CN113105557A - 一种酿酒酵母表达人rVEGF-HSA融合蛋白及其标准品的制备方法 - Google Patents
一种酿酒酵母表达人rVEGF-HSA融合蛋白及其标准品的制备方法 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体涉及一种酿酒酵母表达人rVEGF‑HSA融合蛋白的制备方法，通过设计并获得rVEGF‑HSA基因序列和融合蛋白rVEGF‑HSA的氨基酸序列，获得表达载体pYES2/CT‑MFα‑rVEGF‑HSA的构建，培养并获得转化rVEGF‑HSA融合蛋白工程菌，再进一步扩大培养并诱导表达，获得纯化后的酿酒酵母表达人rVEGF‑HSA融合蛋白。本发明还提供了一种酿酒酵母表达人rVEGF‑HSA融合蛋白标准品的制备方法。本发明利用酿酒酵母表达人rVEGF与HSA融合而成的重组蛋白(rVEGF‑HSA)，生产工艺简单、表达稳定。
Description
技术领域
本发明属于生物技术领域，具体涉及一种酿酒酵母表达人rVEGF-HSA融合蛋白及其标准品的制备方法。
背景技术
血管内皮生长因子(vascular endothelial growth factor,VEGF)能够特异地作用于血管内皮细胞，是上调血管生成的重要细胞因子。VEGF具有促进胚胎发育、促进新血管形成、增加缺血组织血流量以及降低组织损伤的生理功能。VEGF的基因定位于6p12，由8个外显子和7个内含子交替排列构成，其mRNA以不同剪切方式形成VEGF121、VEGF145、VEGF165、VEGF183、VEGF189和VEGF206 6种不同的异构体，其中VEGF165发挥主要的生物学活性功能。VEGF165包含165个氨基酸残基，是一个二硫键连接的同型二聚体。体外实验证实，乳腺癌细胞株MCF7、人宫颈癌细胞株HeLa以及黑色素瘤细胞株A375等很多肿瘤细胞株分泌VEGF165。
人血清白蛋白(Human serum albumin,HSA)是人体血液中的主要蛋白，由585个氨基酸构成，是人体循环系统内的含量最多的可溶性蛋白，在血液中的浓度为34～54g/L。HSA由肝脏合成，血清半衰期长，可达19天。HSA在调节血液渗透压、营养和促进伤口愈合等方面发挥重要作用，广泛用于肝硬化腹水、烧伤、休克等临床治疗。此外，HSA具有无免疫原性、人体相容性好、组织分布广和无酶活等特性，使其成为非常理想的重组蛋白融合载体。通过构建融合蛋白技术能够增加重组蛋白的分子量从而延长半衰期，有效的提高重组蛋白的稳定性。
VEGF融合HSA表达能有效提高重组蛋白的稳定性，且生产工艺简单、成本低、产物均一、无免疫原性。对于生物制品来说，质量评价的有效性指标常采用生物学方法，而这些方法本身变异性较大，所以标准品是生产中必不可少的组成部分。在生物制品标准化、质量控制和效力评价中，标准品是评价产品质量的重要标尺。
目前VEGF的获得主要通过两条途径：一是从生物的组织细胞中提取；二是运用基因工程技术构建表达载体，利用宿主表达重组VEGF。天然提纯VEGF工艺复杂、成本高，其推广应用受到了限制，而利用基因工程技术表达外源基因已成为获取目的蛋白的高效途径之一，根据表达载体和宿主菌的不同，主要分为原核表达和真核表达。原核表达系统虽成本低廉，但该系统存在容易形成包涵体、获得的蛋白生物学活性较低等缺陷。
发明内容
为克服背景技术中的技术问题，本公司利用酿酒酵母表达人VEGF与HSA融合而成的重组蛋白(rVEGF-HSA)，生产工艺简单、成本低、产物均一，建立了一种经济、高效、稳定表达的高浓度、高活性的长效重组人VEGF标准品蛋白，为建立人rVEGF蛋白质量标准提供依据。
一种酿酒酵母表达人rVEGF-HSA融合蛋白制备方法，包含以下步骤：
(1)酿酒酵母分泌表达载体pYES2/CT-MFα-rVEGF-HSA的构建，具体包括：
设计并获得rVEGF-HSA基因序列和融合蛋白rVEGF-HSA的氨基酸序列；
根据rVEGF-HSA核苷酸序列设计并扩增目的基因，回收并双酶切PCR产物和pYES2/CT-MFα质粒，用T4 DAN连接酶连接，转入E.coli DH5α感受态细胞培养，获得阳性克隆pYES2/CT-MFα-rVEGF-HSA；
(2)rVEGF-HSA融合蛋白工程菌的制备、转化，具体包括：
酿酒酵母表达体系常用溶液及培养基的配制；
将步骤(1)获得的pYES2/CT-MFα-rVEGF-HSA电转化酿酒酵母INVSc1感受态细胞，通过培养基的培养及PCR扩增、筛选，获得阳性克隆子INVSc1/pYES2/CT-MFα-rVEGF-HSA；
(3)INVSc1/pYES2/CT-MFα-rVEGF-HSA工程菌的诱导表达和纯化，具体包括：
将步骤(2)获得的阳性克隆子INVSc1/pYES2/CT-MFα-rVEGF-HSA，通过培养、诱导表达，再依次经过金属离子亲和层析和阴离子交换层析纯化，即得本发明获得的酿酒酵母表达人rVEGF-HSA融合蛋白。
进一步的，所述步骤(1)中，设计并获得rVEGF-HSA基因序列和融合蛋白rVEGF-HSA的氨基酸序列，具体步骤为：
根据pYES2/CT-MFα载体(图1)的性质和酿酒酵母宿主密码子偏爱性，设计了人rVEGF-HSA基因序列和人rVEGF-HSA的氨基酸序列，人rVEGF-HSA基因序列如序列表1所示，人rVEGF-HSA的氨基酸序列如序列表2所示。
进一步的，所述步骤(1)中，根据rVEGF-HSA核苷酸序列设计并扩增目的基因，回收并双酶切PCR产物和pYES2/CT-MFα质粒，用T4 DAN连接酶连接，转入E.coli DH5α感受态细胞培养，获得阳性克隆pYES2/CT-MFα-rVEGF-HSA，具体步骤为：
