CN107383182A - 大肠杆菌表达的rhFGF7蛋白及应用 - Google Patents
大肠杆菌表达的rhFGF7蛋白及应用 Download PDFInfo
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- CN107383182A CN107383182A CN201710233150.0A CN201710233150A CN107383182A CN 107383182 A CN107383182 A CN 107383182A CN 201710233150 A CN201710233150 A CN 201710233150A CN 107383182 A CN107383182 A CN 107383182A
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Abstract
大肠杆菌表达的rhFGF7蛋白及应用。本发明公开了一种重组的成纤维细胞生长因子Halo‑rhFGF7基因，在于插入表达载体pET14b中，构建表达载体pET14b‑Halo‑rhFGF7，转化到大肠杆菌BL21(DE3)pLysS感受态中；进行培养；收集的菌体重悬于裂解缓冲液，收集上清；洗脱、纯化得Halo‑rhFGF7；用rTEV蛋白酶酶解、纯化，得rhFGF7；实验证明重组的成纤维细胞生长因子Halo‑rhFGF7和重组的成纤维细胞生长因子rhFGF7，对BaF3细胞和L‑O2细胞的促增殖作用，缓解CCL4损伤L‑O2细胞的作用，保护肝细胞急性损伤的信号通路方面均高于rhFGF7标准品；细胞毒性也有所降低。
Description
技术领域
本发明属分子生物学领域，具体涉及大肠杆菌表达的Halo-tag-rhFGF7蛋白及制备治疗急性肝损伤药物中的应用。
背景技术
成纤维细胞生长因子-7（FGF7）是成纤维细胞生长因子家族成员之一，1989年由Rubin等人从人胚胎性肺成纤维细胞首先分离到的。FGF7是一种由间质细胞分泌、以上皮细胞为靶细胞的肝素结合性细胞因子。它只与FGFR2IIIb型受体特异性结合[2],继而引起胞内一系列信号传递和基因表达，在上皮细胞的生长、分化、迁移等过程中起重要作用。随后的研究发现，该蛋白在上皮组织发育和损伤的预防、修复及肿瘤的发生[8]中发挥重要的生物学作用。FGF7促进上皮细胞生长和分化，对肝脏细胞也有强大的影响。证明激活后的HSC表达的FGF7对刺激肝细胞生长因子信号通路和调节肝再生有促进作用。体外实验证明FGF7可以抑制肝细胞调亡，对肝细胞有暂时的保护作用。Bohm等人（2010）报道，重组FGF7的使用可以增加肝组织手术切除处理后的存活率。
FGF7具有组织特异性高、致癌作用低、生物活性强等优点，其重组蛋白产品（商品名为Palifermin）已被美国 FDA 批准用于治疗放化疗引起的急性口腔黏膜炎。FGF7还有很多潜在应用，如治疗糖尿病等代谢性疾病引起的慢性伤口、治疗早产儿的表面活性剂不足和支气管肺发育不良（BPD）、治疗急性肺损伤、急性肝损伤等，均显示了较好的前景。鉴于FGF7在研究和应用方面有重要意义，研究者们围绕FGF7表达进行了大量的工作。重组FGF7及其突变体曾在大肠杆菌和哺乳动物细胞CHO及植物中表达，但是仍存在一定的问题。这些重组FGF7蛋白在表达中表现出对宿主菌的毒性、表达量低、稳定性差、易降解等问题，从而限制了相关机制深入研究和药物研发的进程。前期研究表明，利用Halo-tag与蛋白融合表达，获得高产量。Halo-tag 是分子质量单位为33 kDa的蛋白标签, 由微生物紫红红球菌的脱卤素酶演化而来，该酶可与合成配体共价结合。Halo-tag 可以在原核表达系统（大肠埃希菌表达系统）或真核表达系统中与目的蛋白的N端或C端融合表达且不破坏目的蛋白功能。
发明内容
本发明的目的是为了提高成纤维细胞生长因子-7的活性，而提供一种重组成纤维细胞生长因子-7Halo-rhFGF7。
重组的成纤维细胞生长因子Halo-rhFGF7基因，它的碱基序列如SEQ ID NO.1所示；
重组的成纤维细胞生长因子Halo-rhFGF7蛋白，它是碱基序列如SEQ ID NO.1所示的基因表达的蛋白；
表达载体pET14b-Halo-rhFGF7，它是在表达载体中插入了碱基序列如SEQ ID NO.1所示的基因；
一种大肠杆菌工程菌，它是上述的表达载体pET14b-Halo-rhFGF7转化到大肠杆菌BL21(DE3)pLysS感受态中。
重组的成纤维细胞生长因子rhFGF7，它是由下述方法制备的：
1）将上述的一种大肠杆菌工程菌，在30mL含氨苄青霉素和氯霉素的LB培养基中以37℃、180 rpm的条件进行培养。当OD600达到0.8-1.0时，将培养物转移到300mL含胰蛋白胨（10g/L），酵母粉（10g/L），KH2PO4（1g/L），K2HPO4（3g/L），氯化钠（4g/L）的改良培养基中并以180rpm、37℃为条件进行振荡培养；收集的菌体重悬于裂解缓冲液，收集上清；
