CN111424035A - 基于家蚕丝腺表达具有生物学活性的人结缔组织生长因子的方法及其产品和应用 - Google Patents
基于家蚕丝腺表达具有生物学活性的人结缔组织生长因子的方法及其产品和应用 Download PDFInfo
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Abstract
本发明公开了基于家蚕丝腺表达具有生物学活性的人结缔组织生长因子的方法及其产品和应用，具体方法是将编码人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因在家蚕丝腺表达，结茧后茧壳用尿素提取，纯化，得到具有生物学活性人结缔组织生长因子或N端含透皮肽的人结缔组织生长因子；该蛋白具有易于纯化，具有促进细胞增殖和透明质酸合成等生物学活性，该研究将加速CTGF作为功能性蛋白质药物在医学和医学美容领域中应用的进程，提供一种利用家蚕或其它生物反应器系统设计和生产高效的蛋白质药物的新策略。
Description
技术领域
本发明涉及生物医药领域，具体涉及基于家蚕丝腺表达具有生物学活性的人结缔组织生长因子的方法，还涉及由该方法制得的产品和应用。
背景技术
人结缔组织生长因子，也称为CTGF或CCN2，是一种富含半胱氨酸的分泌型蛋白质，含有349个氨基酸，分子量为34～38KD。CTGF蛋白由四个保守结构域组成，即胰岛素样生长因子结合结构域、chordin样半胱氨酸富集结构域、血小板反应蛋白1型结构域和C端胱氨酸结结构域，因此具有多种生物学功能，参与了血管、皮肤、心脏、肾脏、胰腺、肺和肝等多种慢性疾病的组织纤维化过程。例如，促进损伤后肉芽组织的形成、再上皮化以及基质形成和重塑；通过Ras/MEK/ERK MAPK信号通路促进血管生成中的内皮增殖、迁移、存活和粘附以及伤口愈合；增加成纤维细胞和衰老皮肤组织中细胞外基质蛋白的产生(例如I型胶原、纤连蛋白及其整联蛋白受体)。这些结果表明，CTGF在新组织器官和结构的形成过程中起着重要作用，可以作为功能性药物用于医学和医学美容领域，以帮助受损皮下组织的再生和结构重建，从而迅速恢复其生物学功能。
生物反应器是一种高效、快捷的生产各种有价值的蛋白质药物的重要方法。该方法可以在短时间内获得大量的活性靶蛋白药物，并且具有以下特点：低成本和低工业污染。目前，已经成功开发了大肠杆菌、酵母、中国仓鼠卵巢和其它转基因植物、昆虫和动物的多种生物反应器系统，并成功生产了激素、抗体、疫苗、酶和其它多种蛋白质药物。此前，利用生物反应器的方法也成功生产过CTGF蛋白，例如，大肠杆菌生物反应器系统，该重组CTGF蛋白在I(50)约8pM时显着抑制血清剥夺诱导的鸡胚成纤维细胞凋亡；通过转染人仓鼠卵巢CTGF基因的cDNA改良的中国仓鼠卵巢细胞生物反应器系统，其生产的38kDa CTGF蛋白被忠实地糖基化并进行了有限的蛋白水解，并在体外以及皮下有效地促进了细胞粘附、有丝分裂和上皮细胞转分化。
家蚕的蚕腺是一种理想的可以用于构建生物反应器的组织器官，因为在家蚕幼虫五龄末期该器官能够大量合成蚕丝蛋白。目前，基于家蚕丝腺已成功开发了多种生物反应器系统，包括家蚕后部丝腺FibL表达系统、Fhx表达系统和FibH表达系统，这些系统将靶标蛋白表达于丝纤维的内部丝素层；家蚕前中部丝腺Ser1表达系统和Ser3表达系统，这些系统将靶标蛋白表达于丝纤维的外部丝胶层。因为位于丝纤维外部丝胶层的靶标蛋白易于分离和纯化，因此，家蚕中部丝腺生物反应器得到了快速地发展与优化，目前最优的家蚕中部丝腺表达系统由一个在家蚕神经系统与眼部特异性表达红色荧光蛋白的3xp3-DsRed-SV40转录调控单元、一个来自家蚕核型多角体病毒的Hr3增强子、一个来源于家蚕内源丝胶蛋白1基因的Ser1-P启动子和Ser1PA终止子组成。迄今为止，该系统已经成功地生产了包括人酸性成纤维细胞生长因子、人碱性成纤维细胞生长因子、人血小板衍生生长因子、人乳铁蛋白和人转化生长因子在内的多种蛋白质药物，并且它们均具有生物学活性。
蛋白质药物的细胞跨膜活性与其生物学活性密切相关。因此，提高重组CTGF蛋白的细胞跨膜活性是增强其生物学活性、降低生产成本的重要方法。近年来，透皮肽被认为是一种能够有效地将外源蛋白质药物传递到活细胞内的肽段，包括HIV-1Tat蛋白肽、果蝇触角Antp蛋白肽、单纯疱疹病毒VP22蛋白肽和Pep-1。Pep-1是一种含有21个残基的透皮肽(KETWWETWWTEWSQPKKKRKRKV)，由具有丰富疏水性色氨酸的结构域、具有丰富亲水性赖氨酸的结构域和分隔以上两个结构域的间隔结构域组成。此前的研究已经证实，pep-1通过与蛋白质药物共混与耦合的方式能够将外源蛋白药物递送到人HS-68、鼠NIH/3T3成纤维细胞、Jurkat T和Cos细胞的内部。
发明内容
有鉴于此，本发明的目的在于提供一种家蚕丝腺表达具有生物学活性人结缔组织生长因子或N端含透皮肽的人结缔组织生长因子的方法；本发明的目的之二在于提供有生物学活性的重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子；本发明的目的之三在于提供重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子在制备促进细胞增殖的药物中的应用；本发明的目的之四在于提供重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子在制备促进ERK蛋白磷酸化的药物中的应用；本发明的目的之五在于提供重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子在制备促进透明质酸合成或/和羟脯氨酸合成的药物中的应用；本发明的目的之六在于提供适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因；本发明的目的之七在于提供含有所述适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因的重组表达载体；本发明的目的之八在于提供含有所述适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因的转基因家蚕。