根据rVEGF-HSA核苷酸序列设计扩增引物，其中：
正向引物：5'ATAAGAATGCGGCCGCAATGGCTCCA 3'；
反向引物：5'CTAGTCTAGATTAGTGATGGTGATGG 3'；
进行PCR扩增；
配制1％琼脂糖凝胶，电泳分离PCR扩增产物，紫外灯下快速切下目的条带，用DNA凝胶回收试剂盒回收目的基因PCR扩增产物；
提取DH5α/pYES2/CT-MFα中的pYES2/CT-MFα质粒；
将质粒pYES2/CT-MFα和回收的PCR产物分别用Not I和Xba I进行双酶切，并胶回收双酶切的PCR产物和质粒pYES2/CT-MFα；
将双酶切回收的PCR扩增产物与pYES2/CT-MFα质粒用T4 DNA连接酶在37℃中连接约30min；
将连接产物转化到E.coli DH5α感受态细胞中，经Amp抗性筛选后挑取阳性转化子进行培养，获得阳性克隆pYES2/CT-MFα-rVEGF-HSA。
进一步的，所述步骤(2)中，酿酒酵母表达体系常用溶液及培养基的配制，具体方法为：
YPD培养基：蛋白胨20g，酵母提取物10g，加纯化水定容至900ml，121℃高压灭菌20min，冷却至60℃以下，超净台中加入无菌100ml 20×葡萄糖，固体培养基另加琼脂粉2.0％；
SC-U缺陷培养基：YNB无氨基酸氮源6.70g，0.01％氨基酸混合物I 1g，0.005％氨基酸混合物II 0.5g，加蒸馏水定容至900ml，121℃高压灭菌20min，冷却至60℃以下，超净台中加入无菌100ml 20％葡萄糖，固体培养基另加琼脂粉2.0％；
其中，所述0.01％氨基酸混合物I为精氨酸、亮氨酸、苏氨酸、赖氨酸、色氨酸、半胱氨酸和腺嘌呤；所述0.005％氨基酸混合物II为天冬氨酸、丝氨酸、组氨酸、脯氨酸、异亮氨酸、苯丙氨酸、撷氨酸、酪氨酸和甲硫氨酸；
SC-U诱导培养基：蛋白胨20g，酵母提取物10g，加纯化水定容至700ml，121℃高压灭菌20min，冷却至60℃以下，超净台中加入无菌100ml 20％半乳糖，固体培养基另加琼脂粉2.0％。
进一步的，所述步骤(2)中，获得阳性克隆子INVSc1/pYES2/CT-MFα-rVEGF-HSA的具体步骤为：
采用电转化法将pYES2/CT-MFα-rVEGF-HSA转化至酿酒酵母INVSc1感受态细胞：
将10μl pYES2/CT-MFα-rVEGF-HSA质粒加到80μl酿酒酵母INVScl感受态细胞中，吹吸使其混合均匀，然后转移到预冷的电击杯中，冰浴5min，擦干电击杯外壁；
将Bio-Rad电转化仪调至真菌档，PIC选项，电击杯置于Bio-Rad电转化仪上电击，迅速向电击杯中加入500μl预冷的1M山梨醇溶液，混合均匀，涂SC-U板；
30℃恒温倒置培养，直至长出单克隆；
挑去转化子接种到SC-U液体培养基中，30℃、200rpm恒温培养；
以培养的菌液为模板进行PCR反应，筛选INVSc1/pYES2/CT-MFα-rVEGF-HSA阳性克隆子。
进一步的，所述步骤(3)中，INVSc1/pYES2/CT-MFα-rVEGF-HSA工程菌的诱导表达，具体步骤为：
挑取INVSc1/pYES2/CT-MFα-rVEGF-HSA单菌落接种于20ml SC-U选择培养基，经30℃、220rpm震荡培养过夜，测定其OD600nm吸光值，计算好相应体积的菌液转接至于100ml SC-U诱导培养基中，使得初始OD600nm达到0.4，诱导时间为24h；
诱导表达的上清液经离心并通过0.22μm滤膜过滤，收集过滤液。
进一步的，所述步骤(3)中，金属离子亲和层析的步骤为：
取离心并过0.22μm滤膜过滤后的过滤液，使用GE Healthcare公司ChelatingSepharose TM Fast Flow镍离子螯合亲和层析填料自行装柱，用3个柱体积的纯化水清洗Ni2+螯合亲和层析柱，再用PBS平衡2-3个柱体积；
在线检测电导率值及280nm波长吸收值，待两者都稳定后开始上样，设置样品经过泵过层析柱的流速5-6ml/min；
再用PBS过层析柱，洗去未与层析柱结合的杂蛋白，直到OD280nm稳定，再以含500mM咪唑PBS缓冲液过层析柱，洗脱并收集洗脱峰对应的蛋白，即得到经过金属离子亲和层析后的人rVEGF-HSA蛋白原液。
进一步的，所述步骤(3)中，阴离子交换层析的步骤为：
将金属离子亲和层析纯化后收集的蛋白原液置换到Tris缓冲液中后上样，通过用Tris缓冲液平衡好的DEAE阴离子交换层析柱，收集VEGF-HSA蛋白峰，Tris-NaCl洗脱液洗脱，洗去杂蛋白，即为本发明获得的纯化后的人rVEGF-HSA融合蛋白。
一种人rVEGF-HSA融合蛋白标准品的制备方法，包括以下步骤：
取制得的人rVEGF-HSA融合蛋白溶液，用0.22μm滤膜过滤除菌，用10mM磷酸缓冲液稀释后加入终浓度为10％甘油、0.12g/ml甘露醇、0.025g/ml蔗糖冻干保护剂，进行冷冻真空干燥，干燥后即为重组人VEGF-HSA融合蛋白标准品。
与现有技术相比，本发明的有益效果是：
本发明利用酿酒酵母分泌表达的血管内皮生长因子与HSA融合而成的重组蛋白(rVEGF-HSA)，提高了重组蛋白的稳定性。酿酒酵母分泌表达系统为真核表达系统，能够高水平表达蛋白质分泌到培养基中，产品生产工艺简单、成本低、产物均一、无免疫原性。
本发明同时制备了检测标准品，在生物制品标准化、质量控制和效力评价中，本发明所建立的经济、高效的rVEGF-HSA标准品制备方法具有十分积极的意义。
附图说明：
图1质粒pYES2/CT-MFα图谱；
图2SDS-PAGE鉴定纯化后人rVEGF-HSA蛋白