2）洗脱、纯化；
3）用rTEV蛋白酶酶解、纯化，得rhFGF7。
重组的成纤维细胞生长因子Halo-rhFGF7或重组的成纤维细胞生长因子rhFGF7制备治疗急性肝损伤药物中的应用。
本发明提供了重组的成纤维细胞生长因子Halo-rhFGF7基因，插入表达载体pET14b中，构建表达载体pET14b-Halo-rhFGF7， 转化到大肠杆菌BL21(DE3)pLysS感受态中；进行培养；收集的菌体重悬于裂解缓冲液，收集上清；洗脱、纯化得Halo-rhFGF7；用rTEV蛋白酶酶解、纯化，得rhFGF7。实验证明重组的成纤维细胞生长因子Halo-rhFGF7和重组的成纤维细胞生长因子rhFGF7，对BaF3细胞和L-O2细胞的促增殖作用，缓解CCL4损伤L-O2细胞的作用，保护肝细胞急性损伤的信号通路方面均高于rhFGF7标准品；细胞毒性也有所降低。
附图说明：
图1FGF7目的基因和重组载体双酶切鉴定；
图2重组Halo-rhFGF7蛋白表产物的鉴定；
图3 重组Halo-rhFGF7蛋白的检测及纯化；
图4 重组Halo-rhFGF7蛋白的酶切和rhFGF7蛋白的纯化；
图5 rhFGF7对BaF3细胞和L-O2细胞的促增殖作用；
图6 hFGF7缓解CCL4损伤L-O2细胞的作用；
图7 hFGF7缓解CCL4损伤L-O2的镜下细胞形态；
图8rhFGF7保护肝细胞急性损伤的信号通路。
具体实施方式：
实施例1 重组Halo-rhFGF7表达载体的构建及表达产物的检测
（1）FGF7基因序列合成与重组表达载体构建
根据Genbank(NM002009.3)中人FGF7cDNA基因序列进行密码子偏好性优化，上游引入Nco I酶切位点，下游引入BamH I酶切位点。全序列由金维智生物科技有限公司合成。
上游引物：CCATGGCATGCAATGATATGAC
下游引物：CCTAGGAATCCATTACCGGTAG
合成的质粒与pET14b-Halo-tag表达载体分别用Nco I和BamH I内切酶进行酶切，回收目的片段（如图1a所示），采用常规T4DNA连接酶连接，连接产物采用CaCl2法转化到大肠杆菌DH5α感受态中。用氨苄青霉素和氯霉素筛选出阳性克隆，进一步扩增后，提取质粒，进行双酶切鉴定和测序鉴定，测序鉴定由Invitrogen英潍捷基（上海）贸易有限公司完成。结果表明，成功构建了重组表达载体pET14b-Halo-rhFGF7，如图1b所示。Halo-rhFGF7碱基序列如SEQ ID NO.1所示；rhFGF7碱基序列如SEQ ID NO.2所示，Halo-rhFGF7氨基酸序列如SEQ IDNO.2所示，rhFGF7氨基酸序列如SEQ ID NO.2所示。
（2）重组表达菌株的获得及表达产物检测
采用CaCl2法，将表达载体pET14b-Halo-rhFGF7转化到大肠杆菌BL21(DE3)pLysS感受态中。挑取单菌落接种至含氨苄青霉素（100μg/mL）和氯霉素（34μg/mL）的5 mL LB培养基中，37℃、180 rpm摇床培养过夜后按1%比例接种至新鲜含氨苄青霉素和氯霉素的5 mLLB培养基中，37℃、180 rpm培养至菌液OD600达到0.6-0.8时，加入终浓度为1 mmol/L的异丙基硫代-β-D-半乳糖苷（IPTG），诱导表达4h后离心收集菌体。分别取诱导前和诱导后的菌液进行12% SDS-PAGE电泳分析。
结果表明，与诱前相比，诱导后可见明显的蛋白表达条带，分子量约为54kDa（如图2a所示），与预期大小相符。通过Western blot对该重组蛋白进行鉴定，结果显示所表达的蛋白具有与FGF7单克隆抗体特异性结合的能力，说明利用该表达载体成功获得了重组Halo-rhFGF7蛋白，如图2b所示。
实施例2 重组Halo-rhFGF7的大量发酵及蛋白纯化
（1）重组表达菌株大量发酵体系建立
将上述获得的重组表达菌在30mL含氨苄青霉素和氯霉素的LB培养基中以37℃、180rpm的条件进行培养。当OD600达到0.8-1.0时，将培养物转移到300mL含胰蛋白胨（10g/L），酵母粉（10g/L），KH2PO4（1g/L），K2HPO4（3g/L），氯化钠（4g/L）的改良培养基中并以180rpm、37℃为条件进行振荡培养。当菌液OD600达到0.8-1.0时将培养物转移到600mL上述改良培养基中培养，至菌液OD600达到0.8-1.2时，加入终浓度为1mmol/L的异丙基硫代-β-D-半乳糖苷（IPTG），16℃、180rpm诱导过夜后，收集菌液在4℃、9000rpm的条件下离心10min收集菌体。
将上述收集的菌体按1:10（g/mL）比例重悬于裂解缓冲液（50mmol/L Tris-HCl，100 mmol/L NaCl，5 mmol/L EDTA ，1%Triton-X-100，1mmol/L PMSF，5mmol/L DTT，pH7.8）中，充分混匀后冰浴超声破碎（300w, 10s/10s, 20min），20000rpm，4℃离心30min，收集上清和沉淀进行12% SDS-PAGE电泳分析，如图3a所示，目的蛋白主要以可溶的形式在重组菌种中表达。