为达到上述目的，本发明提供如下技术方案：
1、家蚕丝腺表达具有生物学活性人结缔组织生长因子或N端含透皮肽的人结缔组织生长因子的方法，优化人结缔组织生长因子基因密码子获得适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因，然后将改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因在家蚕丝腺表达，结茧后茧壳用尿素提取，纯化，得到具有生物学活性人结缔组织生长因子或N端含透皮肽的人结缔组织生长因子；所述改造人结缔组织长因子基因的核苷酸序列如SEQ ID NO.1所示；所述N端含透皮肽的人结缔组织生长因子基因的核苷酸序列如SEQ ID NO.3所示。
优选的，家蚕丝腺表达改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因的方法如下：构建含有所述适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因的转基因表达载体，将所述转基因表达载体注射已解除滞育的家蚕蚕卵，培育转基因家蚕即可。
优选的，所述转基因表达载体含有顺序连接的增强子hr3，分泌型丝胶1基因启动子、改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因、丝胶1基因的终止子序列。
更优选的，所述转基因表达载体由顺序连接的增强子hr3，分泌型丝胶1基因启动子、改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因、丝胶1基因的终止子序列通过AscI酶切后连入pBac[3×p3EGFPaf]载体的AscI位点而得。
更优选的，所述尿素提取是用将茧壳粉碎后用提取缓冲液萃取，离心收集总茧壳蛋白粗提物，所述提取缓冲液为pH7.0、含50mM NaCl、50mM Tris-HCl和8M尿素的溶液。
更优选的，所述纯化为将总茧壳蛋白粗提物加入咪唑至终浓度为5mM，然后上样到Ni-Sepharose FF柱，然后依次使用分别使用含有60mM、80mM与1000mM咪唑浓度的洗脱缓冲液洗涤，得到重组人结缔组织生长因子。
更优选的，所述洗脱缓冲液pH为7.4，还含有135mM NaCl、2.7mM KCl、1.5mMKH2PO4、8mM K2HPO4。
2、具有生物学活性的重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子，由如SEQ ID NO.1或SEQ ID NO.3所示的核苷酸序列于在家蚕丝腺表达，结茧后茧壳用尿素提取，纯化制得。
3、所述的重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子在制备促进细胞增殖的药物中的应用，所述重组人结缔组织生长因子的氨基酸序列如SEQ IDNO.2所示；所述N端含透皮肽的人结缔组织生长因子的氨基酸序列如SEQ ID NO.4所示。
4、所述的重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子在制备促进ERK蛋白磷酸化的药物中的应用，所述重组人结缔组织生长因子的氨基酸序列如SEQ ID NO.2所示；所述N端含透皮肽的人结缔组织生长因子的氨基酸序列如SEQ ID NO.4所示。
5、所述的重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子在制备促进透明质酸合成或/和羟脯氨酸合成的药物中的应用，所述重组人结缔组织生长因子的氨基酸序列如SEQ ID NO.2所示；所述N端含透皮肽的人结缔组织生长因子的氨基酸序列如SEQ ID NO.4所示。
6、适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因，所述改造人结缔组织生长因子基因的核苷酸序列如SEQ ID NO.1所示；所述N端含透皮肽的人结缔组织生长因子基因如SEQ ID NO.3所示。
7、含有所述适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因的重组表达载体。
8、含有所述适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因的转基因家蚕。
本发明的有益效果在于：本发明公开了家蚕丝腺表达具有生物学活性人结缔组织生长因子或N端含透皮肽的人结缔组织生长因子的方法，利用家蚕中部丝腺生物反应器系统生产透皮肽偶联的CTGF蛋白，该蛋白具有易于纯化、以及增强的促进细胞增殖和透明质酸合成的活性。该研究将加速CTGF作为功能性蛋白质药物在医学和医学美容领域中应用的进程，提供一种利用家蚕或其它生物反应器系统设计和生产高效的蛋白质药物的新策略。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为培育转基因家蚕(A：pR-CT8ht和pR-PCT8h转基因表达载体；表达载体中含有一个在家蚕眼部和神经系统中特异性表达红色荧光蛋白的3xp3-DsRed-SV40调控单元、来自家蚕核型多角体病毒的Hr3增强子、源于家蚕内源sericin1基因的Ser1-P启动子和Ser1PA终止子；Pep-1为一种透皮肽，CTGF-8ht为与增强型his标签相连的人CTGF基因；B：转基因P-CT和P-PCT家蚕品系的筛选；比例尺为2毫米；C和D：分别为在P-CT和P-PCT家蚕基因组中CTGF-8ht和pepCTGF-8ht基因的插入位置)。