序列表说明：
序列表1本发明人rVEGF-HSA的核苷酸序列
序列表2本发明人rVEGF-HSA的氨基酸序列
为了更好地理解本发明，下面结合实施例进一步阐明本发明的内容，但本发明的内容不仅仅局限于下面的实施例。
具体实施方式：
实施例1：酿酒酵母分泌表达载体pYES2/CT-MFα-rVEGF-HSA的构建
1.1血管内皮生长因子和人血清白蛋白因子的人工优化和获得：
通过GenBank查询获得以下相关基因和氨基酸序列，本次试验将利用目的基因表达的氨基酸序列优化基因序列，再进行人工合成两段基因来构建表达载体。根据pYES2/CT-MFα载体(图1)的性质和酿酒酵母宿主密码子偏爱性，设计人rVEGF-HSA基因序列和人rVEGF-HSA的氨基酸序列，人rVEGF-HSA基因序列如序列表1所示，人rVEGF-HSA的氨基酸序列如序列表2所示。
序列表1中，GCGGCCGC为Not I酶切位点，TCTAGA为Xba I酶切位点；ATG为起始密码子，TAA为终止密码子；GCAGAGGCGGCGGCTAAGGAAGCTGCAGCCAAAGCC为连接VEGF和HSA序列的Linker所对应的碱基序列；CATCACCATCACCATCAC为6×His标签序列。
1.2pYES2/CT-MFα-rVEGF-HSA表达载体的构建
根据rVEGF-HSA核苷酸序列设计扩增引物，其中：
正向引物：5'ATAAGAATGCGGCCGCAATGGCTCCA 3'；
反向引物：5'CTAGTCTAGATTAGTGATGGTGATGG 3'。
PCR条件：95℃预变性5min；95℃变性1min，60℃退火1min，72℃延伸2.5min，共29个循环；72℃最终延伸10min。
配制1％琼脂糖凝胶，电泳分离PCR扩增产物，紫外灯下快速切下目的条带，用DNA凝胶回收试剂盒回收目的基因PCR扩增产物。
在37℃金属浴中酶切3h。酶切后以2％琼脂糖凝胶进行电泳，并胶回收双酶切的PCR产物和质粒。
将双酶切回收的PCR扩增产物与pYES2/CT-MFα质粒用T4 DNA连接酶在37℃中连接约30min。连接体系(10μl)：双酶切回收PCR产物5μl、双酶切回收pYES2/CT-MFα片段3μl、T4DNA ligase和10×ligase buffer各1μl。
将连接产物转化到E.coli DH5α感受态细胞中，经Amp抗性筛选后挑取阳性转化子进行培养。利用菌液PCR鉴定基因已成功导入载体，测序获得pYES2/CT-MFα-rVEGF-HSA载体。
实施例2：rVEGF-HSA融合蛋白工程菌的制备、转化
2.1酿酒酵母表达体系常用溶液及培养基的配制
YPD培养基：蛋白胨20g，酵母提取物10g，加纯化水定容至900ml，121℃高压灭菌20min，冷却至60℃以下，超净台中加入无菌100ml 20×葡萄糖，固体培养基另加琼脂粉2.0％；
SC-U缺陷培养基：YNB无氨基酸氮源6.70g，0.01％氨基酸混合物I1g，0.005％氨基酸混合物II 0.5g，加蒸馏水定容至900ml，121℃高压灭菌20min，冷却至60℃以下，超净台中加入无菌100ml 20％葡萄糖，固体培养基另加琼脂粉2.0％；
其中，所述0.01％氨基酸混合物I为精氨酸、亮氨酸、苏氨酸、赖氨酸、色氨酸、半胱氨酸和腺嘌呤；所述0.005％氨基酸混合物II为天冬氨酸、丝氨酸、组氨酸、脯氨酸、异亮氨酸、苯丙氨酸、撷氨酸、酪氨酸和甲硫氨酸；
SC-U诱导培养基：蛋白胨20g，酵母提取物10g，加纯化水定容至700ml，121℃高压灭菌20min，冷却至60℃以下，超净台中加入无菌100ml 20％半乳糖，固体培养基另加琼脂粉2.0％。
2.2pYES2/CT-MFα-rVEGF-HSA转化酿酒酵母
采用电转化法将pYES2/CT-MFα-rVEGF-HSA转化酿酒酵母INVSc1感受态细胞。
将10μl pYES2/CT-MFα-rVEGF-HSA质粒加到80μl酿酒酵母INVScl感受态细胞中，吹吸使其混合均匀，然后转移到预冷的电击杯中。冰浴5min，擦干电击杯外壁。将Bio-Rad电转化仪调至真菌档，电击杯置于Bio-Rad电转化仪上电击。迅速向电击杯中加入500μl预冷的1M山梨醇溶液，混合均匀，涂SC-U固体平板，30℃恒温倒置培养，直至长出单克隆。
挑取转化子接种到SC-U液体培养基中，30℃、200rpm恒温培养。以菌液为模板进行PCR反应，鉴定筛选阳性克隆，由此获得工程菌INVSc1/pYES2/CT-MFα-rVEGF-HSA。
实施例3：INVSc1/pYES2/CT-MFα-rVEGF-HSA工程菌的诱导表达、检定和纯化
3.1工程菌的诱导表达、检定
挑取INVSc1/pYES2/CT-MFα-rVEGF-HSA单菌落接种于20ml SC-U选择培养基，经30℃、220rpm震荡培养过夜，测定其OD600nm吸光值，计算好相应体积的菌液转接至于100ml SC-U诱导培养基中，使得初始OD600nm达到0.4，诱导时间为24h。
INVSc1/pYES2/CT-MFα-rVEGF-HSA诱导表达的上清液经SDS-PAGE电泳可观察到约78kDa左右的特异性条带，而含有pYES2/CT-MFα空载质粒的酿酒酵母菌株的诱导表达上清液则无特异性条带(图2，其中，M，Marker；1，经浓缩的诱导上清；2，含有空载质粒酿酒酵母菌株的诱导上清)。
3.2重组人VEGF-HAS融合蛋白的纯化
3.2.1试剂配制
(1)PBS缓冲液：
称上述成分加纯化水溶解，调pH至8.0，定容至1L。
(2)PBS洗脱液：
称上述成分加纯化水溶解，调pH至8.0，定容至1L。
(3)Tris缓冲液：