（2）重组目的蛋白rhFGF7的洗脱及纯化
为获得大量可溶性的Halo-rhFGF7，发酵上清依次采用SP柱、肝素柱纯化、TEV蛋白酶酶解、再过肝素柱的方法。
①SP柱洗脱。SP(SP-sepharose)柱用buffer A（50mmol/L Tris-HCl，100mmol/LNaCl，pH 7.2）平衡。将可溶性样品上样后分别用buffer B（50mmol/L Tris-HCl，300mmol/LNaCl，pH 7.2)、buffer C（50mmol/L Tris-HCl，600mmol/L NaCl，pH 7.2)、buffer D（50mmol/L Tris-HCl，1mol/L NaCl，pH 7.2)梯度洗脱，根据280 nm紫外吸收值收集洗脱峰。取各个梯度的收集液进行12%SDS-PAGE分析。结果表明，目的蛋白主要在600mmol/LNaCl被洗脱下来，如图3b所示，为进一步除去杂带，进行肝素柱洗脱。
②肝素柱洗脱。将肝素柱先用buffer A平衡，按照如上洗脱梯度对含有目的蛋白的收集液进行肝素柱纯化。取各个梯度的收集液进行12%SDS-PAGE分析。纯化后的蛋白经超滤管(15kDa)在6000g、4℃的条件下超滤除盐。结果表明，600mmol/L NaCl的洗脱液，经肝素柱洗脱后，杂带几乎除尽，如图3c所示。洗脱的蛋白超滤后经薄层扫描分析表明，其纯度在98%以上，如图3d所示。经BCA试剂盒测定纯化的Halo-rhFGF7蛋白，其浓度为400μg/mL。
③rTEV蛋白酶酶解及纯化。取上述重组蛋白液2.5mL，参照rTEV Protease重组烟草蚀纹病毒蛋白酶使用说明书进行酶切，经12%SDS-PAGE电泳分析后，确认蛋白被充分切开，如图4a所示，将酶切产物按照如上所述的洗脱梯度进行肝素柱纯化，取各个梯度的收集液进行12%SDS-PAGE分析。结果表明，目的蛋白主要在1mol/L NaCl被洗脱下来，如图4b所示。洗脱后的蛋白经超滤管(3000kDa)在6000g、4℃的条件下超滤除盐后，经薄层扫描分析表明，其纯度在96%以上，如图4c所示。
实施例3重组rhFGF7促增殖活性检测
采用DMEM培养基（10 %FBS、1%双抗）培养人胚胎正常肝细胞株L-O2细胞。取对数生长期L-O2细胞，稀释至5×104 /mL的单细胞悬液。以100 μL/孔接种于96孔细胞培养板中，于37℃、5%CO2的培养箱中培养24 h后，更换为含0. 5%FBS的饥饿培养基培养24h，取出培养板，加入不同浓度的Halo-rhFGF7、rhFGF7和FGF7标准品作用48h后，每孔加入20μLMTT继续培养，4h后吸弃孔内培养上清液，每孔加入150uL DMSO，反复吹打，使结晶物充分溶解。检测490nm下各孔吸光度。结果表明，Halo-rhFGF7、rhFGF7和FGF7标准品对L-O2细胞和小鼠BaF3细胞具有明显的促增殖作用，且存在剂量依赖性，在浓度为2nmol/L时作用达到最高。三种蛋白活性比较，rhFGF7蛋白最高，Halo-rhFGF7蛋白次之，FGF7标准品最低，如图5a所示。
实施例4重组rhFGF7促有丝分裂活性检测
采用1640培养基（含10%FBS、1%双抗，2.5ng/mLIL-3，600μg/mLG-418），37℃、5% CO2培养小鼠BaF3细胞(转FGFR2IIIb型受体)。取96孔板每孔加入90uL含0.5%FBS的饥饿培养基，加入不同终浓度的Halo-rhFGF7、rhFGF7和FGF7标准品。再取对数生长期的BaF3细胞，稀释至3×105 /mL的单细胞悬液，并以50 μL/孔接种于96孔细胞培养板中，于37℃、5% CO2条件下培养48h后，每孔加入15μL的CCK-8再培养2h，在450nm下测其吸光度。结果表明，Halo-rhFGF7、rhFGF7和FGF7标准品对小鼠BaF3细胞无明显的促有丝分裂作用，如图5b所示。
实施例5重组rhFGF7缓解CCL4损伤L-O2细胞的作用
（1）L-O2损伤条件的建立
取对数生长期L-O2细胞接种于96孔板，按实施例4所述培养24 h后更换为饥饿培养基培养24h，取出培养板，加入不同浓度CCL4（分别为正常对照组、CCL4模型组5 mmol/L、CCL4模型组10 mmol/L、CCL4模型组15 mmol/L、CCL4模型组20 mmol/L、CCL4模型组25 mmol/L、CCL4模型组30 mmol/L），在37℃、5%CO2条件下分别培养3h、6h、9h、12 h、24h 后，利用MTT法测定各孔吸光度。根据如下公式计算存活率。
存活率（%）=（处理组吸光度-空白对照组吸光度）/（正常对照组吸光度-空白对照组吸光度）×100%
结果表明，25mmol/L的CCL4损伤12h细胞的存活率约为50%（P<0.01）。因此，选用25mmol/LCCL4作用12h作为L-O2细胞的损伤模型，如图6a所示。
（2）重组rhFGF7缓解CCL4损伤L-O2