图2为茧壳中重组CTGF-8ht和pepCTGF-8ht蛋白的鉴定((A)与(B)分别为在P-CT和P-PCT茧壳中重组CTGF-8ht和pepCTGF-8ht蛋白的表达情况。红色和绿色箭头分别表示重组CTGF-8ht和pepCTGF-8ht蛋白)。
图3为重组CTGF蛋白的表达与纯化(A-D：分别为在P-CT和P-PCT家蚕中CTGF-8ht和pepCTGF-8ht基因在mRNA水平上的表达情况；E：在P-CT和P-PCT茧壳中CTGF-8ht和pepCTGF-8ht蛋白的表达情况；F：重组CTGF-8ht和pepCTGF-8ht蛋白的纯化。TP、FP、E60、E80和E1000分别表示从P-CT和P-PCT茧壳中提取的蛋白粗提物、未能结合到Ni-Sepharose FF柱的蛋白以及分别含有60mM、80mM和1000mM咪唑浓度的洗脱缓冲液。红色和绿色箭头分别表示重组CTGF-8ht和pepCTGF-8ht蛋白)。
图4为重组CTGF蛋白具有促进细胞增殖活性(A：重组CTGF-8ht、pepCTGF-8ht蛋白和CTGFstd标准品与NIH/3T3细胞共培养2d后细胞生长情况；B-C：分别为4ng/ml重组CTGF-8ht、pepCTGF-8ht蛋白与CTGFstd标准品与NIH/3T3细胞共培养3天后的白光结果、Live/Dead细胞染色结果，比例尺为400μm；D：重组CTGF-8ht、pepCTGF-8ht蛋白和CTGFstd标准品与NIH/3T3细胞共培养1d、3d后细胞的生长情况；E-F：分别为重组pepCTGF-8ht蛋白和CTGFstd标准品与NIH/3T3细胞共培养5min、10min、20min和40min后p-ERK的表达情况)。
图5为重组CTGF蛋白具有促进透明质酸和羟脯氨酸合成的活性(A：重组CTGF-8ht、pepCTGF-8ht蛋白鱼CTGFstd标准品与HDF细胞共培养2天后细胞培养基中透明质酸的含量；B-C：分别为31.25ng/ml CTGF-8ht、pepCTGF-8ht蛋白和CTGFstd标准品与HDF细胞共培养2d后细胞培养基中的透明质酸和羟脯氨酸的含量；D：pepCTGF-8ht蛋白和CTGFstd标准品与HDF细胞共培养2d后细胞中透明质酸合酶3的表达情况)。
图6为透皮肽增强重组CTGF蛋白的跨膜能力(重组CTGF-8ht、pepCTGF-8ht蛋白和CTGFstd标准品与NIH/3T3细胞共培养1h，然后用PBS缓冲液(pH＝7.4)洗涤，并用CTGF抗体进行细胞免疫荧光实验，比例尺为800μm)。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明实施例使用的细胞系如下：
小鼠胚胎成纤维细胞NIH/3T3细胞(ATCC)，使用含有10％胎牛血清(FBS)、50mg/mL链霉素和100mg/mL氨苄青霉素的Dulbecco改良Eagle培养基(DMEM，均来自Gibco)培养；
人真皮成纤维细胞(HDF，Procell，中国)，使用人真皮成纤维细胞完全培养基(Procell，中国)培养。
实施例1、转基因表达载体的构建及转基因家蚕培育
构建转基因表达载体的方法参考此前的报道(Wang,Y.,et al.,2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyxmori.Sci Rep,2015.5:p.16273.Wang,Y.,et al.,Genetically engineered bi-functional silk material with improved cell proliferation and anti-inflammatory activity for medical application.Acta Biomater,2019.86:p.148-157.)。首先，根据家蚕丝腺密码子偏好性设计并合成了一个在C端带有8个组氨酸的增强型his标签(8ht)的CTGF-8ht基因(核苷酸如SEQ ID NO.1所示，氨基酸如SEQ ID NO.2所示)与一个在C端带有8ht、N端带有透皮肽(pep-1)的pepCTGF-8ht基因(核苷酸如SEQ ID NO.3所示，氨基酸如SEQ ID NO.4所示)，同时，CTGF-8ht与pepCTGF-8ht基因两侧分别融合了BamHI和NotI限制性核酸内切酶酶切位点。然后，利用BamH I和Not I双酶切目标基因，将CTGF-8ht和pepCTGF-8ht基因片段亚克隆到pSL1180[h Ser1sp DsRed Ser1](Feng Wang,HanfuXu,Lin Yuan,Sanyuan Ma,Yuancheng Wang,Xiaoli Duan,Jianping Duan,ZhonghuaiXiang,Qingyou Xia*.An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenicsilkworms.Transgenic Res.2013 Oct；22(5):925-38.)中间载体，该载体含有家蚕核型多角体病毒增强子Hr3、家蚕sericin1基因启动子Ser1-P和3'-UTR终止序列Ser1PA。之后，利用AscI单酶切中间载体，将含有CTGF-8ht和pepCTGF-8ht基因片段亚克隆到pBac[3×p3DsRedaf](徐汉福，适用于实用家蚕品种转基因的蚕卵滞育解除方法，2011.蚕业科学)转基因表达载体，获得转基因表达载体p[3×p3 DsRed SV40；Hr3 Ser1-P CTGF-8ht Ser1PA]和p[3×p3 DsRed SV40；Hr3 Ser1-P pepCTGF-8ht Ser1PA]，分别命名为pR-CT8ht和pR-PCT8ht，结构如图1中A所示。在p[3xp3 DsRed SV40]转基因表达载体中含有一个3xp3-DsRed-SV40转录调控单元，能够在家蚕的眼部和神经系统中特异性地表达红色荧光蛋白，发出红色荧光信号。