称上述成分加纯化水溶解，调pH至8.5，定容至1L。
(4)Tris-NaCl洗脱液：
称上述成分加纯化水溶解，调pH至8.5，定容至1L。
3.2.2金属离子亲和层析
离心收集培养液上清，用0.22μm滤膜过滤用以上样。使用GE Healthcar e公司Chelating Sepharose TM Fast Flow镍离子螯合亲和层析填料自行装柱，用3个柱体积的纯化水清洗Ni2+螯合亲和层析柱，再用PBS缓冲液平衡2-3个柱体积。在线检测电导率值及280nm波长吸收值，待两者都稳定后开始上样，设置样品经过泵过层析柱的流速5-6ml/min。再用PBS缓冲液过层析柱，洗去未与层析柱结合的杂蛋白，直到OD280nm稳定。再以PBS洗脱液过层析柱，洗脱并收集洗脱峰对应的蛋白，即得到rVEGF-HSA蛋白原液。
3.2.2阴离子交换层析
将经过金属离子亲和层析的蛋白原液置换到Tris缓冲液中后上样，通过用Tris缓冲液平衡好的DEAE阴离子交换层析柱，收集rVEGF-HSA蛋白峰。Tris-NaCl洗脱液洗脱，洗去杂蛋白，即得本发明获得的酿酒酵母表达人rVEGF-HSA融合蛋白。
实施例4：rVEGF-HSA标准品的制备和检测
4.1、rVEGF-HSA标准品的制备
将rVEGF-HSA纯化后蛋白溶液用0.22μm滤膜过滤除菌，用10mM磷酸缓冲液稀释后加入终浓度为10％甘油、0.12g/ml甘露醇、0.025g/ml蔗糖冻干保护剂，进行冷冻真空干燥。
4.2VEGF-HSA标准品的检测
4.2.1蛋白质含量测定
对上述纯化后的rVEGF-HSA标准品用Lowry法检测蛋白浓度，其蛋白含量为2.0mg/ml。
4.2.2SDS-PAGE进行纯度鉴定
对rVEGF-HSA标准品进行SDS-PAGE鉴定纯度，其纯度为98％，相对分子量为78kDa。
4.2.3HPLC纯度鉴定
VEGF-HSA标准品经μRPC C18 ST 4.6/100反相层析柱进行分析只得到一个主吸收峰，有2个峰为杂峰。
通过计算峰面积得出主峰面积占总面积的99.47％。
4.2.4N-端氨基酸序列测定
将rVEGF-HSA蛋白样品SDS-PAGE电泳，上样前先将配置好的聚丙烯酰胺凝胶装到垂直电泳仪上，用50V恒压空跑30min。将蛋白电转到PVDF(聚偏氟乙烯)膜上，电转缓冲液用CAPS缓冲液。将PVDF膜放到丽春红染液中染色30min，用水洗去背景颜色。将目的条带剪下放于EP管中，-20℃保存，用Edman降解法进行N端氨基酸测序。
N-端氨基酸序列为EAEAYVEFPRAAAMAPMAEG，说明纯化后的目的蛋白确实为rVEGF-HSA。
综上，本发明利用酿酒酵母表达的长效重组人VEGF与HSA融合而成的重组蛋白(rVEGF-HSA)，克服了现有技术中，采用大肠杆菌、乳酸杆菌、短芽孢杆菌及毕赤酵母表达系统容易形成包涵体、获得的蛋白生物学活性较低的缺陷，生产工艺简单、成本低、产物均一；同时，提供了一种重要的人rVEGF-HSA标准品，在生物制品标准化、质量控制和效力评价中，起着非常重要的作用。本发明制备人rVEGF-HSA融合蛋白，具有更好的均一性、稳定性和更好的回收率，可显著降低生产成本。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。
序列表
<110> 芜湖英特菲尔生物制品产业研究院有限公司
<120> 一种酿酒酵母表达人rVEGF-HSA融合蛋白及其标准品的制备方法
<160> 2
<170> SIPOSequenceListing 1.0
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tttatggacg tgtatcaaag gtcctactgc cacccgatag aaacacttgt ggacattttt 120
caggaatatc ctgatgaaat tgaatacatt ttcaaacctt cttgcgttcc cttgatgcgt 180
tgcggcggat gctgtaacga tgagggtttg gaatgcgtac caactgaaga gtctaatatc 240
accatgcaaa ttatgagaat taagccccat caaggccagc atataggaga aatgtccttc 300
ttgcaacaca ataagtgtga atgtagacca aagaaagata gggctagaca agaaaatcct 360
tgcggtccat gttctgaacg tcgtaagcat ttattcgtcc aagatccaca aacgtgcaag 420
tgtagttgta agaatacaga ttcaagatgc aaggctagac aactagagtt aaacgaaaga 480
acttgtaggt gtgataaacc acgtagagca gaggcggcgg ctaaggaagc tgcagccaaa 540
gccatgaagt gggttacgtt tatctcccta ttatttctgt tctcatccgc ctactccaga 600
ggtgttttca ggagagatgc tcacaaatct gaggttgctc atagattcaa ggatttgggt 660
gaagaaaact ttaaggcctt agtgttaata gctttcgcac aatacctgca acagtgtcct 720
tttgaagacc atgtcaaatt agttaatgaa gtcaccgaat ttgctaagac gtgcgttgct 780