取对数生长期L-O2细胞制成5×104 /mL的细胞悬液，按每孔100 μ L接种于96孔板，孵育24h后，更换为饥饿培养基培养24h，取出培养板，加入不同浓度的Halo-rhFGF7、rhFGF7和FGF7标准品作用4h，再按照25mmol/LCCL4条件对其进行损伤，采用MTT法测定各孔吸光度。结果表明，三种蛋白对损伤后的L-O2细胞都有明显的保护作用，且存在剂量依赖性，但浓度为13.04nmol/L时都达到平台期。三种蛋白的作用比较，rhFGF7最高，Halo-rhFGF7次之，FGF7标准品最低，如图6b所示。
实施例6重组rhFGF7对肝细胞急性损伤的体外保护作用
通过检测谷丙转氨酶（ALT）、谷草转氨酶（AST）、丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH-Px）水平，评价Halo-rhFGF7、rhFGF7和FGF7标准品对肝细胞急性损伤的体外保护作用。
取对数生长期L-O2细胞制备成5×104 /mL的细胞悬液，按每孔2mL接种于6孔板中，孵育48h后，更换为饥饿培养基培养24h，取出培养板，分组如下：（1）正常对照组：无CCL4处理；（2）CCL4模型组；（3）Halo-rhFGF7组；（4）rhFGF7组；（5）FGF7标准品组。各组加药作用4h后，除对照组外，其余各组按照一定条件对其进行损伤。显微镜下观察各组细胞形态后，取各组上清按照ALT、AST的说明书进行酶活性测定。收集细胞，破碎后按MDA和GSH-PX试剂盒说明书进行各组的测定。
结果表明，正常组L-O2细胞状态良好，贴壁生长，呈多边形，分界清楚，表面光滑。与正常组比较，CCL4模型组细数明显减少，细胞状态明显不佳，可见细胞凋亡。各给药组细胞生长状态明显优于模型组，细胞结构较清晰，皱缩程度较轻，脱落减少，尤其rhFGF7组作用最明显，如图7所示。
如表1显示，与正常对照组相比，CCL4模型组的ALT、AST和MDA水平显著增加（P＜0.01），GSH-Px活力显著降低（P＜0.01）。与模型组相比，三个给药组的ALT、AST和MDA水平显著降低（P＜0.05），GSH-Px活力显著升高（P＜0.05），且Halo-rhFGF7、rhFGF7和FGF7标准品组间ALT、AST、MDA和GSH-Px活力含量有显著差异（P＜0.05）。其中Halo-rhFGF7作用最强，rhFGF7次之，FGF7标准品最低。
实施例7重组rhFGF7保护肝细胞急性损伤的信号通路
为阐明重组rhFGF7保护肝细胞急性损伤的作用机制，通过Western blot检测了Halo-rhFGF7、rhFGF7和FGF7标准品对保护作用的指标p38 MAPK和ERK1/2的磷酸化水平，以及细胞凋亡的关键指标Caspase 3、Bax、Bcl-2水平。
采用Halo-rhFGF7、rhFGF7和FGF7标准品如上所述处理急性损伤的肝细胞，常规方法获取细胞蛋白裂解液，经12% SDS-PAGE电泳分离后，以恒流300mA转膜90min到PVDF膜上。取下PVDF膜，用TBST(8g/L NaCl，0.2g/L KCl，3g/LTris-Cl，0.1% (v/v) Tween 20，pH =7.4)配制的5%脱脂牛奶37℃封闭1.5h，洗膜后，与兔抗人一抗FGF7、Bcl-2、Bax、Caspase-3、p-ERK、ERK、p-p38、p-38和β-actin（1：1000）4℃摇床孵育过夜，洗膜后与HRP标记的羊抗兔二抗 ( 1:8000）常温孵育1 h，用TBST充分洗膜后，滴加混匀的ECL化学发光剂，置凝胶成像系统显影，实验结果采用Image Lab软件进行灰度值分析。
结果表明，CCL4损伤组的Caspase 3蛋白表达明显提高，而Halo-rhFGF7、rhFGF7和FGF7标准品三个加药组的Caspase 3蛋白表达明显下降，其中rhFGF7效果最好，如图8a所示。如图8b所示，三个加药组的ERK1/2和p38 MAPK总蛋白的表达无明显变化，但磷酸化的ERK1/2和p38 MAPK水平都有提高，其中rhFGF7的作用最强。如图8c所示，rhFGF7的Bcl-2表达水平最高、Bax表达水平最低。综上表明，rhFGF7的保护作用最强，其次是Halo-rhFGF7和FGF7标准品。
<110> 温州医科大学；李校堃
<120> 大肠杆菌表达的rhFGF7蛋白及应用
<160> 4
<210> 1
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<212> DNA
<213> 人工
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atgccagaaa tcggtactgg ctttccattc gacccccatt atgtggaagt cctgggcgag 60
cgcatgcact acgtcgatgt tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt 120