培育转基因家蚕品系的方法现有的方法。首先，将转基因表达载体pR-CT8ht和pR-PCT8ht与辅助质粒Helper以1：1的比例混合，然后，显微注射到200粒刚产下2h内的非滞育家蚕蚕卵中；用桑叶精心饲养经催青成功孵化的G0代家蚕幼虫，分别孵化了55(28％)、32(16％)粒蚕卵，通过自交的方式获得了G1代蚕卵得到了8(16％)、4(33％)个阳性转基因个体；通过观察G1代家蚕胚胎期眼中的红色荧光信号筛选阳性转基因个体，分别命名为P-CT与P-PCT(图1，B和表1)。
表1、转基因载体注射统计
实施例2、转基因家蚕检测
1、反向PCR
反向PCR的方法参考此前的报道。首先，利用基因组DNA提取试剂盒抽提P-CT和P-PCT家蚕的基因组DNA，然后用HaeIII酶消化1μg基因组DNA，并用苯酚/氯仿法纯化DNA片段，再利用T4 DNA连接酶连接环化16℃过夜。以100ng环化的DNA片段为模板，利用重叠在piggyBac载体上的引物(表2)扩增piggyBac载体两端的基因组序列，通过琼脂糖凝胶电泳分离、凝胶提取试剂盒回收扩增片段，并将其克隆到pEASY-T5载体中进行测序。基于家蚕基因组数据库SilkDB 3.0(https://silkdb.bioinfotoolkits.net/)分析靶标基因的插入位置。结果显示，在P-CT和P-PCT家蚕基因组中，CTGF-8ht和pepCTGF-8ht基因分别位于8号染色体上的Bm_scaf58和26号染色体上的Bm_scaf25(图1，C和D)；
表2、转基因家蚕检测使用的引物
1,2,3引物分别用于检测家蚕丝腺中CTGF-8ht、pepCTGF-8ht、Ser1和SW22934基因在mRNA水平上的表达情况；4,5引物分别用于扩增P-CT和P-PCT家蚕piggyBac转座子左臂和右臂侧翼的基因组片段；6,7引物分别用于检测HDF细胞中HAS3和GUS基因在mRNA水平上的表达情况。
2、定量PCR(qPCR)
qPCR的方法参考此前的报道。以五龄第七天的P-CT和P-PCT家蚕的中腺丝腺与人真皮成纤维细胞(HDF)为原料，利用总RNA抽提试剂盒II(Omega，Switzerland)抽提总RNA，然后利用M-MLV逆转录酶(Promega，美国)将1.0μg RNA转录成cDNA模板。利用SYBR qPCRSuperMix Plus试剂(Novoprotein，中国)在ABI Prism 7000检测仪(美国AppliedBiosystems)上分析SW22934、Ser1、CTGF-8ht、pepCTGF-8ht、透明质酸合酶3(HAS3)和β-葡萄糖醛酸苷酶(β-GUS)mRNA的表达水平(表2)。SW22934和Ser1是家蚕的内源基因；β-GUS是小鼠的内源基因。结果显示，在P-CT和P-PCT家蚕中，CTGF-8ht和pepCTGF-8ht基因相对于内源丝胶1基因的表达量分别为55.3±7.4％和42.3±4.2％(图3，A-D)
3、十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白免疫印迹(Westernbloting)
SDS-PAGE和Western blotting的方法参考此前的报道。首先，在液氮中粉碎P-CT和P-PCT蚕茧，然后在80℃条件下利用蛋白提取缓冲液(50mM Nacl、50mM tris-hcl和8M尿素，pH＝7.4)萃取30min，并在4℃条件下以13,400rpm离心5min收集茧壳总蛋白样品。利用增强型BCA蛋白质浓度测定试剂盒测定总茧壳蛋白样品浓度，然后进行12％SDS-PAGE电泳，用考马斯亮蓝R-250染色。在Western blotting实验中，将SDS-PAGE凝胶中的蛋白质样品转移到聚偏二氟乙烯(PVDF)膜上，依次用5％脱脂奶粉缓冲液封闭1h、CTGF一抗缓冲液(CellSignaling Technology，USA)孵育1小时、TBST缓冲液洗涤5次、IgG二抗缓冲液(中国，Beyotime)孵育1小时、TBST缓冲液洗涤5次。使用Chemiscope Series仪(Clinx ScienceInstruments，China)通过自动曝光的方式拍照记录结果，并使用ImageJ软件分析条带信号强度。分析茧壳蛋白结果发现，在转CTGF-8ht基因家蚕品系1与转pepCTGF-8ht基因家蚕品系3的茧壳中重组CTGF-8ht与pepCTGF-8ht蛋白的含量最高(图2)，因此，分别命名为P-CT和P-PCT。SDS-PAGE与Western blotting结果显示，在P-CT和P-PCT的茧壳中，成功地检测到了重组CTGF-8ht和pepCTGF-8ht蛋白的特异性条带(图3，E)。以上结果说明，在P-CT和P-PCT家蚕的中部丝腺与茧壳中成功地表达了CTGF-8ht和pepCTGF-8ht基因。在P-CT和P-PCT茧壳中，重组CTGF-8ht和pepCTGF-8ht蛋白的含量分别占茧壳重量的4.2±0.7％、0.9±0.2％。