gatgagtctg ccgaaaattg tgacaaatca ctgcatacat tgttcggtga taagctatgt 840
accgttgcaa ctcttagaga aacgtacgga gagatggcgg actgttgtgc taaacaagaa 900
cctgaaagaa atgaatgttt tttgcaacac aaagatgata atccaaactt gccaagattg 960
gtaagaccag aagttgacgt tatgtgtacc gcttttcatg ataatgaaga aacatttttg 1020
aaaaagtatc tttatgaaat agcaaggagg catccttact tctacgctcc agagttatta 1080
ttttttgcaa aaagatataa ggcagctttt actgaatgtt gtcaggctgc ggataaagcc 1140
gcatgtctgt tacccaaatt ggatgaattg agagacgagg gcaaagctag tagtgccaaa 1200
caaagattaa aatgcgcttc attacaaaaa tttggagaaa gagcgtttaa ggcttgggcc 1260
gtagcaagat tgtctcagag attcccgaaa gccgaatttg cagaagtgag taaactggtc 1320
acagatttga cgaaagttca cacagaatgt tgtcacggag atttattgga atgcgctgac 1380
gatagggctg acttagctaa atacatatgc gagaatcaag attccatatc atcaaaattg 1440
aaagaatgtt gtgagaaacc attattagaa aaatcccact gtatagctga agttgagaac 1500
gatgaaatgc ccgcggattt accctccctt gcggctgact tcgttgagtc aaaggatgtt 1560
tgcaagaatt acgcggaggc caaggatgtt tttcttggca tgtttttata tgagtatgcc 1620
agacgtcatc cggattattc tgtagttcta ctgttaaggc ttgccaagac atacgaaact 1680
accttagaaa aatgttgtgc ggctgccgat ccacatgaat gttacgcaaa agtttttgat 1740
gaattcaagc cgcttgtcga ggagccacaa aatttaatta aacaaaactg tgaattattt 1800
gaacaattag gtgaatataa attccaaaac gcattattgg tcagatatac aaaaaaagta 1860
cctcaggttt ccacaccaac tttagtggaa gtgtcacgta acctaggcaa ggttggtagt 1920
aagtgctgta aacacccaga agctaagaga atgccatgcg ctgaagatta tctatcagtc 1980
gtacttaatc aactgtgtgt cctacacgag aagactcctg tcagtgacag agtgacaaaa 2040
tgttgcaccg agagcttagt taatagaaga ccgtgttttt cagcgctgga agttgatgaa 2100
acctatgttc caaaggagtt caatgcagaa acattcacct tccatgctga tatatgtact 2160
cttagtgaaa aagaaaggca gatcaaaaaa caaactgccc tggtcgaatt agtcaaacat 2220
aaacctaaag caacgaagga acagttgaag gccgtaatgg atgatttcgc agctttcgtt 2280
gaaaaatgtt gcaaggctga tgacaaagag acatgttttg ctgaagaggg aaaaaaattg 2340
gtggcagctt ctcaagccgc tttagggtta catcaccatc accatcacta atctaga 2397
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Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr
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Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe
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Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp
50 55 60
Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln
65 70 75 80
Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser
85 90 95
Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala
100 105 110