aacccgacct cctcctacgt gtggcgcaac atcatcccgc atgttgcacc gacccatcgc 180
tgcattgctc cagacctgat cggtatgggc aaatccgaca aaccagacct gggttatttc 240
ttcgacgacc acgtccgctt catggatgcc ttcatcgaag ccctgggtct ggaagaggtc 300
gtcctggtca ttcacgactg gggctccgct ctgggtttcc actgggccaa gcgcaatcca 360
gagcgcgtca aaggtattgc atttatggag ttcatccgcc ctatcccgac ctgggacgaa 420
tggccagaat ttgcccgcga gaccttccag gccttccgca ccaccgacgt cggccgcaag 480
ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg 540
ctgactgaag tcgagatgga ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag 600
ccactgtggc gcttcccaaa cgagctgcca atcgccggtg agccagcgaa catcgtcgcg 660
ctggtcgaag aatacatgga ctggctgcac cagtcccctg tcccgaagct gctgttctgg 720
ggcaccccag gcgttctgat cccaccggcc gaagccgctc gcctggccaa aagcctgcct 780
aactgcaagg ctgtggacat cggcccgggt ctgaatctgc tgcaagaaga caacccggac 840
ctgatcggca gcgagatcgc gcgctggctg tcgacgctcg agatttccgg cgagccaacc 900
actgaggatc tgtactttca gagctccatg gcatgcaatg atatgacccc ggagcagatg 960
gccaccaatg tgaattgcag cagcccggaa cgccataccc gcagctatga ctacatggaa 1020
ggcggtgata tccgcgtgcg ccgcctgttt tgccgtaccc agtggtatct gcgcatcgat 1080
aagcgcggca aagtgaaagg cacccaggaa atgaaaaata attacaatat tatggaaatt 1140
cgcaccgtgg ccgtgggcat tgtggccatc aaaggcgtgg aaagcgagtt ctacctggcc 1200
atgaacaaag aaggtaaact gtacgccaag aaggagtgca acgaagattg caattttaaa 1260
gaactgattc tggaaaatca ttacaatacc tatgcaagcg ccaaatggac ccacaatggc 1320
ggcgaaatgt tcgtggccct gaatcagaaa ggcattccgg tgcgcggcaa gaagaccaag 1380
aaagagcaga agaccgccca ttttctgccg atggccatta cctaaggatc c 1431
<210> 2
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<212> DNA
<213> 人工
<400> 2
ccatggcatg caatgatatg accccggagc agatggccac caatgtgaat tgcagcagcc 60
cggaacgcca tacccgcagc tatgactaca tggaaggcgg tgatatccgc gtgcgccgcc 120
tgttttgccg tacccagtgg tatctgcgca tcgataagcg cggcaaagtg aaaggcaccc 180
aggaaatgaa aaataattac aatattatgg aaattcgcac cgtggccgtg ggcattgtgg 240
ccatcaaagg cgtggaaagc gagttctacc tggccatgaa caaagaaggt aaactgtacg 300
ccaagaagga gtgcaacgaa gattgcaatt ttaaagaact gattctggaa aatcattaca 360
atacctatgc aagcgccaaa tggacccaca atggcggcga aatgttcgtg gccctgaatc 420