4、重组CTGF蛋白的纯化
纯化P-CT和P-PCT茧中重组CTGF-8ht和pepCTGF-8ht蛋白的方法参考此前的报道。首先，在液氮中粉碎P-CT和P-PCT转基因茧壳，然后取3g茧粉浸入150ml的提取缓冲液(50mMNacl、50mM Tris-Hcl和8M尿素，pH 7.0)，在80℃条件下萃取1小时，并在13,400rmp离心10分钟收集总茧壳蛋白粗提物。将含有5mM咪唑的总茧壳蛋白粗提物缓慢上样到Ni-Sepharose FF柱(GE Healthcare，美国)，然后依次使用300ml洗脱缓冲液1(135mM NaCl、2.7mM KCl、1.5mM KH2PO4、8mM K2HPO4与60mM咪唑，pH 7.4)、10ml洗脱缓冲液2(135mMNaCl、2.7mM KCl、1.5mM KH2PO4、8mM K2HPO4和80mM咪唑，pH 7.4)和10ml洗脱缓冲液3(135mM NaCl、2.7mM KCl、1.5mM KH2PO4、8mM K2HPO4和1000mM咪唑，pH 7.4)洗脱。利用12％SDS-PAGE分析洗脱缓冲液1、洗脱缓冲液2和洗脱缓冲液3中的蛋白质样品，利用Image J软件分析PVDF膜上条带的灰度来计算重组CTGF-8ht和pepCTGF-8ht蛋白的纯度。
为了从转基因茧壳中纯化重组CTGF-8ht和pepCTGF-8ht蛋白，首先，将P-CT和P-PCT茧壳在液氮中粉碎成粉，然后在80℃条件下，利用含有8M尿素的提取缓冲液浸提蚕丝粉1h，并以13,400rpm离心15分钟收集上清液，得到蛋白粗提液。将含有5mM咪唑的蛋白粗提液缓慢地加载到Ni-Sepharose FF柱，分别使用含有60mM、80mM与1000mM咪唑浓度的洗脱缓冲液洗涤Ni-Sepharose FF柱，重组CTGF-8ht和pepCTGF-8ht蛋白大量集中于含有1000mM咪唑浓度的洗脱缓冲液(图3，F)。经计算，从1g P-CT和P-PCT茧壳中分别能够纯化得到178.6±23.2ng CTGF-8ht和114.4±19.1ng pepCTGF-8ht蛋白，纯度为97.8％、92.4％。
实施例3、细胞增殖实验
细胞增殖实验的方法参考此前的报道。在96孔板中加入500个NIH/3T3细胞、100μl含0.25％FBS的DMEM培养基，过夜培养，然后，更换100μl新鲜的含有重组CTGF-8ht和pepCTGF-8ht蛋白、0.25％FBS的DMEM培养基培养3天。以等量的购买的CTGF标准蛋白(CTGFstd，R&D Systems，美国)为阳性对照(下同)。使用荧光显微镜(Nikon,Japan)观察和拍照NIH/3T3细胞；使用Live/Dead dying Viability/Cytotoxicity kit(LifeTechnologies,USA)和AlamarBlueTM Cell Viability Reagent(Invitrogen,USA)检测细胞的生长情况。
在24孔板中加入1×104NIH/3T3细胞、500μl含10％FBS(w/v)的DMEM培养基，过夜培养，然后更换500μl新鲜的含有重组pepCTGF-8ht蛋白和等量CTGFstd标准品的、10％FBS的DMEM培养基培养5min、10min、20min和40min。使用含有不含EDTA的蛋白酶抑制剂(Sigma，USA)的RIPA裂解缓冲液(Beyotime，China)提取NIH/3T3细胞中的总蛋白，然后进行12％SDS-PAGE和Western blotting。在Western blotting实验中，使用的一抗包括anti-ERK1/2抗体、anti-磷酸化ERK1/2抗体(Cell Signaling Technology，USA)、anti-tubulin抗体(Beyotime，China)；使用的二抗是抗兔IgG抗体。
结果显示，低浓度的CTGF-8ht和pepCTGF-8ht蛋白可以促进NIH/3T3细胞增殖和生长，当细胞培养基中含有4ng/ml重组pepCTGF-8ht蛋白时，细胞增殖活性最好(图4，A)。接下来，在0.25％FBS的DMEM培养基中添加4ng/ml重组CTGF-8ht、pepCTGF-8ht蛋白与CTGFstd标准品，然后与NIH/3T3细胞共培养后检测细胞生长情况。Live/Dead细胞染色结果显示，与PBS组相比，CTGFstd、CTGF8-ht和pepCTGF-8ht组中具有绿色荧光信号的活细胞的数量显著增加，pepCTGF-8ht组甚至优于CTGFstd与CTGF8-ht(图4，B-C)；CCK-8试剂盒检测样品在450nm处吸光度的结果显示，当与NIH/3T3细胞共培养1天和3天后，与PBS组相比，CTGFstd、CTGF-8ht和CTGF-8ht组中的细胞数量显著增加，相似地，pepCTGF-8ht组的吸光度亦显著高于CTGF-8ht组(图4，D)。以上结果表明，与CTGF-8ht蛋白相比，重组pepCTGF-8ht蛋白具有更好的细胞增殖活性。
此外，还检测了ERK通路中关键因子ERK蛋白的磷酸化水平，结果显示，与PBS组相比，当重组pepCTGF-8ht与CTGFstd蛋白与NIH/3T3细胞共培养5min、10min、20min和40min后，p-ERK的表达显着增加(图4，E-F)，表明pepCTGF-8ht蛋白通过激活ERK途径促进细胞增殖。
实施例4、重组CTGF蛋白促进透明质酸和羟脯氨酸合成活性
