合能力，还可以降低融合蛋白分子量，增加组织渗透能力，维持被融合蛋白活性；同时本发明在增强FSH的体内半衰期的同时，将α亚基游离或使其C端暴露出来，最大程度的保留FSH的活性。
附图说明
图1为本发明的重组长效人促卵泡激素融合蛋白结构示意图。
图2为本发明的重组长效人促卵泡激素融合蛋白与HEK293-FSHR细胞表面受体FSHR的亲和力检测图谱。
图3为本发明的重组长效人促卵泡激素融合蛋白用HEK293-FSHR-luciferase报告基因细胞株的活性检测图。
具体实施方式
参见图1，为本发明的重组长效人促卵泡激素融合蛋白结构示意图，融合蛋白用以下形式表示：Y-L-α:β和α:β-L-Y。
式中，“α”为人促卵泡激素α亚基(FSHα)，其氨基酸包括SEQ ID NO:1或其至少一部分；“β”为人促卵泡激素β亚基(FSHβ)，其氨基酸包括SEQ IDNO:2或其至少一部分；“Y”为单链Fc(sFc)，其氨基酸序列包括在SEQ ID NO:3中或上述任一序列的至少一部分；“β-L-Y”表示单链Fc通过连接肽共价链接到FSHβ氨基酸序列的N端或C端；“Y-L-α”表示单链Fc通过连接肽共价链接到FSHα氨基酸序列的N端。
通过非限制性实施例对本发明进行进一步说明，应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1制备重组长效人促卵泡激素融合蛋白表达载体。
人促卵泡激素序列来源于Uniprot ID P01215及Uniprot ID P01225，本项目分子所用sFc来自于第三方合法的专利技术(专利名称【一种IgG1 Fc单体及应用】，国际申请号PCT/CN2018/111577，国际公开号2019/080858A1)；本项目分子所用其它Fc为在天然IgG4或IgG1基础上进行氨基酸突变或替换所得；本项目所用linker为常用(GGGGS)n linker(其中n＝1、2、3或4)或DKTHTGP；对密码子进行优化，在人种属和宿主细胞易于表达方面进行优化，最终合成基因质粒，进行表达。
实施例2重组长效人促卵泡激素融合蛋白表达。
利用Expi CHO-S(Gibco,A29133)作为宿主细胞，采用化学转染试剂Polyplus-FectoPRO(polyplus，116-010)，瞬时表达重组长效人促卵泡激素融合蛋白。
实施例3重组长效人促卵泡激素融合蛋白纯化。
将得到的CHO细胞发酵液进行二级离心(一级：3,000×g,30min；二级：12,000×g,20min)，收集上清液经0.2μm滤器过滤，待用；Protein A亲和层析：将滤液上样到经20mMPB,150mM NaCl,pH 7.2预平衡的Protein A柱上，保留时间为5min。再平衡，并分别利用50mM NaAc-HAc,pH 4.5,50mM NaAc-HAc,pH 4.0和50mM NaAc-HAc,pH 3.5进行洗脱，收峰范围为50mAU-peak-50mAU,得到SEC纯度符合要求(＞90.0)的融合蛋白。
实施例4重组长效人促卵泡激素融合蛋白体外生物学活性测定。
参见图2，测试例1融合蛋白对受体结合试验。
将HEK293-FSHR细胞用1％BSA-PBS密度调整至1*106cells/mL,以100μL/孔加入U底96孔板。将重组长效人促卵泡激素融合蛋白稀释至200μg/mL，再4倍稀释10个梯度，100μL/孔加入U底96孔板中；4℃孵育1小时，离心后弃上清，每孔加入300μL的1％BSA-PBS，洗涤2次后加入1％BSA-PBS稀释200倍的R-Phycoerythrin AffiniPure Goat Anti-Human IgG,FcγFragment Specific，100μL/孔加样；室温避光孵育0.5小时后，离心后弃上清，每孔加入300μL的1％BSA-PBS，洗涤3次，加入1％BSA-PBS，每孔100μL，重悬细胞沉淀，流式检测荧光信号。
结果：
表1.重组长效人促卵泡激素融合蛋白对人FSHR结合活性
| Sample NO. | Sample name | EC50(nM) |
| 1 | SEQ ID NO:9 | 1.621 |
| 2 | SEQ ID NO:10 | 1.467 |
| 3 | SEQ ID NO:11 | 0.998 |