Arg Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu
115 120 125
Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp
130 135 140
Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg
145 150 155 160
Cys Asp Lys Pro Arg Arg Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala
165 170 175
Lys Ala Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser
180 185 190
Ser Ala Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu
195 200 205
Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu
210 215 220
Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp
225 230 235 240
His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val
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Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe
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Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu
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Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe
290 295 300
Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro
305 310 315 320
Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe
325 330 335
Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr
340 345 350
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr
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Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu
370 375 380
Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu
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Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp
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Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu
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His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys
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Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys
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Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val
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Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe
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Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser
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Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu
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Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe
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Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln
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Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala
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Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr
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Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys
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Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser
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Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser
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Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro
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Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe
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Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu
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Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys
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His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp
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Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr
755 760 765
Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala
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Leu Gly Leu His His His His His His
785 790
Claims (9)
1.一种酿酒酵母表达人rVEGF-HSA融合蛋白制备方法，其特征在于，包含以下步骤：
(1)酿酒酵母分泌表达载体pYES2/CT-MFα-rVEGF-HSA的构建，具体包括：
设计并获得rVEGF-HSA基因序列和融合蛋白rVEGF-HSA的氨基酸序列；
根据VEGF-HSA核苷酸序列设计并扩增目的基因，回收并双酶切PCR产物和pYES2/CT-MFα质粒，用T4 DAN连接酶连接，转入E.coli DH5α感受态细胞培养，获得阳性克隆pYES2/CT-MFα-rVEGF-HSA；
(2)rVEGF-HSA融合蛋白工程菌的制备、转化，具体包括：
酿酒酵母表达体系常用溶液及培养基的配制；
将步骤(1)获得的pYES2/CT-MFα-rVEGF-HSA电转化酿酒酵母INVSc1感受态细胞，通过培养基的培养及PCR扩增、筛选，获得阳性克隆子INVSc1/pYES2/CT-MFα-rVEGF-HSA；
(3)INVSc1/pYES2/CT-MFα-rVEGF-HSA工程菌的诱导表达和纯化，具体包括：
将步骤(2)获得的阳性克隆子INVSc1/pYES2/CT-MFα-rVEGF-HSA，通过培养、诱导表达，再依次经过金属离子亲和层析和阴离子交换层析纯化，即得本发明所需酿酒酵母表达人rVEGF-HSA融合蛋白。
2.根据权利要求1所述的一种酿酒酵母表达人rVEGF-HSA融合蛋白制备方法，其特征在于，所述步骤(1)中，设计并获得rVEGF-HSA基因序列和融合蛋白rVEGF-HSA的氨基酸序列，具体步骤为：
根据pYES2/CT-MFα载体的性质和酿酒酵母宿主密码子偏爱性，设计了人rVEGF-HSA基因序列和人rVEGF-HSA的氨基酸序列，人rVEGF-HSA基因序列如序列表1所示，人rVEGF-HSA的氨基酸序列如序列表2所示。
3.根据权利要求1所述的一种酿酒酵母表达人rVEGF-HSA融合蛋白制备方法，其特征在于，所述步骤(1)中，根据rVEGF-HSA核苷酸序列设计并扩增目的基因，回收并双酶切PCR产物和pYES2/CT-MFα质粒，用T4 DAN连接酶连接，转入E.coli DH5α感受态细胞培养，获得阳性克隆pYES2/CT-MFα-rVEGF-HSA，具体步骤为：