透明质酸和羟脯氨酸合成实验的方法参考此前的报道。在24孔板中加入1×104个HDF细胞、500μl含10％FBS的DMEM培养基，过夜培养，然后，更换500μl新鲜的含有重组CTGF-8ht、pepCTGF-8ht蛋白与CTGFstd标准品的、10％FBS的DMEM培养基培养3天，收集细胞培养基测定透明质酸和羟脯氨酸的含量。分别使用Hyaluronan Quantikine ELISA Kit(R&Dsystem,USA)与Mouse Hydroxyproline ELISA Kit(CUSABIO,China)测定培养基中的透明质酸和羟脯氨酸的含量。此外，使用Total RNA Kit II(Omega，瑞士)提取HDF细胞的总mRNA，转录成cDNA模板，然后通过qPCR的方法分析透明质酸合酶3的表达情况。
结果显示，与PBS组相比，当添加的CTGF蛋白的浓度在4～1000ng/ml时，CTGF-8ht、pepCTGF-8ht蛋白与CTGFstd标准品显著地增加了HDF合成透明质酸和羟脯氨酸的活性，pepCTGF-8ht蛋白在浓度为4～500ng/ml时具有最好的活性(图5，A)。接下来，在DMEM培养基中添加31.25ng/ml重组CTGF-8ht、pepCTGF-8ht蛋白与CTGFstd标准品，然后与HDF共培养3天后检测培养基中透明质酸和羟脯氨酸的含量，结果显示，pepCTGF-8ht组细胞培养基中的透明质酸和羟脯氨酸含量均显著高于PBS组，甚至高于CTGF-8ht和CTGFstd组(图5，B-C)，表明，与CTGF-8ht蛋白相比，pepCTGF-8ht蛋白具有更好的促透明质酸和羟脯氨酸合成的活性。此外，qPCR结果显示，与PBS组相比pepCTGF-8ht蛋白与CTGFstd蛋白处理后的HDF细胞中透明质酸合酶3的表达明显增加(图5，D)，说明，pepCTGF-8ht蛋白通过调节透明质酸合酶3的表达来促进透明质酸和羟脯氨酸的合成。
实施例5、透皮肽增强CTGF蛋白的跨膜能力
细胞免疫荧光实验的方法参考此前的报道。在24孔板中加入1×104NIH/3T3细胞、500μl含10％FBS的DMEM培养基，过夜培养，然后更换500μl含0.25％FBS的培养基饥饿培养2h，再更换含有重组CTGF-8ht、pepCTGF-8ht蛋白和CTGFstd标准品的、0.25％FBS的DMEM培养基培养30min。接下来，分别用1ml PBS缓冲液(pH＝7.4)清洗NIH/3T3细胞3次，用4％多聚甲醛室温固定10分钟，用0.1％Triton-X100渗透5分钟，用PBS缓冲液(pH＝7.4)洗涤3次。然后，将细胞依次与5％BSA缓冲液温育1h、PBS缓冲液(pH＝7.4)洗涤3次、与CTGF一抗缓冲液温育1h、PBS缓冲液(pH＝7.4)洗涤5次、红色荧光标记的兔IgG抗体缓冲液孵育1小时、PBS缓冲液(pH＝7.4)洗涤五次。最后，用4',6-Diamidino-2-Phenylindole(DAPI,Thermo FisherScientific,USA)对细胞核染色，并用Olympus SZX12荧光显微镜(Olympus，日本)拍照记录结果。结果显示，与CTGF-8ht和PBS组相比，pepCTGF-8ht组中观察到更多具有红色荧光信号的细胞，并且它们具有更强的荧光强度(图6)，说明，透皮肽pep-1显著地增强了重组pepCTGF-8ht蛋白的跨膜能力，因此，pepCTGF-8ht蛋白展现出更好的生物学活性。
综上所述，本研究旨在利用家蚕丝腺生物反应器系统生产一种人工设计的重组人pepCTGF-8ht蛋白，该蛋白的N端与C端分别含有透皮肽pep-1和拥有8个组氨酸残基的增强型his标签。这种重组pepCTGF-8ht蛋白不仅易于从转基因茧壳中纯化，而且与天然的CTGF蛋白相比，具有更强的细胞增殖能力和透明质酸合成活性。该研究将指导未来利用家蚕丝腺生物反应器系统生产易于纯化、高效的蛋白质药物，同时，也将促进其它生物反应器系统的设计和优化研究。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 西南大学
<120> 基于家蚕丝腺表达具有生物学活性的人结缔组织生长因子的方法及其产品和应用
<160> 18
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agagtgtgtg ctaagcagct gggtgaattg tgcactgaaa gagacccgtg tgatccccac 180
aaaggcttgt tctgcgactt cggttcaccg gccaacagaa agatcggagt ttgcaccgct 240
aaagatggcg ccccctgtat tttcggtgga acagtctaca gatcgggcga aagtttccaa 300
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tcgatggacg ttagactgcc gagtcccgat tgccctttcc caagaagagt caagttgcct 420
ggtaaatgct gtgaagaatg ggtgtgtgac gaacctaaag atcaaacagt ggttggacca 480
gctttggctg cctacagact cgaagacact ttcggccctg accctaccat gatcagagct 540
aattgcctcg tgcagacaac tgaatggtca gcctgctcta agacttgtgg aatgggcatt 600
tcaactagag tgaccaacga caatgcttca tgcagactgg aaaaacaatc tagattgtgc 660
atggttagac catgtgaagc cgatctcgaa gaaaacataa aaaagggaaa aaagtgcatc 720