| 4 | SEQ ID NO:12 | 1.345 |
| 5 | SEQ ID NO:13 | 1.101 |
| 6 | SEQ ID NO:14 | 0.8870 |
| 对照分子 | 果纳芬 | 1.211 |
参见图3，测试例2重组长效人促卵泡激素融合蛋白和报告基因细胞株生物学活性实验。
收集报告基因细胞(甲贝医药自主构建)并以每毫升5*10^5个细胞的浓度悬浮在培养基(DMEM培养基，含10％FBS)。将每份50μL细胞样品加到96孔细胞培养板的各孔中，培养箱放置过夜。第二天用50μL含有4000ng/mL-0.183ng/mL各种浓度重组长效人促卵泡激素融合蛋白以及对照样品(果纳芬)的分析培养基培养这些细胞。在37℃，5％CO2湿润培养箱中培养该细胞板6小时，然后在各孔中添加100μL Nano-Glo Luciferase Assay(Promega，N1120)。10分钟后，酶标仪检测化学发光信号。由所得剂量反应曲线可测定重组长效人促卵泡激素融合蛋白的生物活性。
结果：
表2.重组长效人促卵泡激素融合蛋白活性(报告基因细胞株实验)
| Sample NO. | Sample name | 相对果纳芬活性 |
| 1 | SEQ ID NO:9 | 99％ |
| 2 | SEQ ID NO:10 | 101％ |
| 3 | SEQ ID NO:11 | 97％ |
| 4 | SEQ ID NO:12 | 93％ |
| 5 | SEQ ID NO:13 | 70％ |
| 6 | SEQ ID NO:14 | 95％ |
| 对照分子 | 果纳芬 | 100％ |
结果证明，本发明重组长效人促卵泡激素融合蛋白活性对照样品(果纳芬)相当。
以上所述，仅为此发明的具体实施方式，但本发明的保护范围不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案和新型的构思加于等同替换或改变，都应涵盖在本发明的保护范围之内。
Claims (11)
1.一种新型、长效且高活性的促卵泡激素融合蛋白，其特征在于结构式为：Y-L-α:β或α:β-L-Y；
式中，“:”为非共价键，表示α亚基与β亚基之间通过非共价键相互作用；“-”为肽键；
“α”为人促卵泡激素α亚基(FSHα)，其氨基酸序列为SEQ ID NO:1；“β”为人促卵泡激素β亚基(FSHβ)，其氨基酸序列为SEQ ID NO:2；“L”为连接肽；
“Y”为单链Fc(sFc)，其氨基酸序列为SEQ ID NO:3；
“β-L-Y”表示单链Fc通过连接肽共价链接到FSHβ氨基酸序列的N端或C端；
“Y-L-α”表示单链Fc通过连接肽共价链接到FSHα氨基酸序列的N端；所述连接肽的氨基酸序列为SEQ ID NO:6或SEQ ID NO:8。
2.一种编码如权利要求1所述融合蛋白的DNA分子，其特征在于DNA分子包含以下序列中的一条或多条：SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:20、SEQ ID NO:22或其密码子优化序列。
3.一种表达载体，其特征在于所述表达载体含有如权利要求2所述的DNA分子。
4.一种宿主细胞，其特征在于所述宿主细胞含有如权利要求3所述的表达载体，或者所述的宿主细胞的基因组中整合有如权利要求2所述的DNA分子。
5.如权利要求4所述的宿主细胞，其特征在于所述宿主细胞包括：原核细胞或真核细胞。
6.如权利要求4所述的宿主细胞，其特征在于所述宿主细胞选自：大肠杆菌、酵母、CHO细胞、BHK、HEK293。
7.一种如权利要求1所述融合蛋白的生产方法，其特征在于
S1.培养如权利要求4所述的宿主细胞，获得含融合蛋白的培养液；
S2.从培养液中分离出如权利要求1所述融合蛋白。
8.一种药物制剂，其特征在于所述药物制剂含有如权利要求1所述的融合蛋白。
9.如权利要求8所述的一种药物制剂，其特征在于所述药物制剂为注射剂。
10.如权利要求8所述的一种药物制剂，其特征在于所述药物制剂为冻干粉针剂。
11.如权利要求8所述的一种药物制剂，其特征在于所述药物制剂为预充针注射剂。
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