agaaaggcat tccggtgcgc ggcaagaaga ccaagaaaga gcagaagacc gcccattttc 480
tgccgatggc cattacctaa ggatcc 506
<210> 3
<211> 1430
<212> PRT
<213> 人工合成
<400> 3
Ala Thr Gly Cys Cys Ala Gly Ala Ala Ala Thr Cys Gly Gly Thr Ala
5 10 15
Cys Thr Gly Gly Cys Thr Thr Thr Cys Cys Ala Thr Thr Cys Gly Ala
20 25 30
Cys Cys Cys Cys Cys Ala Thr Thr Ala Thr Gly Thr Gly Gly Ala Ala
35 40 45
Gly Thr Cys Cys Thr Gly Gly Gly Cys Gly Ala Gly Cys Gly Cys Ala
50 55 60
Thr Gly Cys Ala Cys Thr Ala Cys Gly Thr Cys Gly Ala Thr Gly Thr
65 70 75 80
Thr Gly Gly Thr Cys Cys Gly Cys Gly Cys Gly Ala Thr Gly Gly Cys
85 90 95
Ala Cys Cys Cys Cys Thr Gly Thr Gly Cys Thr Gly Thr Thr Cys Cys
100 105 110
Thr Gly Cys Ala Cys Gly Gly Thr Ala Ala Cys Cys Cys Gly Ala Cys
115 120 125
Cys Thr Cys Cys Thr Cys Cys Thr Ala Cys Gly Thr Gly Thr Gly Gly
130 135 140
Cys Gly Cys Ala Ala Cys Ala Thr Cys Ala Thr Cys Cys Cys Gly Cys
145 150 155 160
Ala Thr Gly Thr Thr Gly Cys Ala Cys Cys Gly Ala Cys Cys Cys Ala
165 170 175
Thr Cys Gly Cys Thr Gly Cys Ala Thr Thr Gly Cys Thr Cys Cys Ala
180 185 190
Gly Ala Cys Cys Thr Gly Ala Thr Cys Gly Gly Thr Ala Thr Gly Gly
195 200 205
Gly Cys Ala Ala Ala Thr Cys Cys Gly Ala Cys Ala Ala Ala Cys Cys
210 215 220
Ala Gly Ala Cys Cys Thr Gly Gly Gly Thr Thr Ala Thr Thr Thr Cys
225 230 235 240
Thr Thr Cys Gly Ala Cys Gly Ala Cys Cys Ala Cys Gly Thr Cys Cys
245 250 255
Gly Cys Thr Thr Cys Ala Thr Gly Gly Ala Thr Gly Cys Cys Thr Thr
260 265 270
Cys Ala Thr Cys Gly Ala Ala Gly Cys Cys Cys Thr Gly Gly Gly Thr
275 280 285
Cys Thr Gly Gly Ala Ala Gly Ala Gly Gly Thr Cys Gly Thr Cys Cys
290 295 300
Thr Gly Gly Thr Cys Ala Thr Thr Cys Ala Cys Gly Ala Cys Thr Gly
305 310 315 320
Gly Gly Gly Cys Thr Cys Cys Gly Cys Thr Cys Thr Gly Gly Gly Thr
325 330 335
Thr Thr Cys Cys Ala Cys Thr Gly Gly Gly Cys Cys Ala Ala Gly Cys
340 345 350
Gly Cys Ala Ala Thr Cys Cys Ala Gly Ala Gly Cys Gly Cys Gly Thr
355 360 365
Cys Ala Ala Ala Gly Gly Thr Ala Thr Thr Gly Cys Ala Thr Thr Thr
370 375 380
Ala Thr Gly Gly Ala Gly Thr Thr Cys Ala Thr Cys Cys Gly Cys Cys
385 390 395 400
Cys Thr Ala Thr Cys Cys Cys Gly Ala Cys Cys Thr Gly Gly Gly Ala
405 410 415