agaacaccta agatctctaa gccaattaaa ttcgaactga gcggctgtac atccatgaag 780
acttacagag ctaaattctg cggtgtttgt acagacggaa gatgctgtac tcctcacaga 840
accacaactc tcccggttga attcaagtgc cccgatggag aagtcatgaa aaagaatatg 900
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atgcaccatc atcaccacca ccaccacaag gaaacttggt gggaaacttg gtggactgaa 60
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gacgaacctg ctccaagatg tcctgccgga gtctccctcg tgctggatgg ctgcggttgc 180
tgtagagtgt gtgctaagca gctgggtgaa ttgtgcactg aaagagaccc gtgtgatccc 240
cacaaaggct tgttctgcga cttcggttca ccggccaaca gaaagatcgg agtttgcacc 300
gctaaagatg gcgccccctg tattttcggt ggaacagtct acagatcggg cgaaagtttc 360
caatcatctt gcaagtacca gtgcacctgt ctggacggcg ctgtgggttg catgcctctc 420
tgttcgatgg acgttagact gccgagtccc gattgccctt tcccaagaag agtcaagttg 480
cctggtaaat gctgtgaaga atgggtgtgt gacgaaccta aagatcaaac agtggttgga 540
ccagctttgg ctgcctacag actcgaagac actttcggcc ctgaccctac catgatcaga 600
gctaattgcc tcgtgcagac aactgaatgg tcagcctgct ctaagacttg tggaatgggc 660
atttcaacta gagtgaccaa cgacaatgct tcatgcagac tggaaaaaca atctagattg 720
tgcatggtta gaccatgtga agccgatctc gaagaaaaca taaaaaaggg aaaaaagtgc 780
atcagaacac ctaagatctc taagccaatt aaattcgaac tgagcggctg tacatccatg 840
aagacttaca gagctaaatt ctgcggtgtt tgtacagacg gaagatgctg tactcctcac 900
agaaccacaa ctctcccggt tgaattcaag tgccccgatg gagaagtcat gaaaaagaat 960
atgatgttca tcaagacttg cgcctgtcac tacaactgtc caggcgacaa tgatatcttc 1020
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<210> 4
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Met His His His His His His His His Lys Glu Thr Trp Trp Glu Thr
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Val Thr Asn Asp Asn Ala Ser Cys Arg Leu Glu Lys Gln Ser Arg Leu
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Cys Met Val Arg Pro Cys Glu Ala Asp Leu Glu Glu Asn Ile Lys Lys
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Gly Lys Lys Cys Ile Arg Thr Pro Lys Ile Ser Lys Pro Ile Lys Phe
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Glu Leu Ser Gly Cys Thr Ser Met Lys Thr Tyr Arg Ala Lys Phe Cys
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Ala
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<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gggaaagggc aatcgggact 20
<210> 7
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atctgaagac ggtttctggt ggt 23
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aactgcctga agtggttgtg c 21