根据人rVEGF-HSA核苷酸序列设计扩增引物，其中：
正向引物：5'ATAAGAATGCGGCCGCAATGGCTCCA 3'；
反向引物：5'CTAGTCTAGATTAGTGATGGTGATGG 3'；
进行PCR扩增；
配制1％琼脂糖凝胶，电泳分离PCR扩增产物，紫外灯下快速切下目的条带，用DNA凝胶回收试剂盒回收目的基因PCR扩增产物；
提取DH5α/pYES2/CT-MFα中的pYES2/CT-MFα质粒；
将质粒pYES2/CT-MFα和回收的PCR产物分别用Not I和Xba I进行双酶切，并胶回收双酶切的PCR产物和质粒pYES2/CT-MFα；
将双酶切回收的PCR扩增产物与pYES2/CT-MFα质粒用T4 DNA连接酶在37℃中连接约30min；
将连接产物转化到E.coliDH5α感受态细胞中，经Amp抗性筛选后挑取阳性转化子进行培养，获得阳性克隆pYES2/CT-MFα-rVEGF-HSA。
4.根据权利要求1所述的一种酿酒酵母表达人rVEGF-HSA融合蛋白制备方法，其特征在于，所述步骤(2)中，酿酒酵母表达体系常用溶液及培养基的配制，具体方法为：
YPD培养基：蛋白胨20g，酵母提取物10g，加纯化水定容至900ml，121℃高压灭菌20min，冷却至60℃以下，超净台中加入无菌100ml 20×葡萄糖，固体培养基另加琼脂粉2.0％；
SC-U缺陷培养基：YNB无氨基酸氮源6.70g，0.01％氨基酸混合物I1g，0.005％氨基酸混合物II 0.5g，加蒸馏水定容至900ml，121℃高压灭菌20min，冷却至60℃以下，超净台中加入无菌100ml 20％葡萄糖，固体培养基另加琼脂粉2.0％；
其中，所述0.01％氨基酸混合物I为精氨酸、亮氨酸、苏氨酸、赖氨酸、色氨酸、半胱氨酸和腺嘌呤；所述0.005％氨基酸混合物II为天冬氨酸、丝氨酸、组氨酸、脯氨酸、异亮氨酸、苯丙氨酸、撷氨酸、酪氨酸和甲硫氨酸；
SC-U诱导培养基：蛋白胨20g，酵母提取物10g，加纯化水定容至700ml，121℃高压灭菌20min，冷却至60℃以下，超净台中加入无菌100ml 20％半乳糖，固体培养基另加琼脂粉2.0％。
5.根据权利要求1所述的一种酿酒酵母表达人rVEGF-HSA融合蛋白制备方法，其特征在于，所述步骤(2)中，获得阳性克隆子INVSc1/pYES2/CT-MFα-rVEGF-HSA的具体步骤为：
采用电转化法将pYES2/CT-MFα-rVEGF-HSA转化至酿酒酵母INVSc1感受态细胞：
将10μl pYES2/CT-MFα-rVEGF-HSA质粒加到80μl酿酒酵母INVScl感受态细胞中，吹吸使其混合均匀，然后转移到预冷的电击杯中，冰浴5min，擦干电击杯外壁；
将Bio-Rad电转化仪调至真菌档，PIC选项，电击杯置于Bio-Rad电转化仪上电击，迅速向电击杯中加入500μl预冷的1M山梨醇溶液，混合均匀，涂SC-U板；
30℃恒温倒置培养，直至长出单克隆；
挑去转化子接种到SC-U液体培养基中，30℃、200rpm恒温培养；
以培养的菌液为模板进行PCR反应，筛选INVSc1/pYES2/CT-MFα-rVE GF-HSA阳性克隆子。
6.根据权利要求1所述的一种酿酒酵母表达人rVEGF-HSA融合蛋白制备方法，其特征在于，所述步骤(3)中，
INVSc1/pYES2/CT-MFα-rVEGF-HSA工程菌的诱导表达，具体步骤为：
挑取INVSc1/pYES2/CT-MFα-rVEGF-HSA单菌落接种于20ml SC-U选择培养基，经30℃、220rpm震荡培养过夜，测定其OD600nm吸光值，计算好相应体积的菌液转接至于100ml SC-U诱导培养基中，使得初始OD600nm达到0.4，诱导时间为24h；
诱导表达的上清液经离心并通过0.22μm滤膜过滤，收集过滤液。
7.根据权利要求1所述的一种酿酒酵母表达人rVEGF-HSA融合蛋白制备方法，其特征在于，所述步骤(3)中，金属离子亲和层析的步骤为：
取离心并过0.22μm滤膜过滤后的过滤液，使用GE Healthcare公司Ch elatingSepharose TM Fast Flow镍离子螯合亲和层析填料自行装柱，用3个柱体积的纯化水清洗Ni2+螯合亲和层析柱，再用PBS平衡2-3个柱体积；
在线检测电导率值及280nm波长吸收值，待两者都稳定后开始上样，设置样品经过泵过层析柱的流速5-6ml/min；
再用PBS过层析柱，洗去未与层析柱结合的杂蛋白，直到OD280nm稳定，再以含500mM咪唑PBS缓冲液过层析柱，洗脱并收集洗脱峰对应的蛋白，即得到经过金属离子亲和层析后的人rVEGF-HSA蛋白原液。
8.根据权利要求1所述的一种酿酒酵母表达人rVEGF-HSA融合蛋白制备方法，其特征在于，所述步骤(3)中，阴离子交换层析的步骤为：
将金属离子亲和层析纯化后收集的蛋白原液置换到Tris缓冲液中后上样，通过用Tris缓冲液平衡好的DEAE阴离子交换层析柱，收集rVEGF-HS A蛋白峰，Tris-NaCl洗脱液洗脱，洗去杂蛋白，即为本发明获得的纯化后的人rVEGF-HSA融合蛋白。
9.一种人rVEGF-HSA融合蛋白标准品的制备方法，其特征在于，包括以下步骤：
取权利要求1-8任一项制备方法制得的人rVEGF-HSA融合蛋白溶液，用0.22μm滤膜过滤除菌，用10mM磷酸缓冲液稀释后加入终浓度为10％甘油、0.12g/ml甘露醇、0.025g/ml蔗糖冻干保护剂，进行冷冻真空干燥，干燥后即为人rVEGF-HSA融合蛋白标准品。
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