Cys Gly Ala Ala Thr Gly Gly Cys Cys Ala Gly Ala Ala Thr Thr Thr
420 425 430
Gly Cys Cys Cys Gly Cys Gly Ala Gly Ala Cys Cys Thr Thr Cys Cys
435 440 445
Ala Gly Gly Cys Cys Thr Thr Cys Cys Gly Cys Ala Cys Cys Ala Cys
450 455 460
Cys Gly Ala Cys Gly Thr Cys Gly Gly Cys Cys Gly Cys Ala Ala Gly
465 470 475 480
Cys Thr Gly Ala Thr Cys Ala Thr Cys Gly Ala Thr Cys Ala Gly Ala
485 490 495
Ala Cys Gly Thr Thr Thr Thr Thr Ala Thr Cys Gly Ala Gly Gly Gly
500 505 510
Thr Ala Cys Gly Cys Thr Gly Cys Cys Gly Ala Thr Gly Gly Gly Thr
515 520 525
Gly Thr Cys Gly Thr Cys Cys Gly Cys Cys Cys Gly Cys Thr Gly Ala
530 535 540
Cys Thr Gly Ala Ala Gly Thr Cys Gly Ala Gly Ala Thr Gly Gly Ala
545 550 555 560
Cys Cys Ala Thr Thr Ala Cys Cys Gly Cys Gly Ala Gly Cys Cys Gly
565 570 575
Thr Thr Cys Cys Thr Gly Ala Ala Thr Cys Cys Thr Gly Thr Thr Gly
580 585 590
Ala Cys Cys Gly Cys Gly Ala Gly Cys Cys Ala Cys Thr Gly Thr Gly
595 600 605
Gly Cys Gly Cys Thr Thr Cys Cys Cys Ala Ala Ala Cys Gly Ala Gly
610 615 620
Cys Thr Gly Cys Cys Ala Ala Thr Cys Gly Cys Cys Gly Gly Thr Gly
625 630 635 640
Ala Gly Cys Cys Ala Gly Cys Gly Ala Ala Cys Ala Thr Cys Gly Thr
645 650 655
Cys Gly Cys Gly Cys Thr Gly Gly Thr Cys Gly Ala Ala Gly Ala Ala
660 665 670
Thr Ala Cys Ala Thr Gly Gly Ala Cys Thr Gly Gly Cys Thr Gly Cys
675 680 685
Ala Cys Cys Ala Gly Thr Cys Cys Cys Cys Thr Gly Thr Cys Cys Cys
690 695 700
Gly Ala Ala Gly Cys Thr Gly Cys Thr Gly Thr Thr Cys Thr Gly Gly
705 710 715 720
Gly Gly Cys Ala Cys Cys Cys Cys Ala Gly Gly Cys Gly Thr Thr Cys
725 730 735
Thr Gly Ala Thr Cys Cys Cys Ala Cys Cys Gly Gly Cys Cys Gly Ala
740 745 750
Ala Gly Cys Cys Gly Cys Thr Cys Gly Cys Cys Thr Gly Gly Cys Cys
755 760 765
Ala Ala Ala Ala Gly Cys Cys Thr Gly Cys Cys Thr Ala Ala Cys Thr
770 775 780
Gly Cys Ala Ala Gly Gly Cys Thr Gly Thr Gly Gly Ala Cys Ala Thr
785 790 795 800
Cys Gly Gly Cys Cys Cys Gly Gly Gly Thr Cys Thr Gly Ala Ala Thr
805 810 815
Cys Thr Gly Cys Thr Gly Cys Ala Ala Gly Ala Ala Gly Ala Cys Ala
820 825 830
Ala Cys Cys Cys Gly Gly Ala Cys Cys Thr Gly Ala Thr Cys Gly Gly
835 840 845
Cys Ala Gly Cys Gly Ala Gly Ala Thr Cys Gly Cys Gly Cys Gly Cys
850 855 860