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ttcgtactgg ctcttctcgt 20
<210> 10
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caaagttgat agcaattccc t 21
<210> 11
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atcagtgaca cttaccgcat tgaca 25
<210> 12
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tgacgagctt gttggtgagg attct 25
<210> 13
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tacgcatgat tatctttaac gta 23
<210> 14
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gtactgtcat ctgatgtacc agg 23
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ccaagaggac cccgaatacc 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ttgctacgcc acacaaagaa 20
<210> 17
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ttggctgggt gtggtatgaa c 21
<210> 18
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gtcagtgtgt tgttgatggc a 21
Claims (10)
1.基于家蚕丝腺表达具有生物学活性人结缔组织生长因子或N端含透皮肽的人结缔组织生长因子的方法，其特征在于：优化人结缔组织生长因子基因密码子获得适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因，然后将改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因在家蚕丝腺表达，结茧后茧壳用尿素提取，纯化，得到具有生物学活性人结缔组织生长因子或N端含透皮肽的人结缔组织生长因子；所述改造人结缔组织长因子基因的核苷酸序列如SEQ ID NO.1所示；所述N端含透皮肽的人结缔组织生长因子基因的核苷酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述的方法，其特征在于：家蚕丝腺表达改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因的方法如下：构建含有所述适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因的转基因表达载体，将所述转基因表达载体注射已解除滞育的家蚕蚕卵，培育转基因家蚕即可。
3.根据权利要求2所述的方法，其特征在于：所述转基因表达载体含有顺序连接的增强子hr3，分泌型丝胶1基因启动子、改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因、丝胶1基因的终止子和piggyBac转座序列。
4.具有生物学活性的重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子，其特征在于：由如SEQ ID NO.1或SEQ ID NO.3所示的核苷酸序列于在家蚕丝腺表达，结茧后茧壳用尿素提取，纯化制得。
5.权利要求5所述的重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子在制备促进细胞增殖的药物中的应用，其特征在于：所述重组人结缔组织生长因子的氨基酸序列如SEQ ID NO.2所示；所述N端含透皮肽的人结缔组织生长因子的氨基酸序列如SEQ ID NO.4所示。
6.权利要求5所述的重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子在制备促进ERK蛋白磷酸化的药物中的应用，其特征在于：所述重组人结缔组织生长因子的氨基酸序列如SEQ ID NO.2所示；所述N端含透皮肽的人结缔组织生长因子的氨基酸序列如SEQ ID NO.4所示。
7.权利要求5所述的重组人结缔组织生长因子或N端含透皮肽的重组人结缔组织生长因子在制备促进透明质酸合成或/和羟脯氨酸合成的药物中的应用，其特征在于：所述重组人结缔组织生长因子的氨基酸序列如SEQ ID NO.2所示；所述N端含透皮肽的人结缔组织生长因子的氨基酸序列如SEQ ID NO.4所示。
8.适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因，其特征在于：所述改造人结缔组织生长因子基因的核苷酸序列如SEQ ID NO.1所示；所述N端含透皮肽的人结缔组织生长因子基因如SEQ ID NO.3所示。
9.含有权利要求8所述适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因的重组表达载体。
10.含有权利要求8所述适用于家蚕表达的改造人结缔组织生长因子基因或N端含透皮肽的人结缔组织生长因子基因的转基因家蚕。
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