Thr Gly Gly Cys Thr Gly Thr Cys Gly Ala Cys Gly Cys Thr Cys Gly
865 870 875 880
Ala Gly Ala Thr Thr Thr Cys Cys Gly Gly Cys Gly Ala Gly Cys Cys
885 890 895
Ala Ala Cys Cys Ala Cys Thr Gly Ala Gly Gly Ala Thr Cys Thr Gly
900 905 910
Thr Ala Cys Thr Thr Thr Cys Ala Gly Ala Gly Cys Thr Cys Cys Ala
915 920 925
Thr Gly Gly Cys Ala Thr Gly Cys Ala Ala Thr Gly Ala Thr Ala Thr
930 935 940
Gly Ala Cys Cys Cys Cys Gly Gly Ala Gly Cys Ala Gly Ala Thr Gly
945 950 955 960
Gly Cys Cys Ala Cys Cys Ala Ala Thr Gly Thr Gly Ala Ala Thr Thr
965 970 975
Gly Cys Ala Gly Cys Ala Gly Cys Cys Cys Gly Gly Ala Ala Cys Gly
980 985 990
Cys Cys Ala Thr Ala Cys Cys Cys Gly Cys Ala Gly Cys Thr Ala Thr
995 1000 1005
Gly Ala Cys Thr Ala Cys Ala Thr Gly Gly Ala Ala Gly Gly Cys Gly
1010 1015 1020
Gly Thr Gly Ala Thr Ala Thr Cys Cys Gly Cys Gly Thr Gly Cys Gly
1025 1030 1035 1040
Cys Cys Gly Cys Cys Thr Gly Thr Thr Thr Thr Gly Cys Cys Gly Thr
1045 1050 1055
Ala Cys Cys Cys Ala Gly Thr Gly Gly Thr Ala Thr Cys Thr Gly Cys
1060 1065 1070
Gly Cys Ala Thr Cys Gly Ala Thr Ala Ala Gly Cys Gly Cys Gly Gly
1075 1080 1085
Cys Ala Ala Ala Gly Thr Gly Ala Ala Ala Gly Gly Cys Ala Cys Cys
1090 1095 1100
Cys Ala Gly Gly Ala Ala Ala Thr Gly Ala Ala Ala Ala Ala Thr Ala
1105 1110 1115 1120
Ala Thr Thr Ala Cys Ala Ala Thr Ala Thr Thr Ala Thr Gly Gly Ala
1125 1130 1135
Ala Ala Thr Thr Cys Gly Cys Ala Cys Cys Gly Thr Gly Gly Cys Cys
1140 1145 1150
Gly Thr Gly Gly Gly Cys Ala Thr Thr Gly Thr Gly Gly Cys Cys Ala
1155 1160 1165
Thr Cys Ala Ala Ala Gly Gly Cys Gly Thr Gly Gly Ala Ala Ala Gly
1170 1175 1180
Cys Gly Ala Gly Thr Thr Cys Thr Ala Cys Cys Thr Gly Gly Cys Cys
1185 1190 1195 1200
Ala Thr Gly Ala Ala Cys Ala Ala Ala Gly Ala Ala Gly Gly Thr Ala
1205 1210 1215
Ala Ala Cys Thr Gly Thr Ala Cys Gly Cys Cys Ala Ala Gly Ala Ala
1220 1225 1230
Gly Gly Ala Gly Thr Gly Cys Ala Ala Cys Gly Ala Ala Gly Ala Thr
1235 1240 1245
Thr Gly Cys Ala Ala Thr Thr Thr Thr Ala Ala Ala Gly Ala Ala Cys
1250 1255 1260
Thr Gly Ala Thr Thr Cys Thr Gly Gly Ala Ala Ala Ala Thr Cys Ala
1265 1270 1275 1280
Thr Thr Ala Cys Ala Ala Thr Ala Cys Cys Thr Ala Thr Gly Cys Ala
1285 1290 1295
Ala Gly Cys Gly Cys Cys Ala Ala Ala Thr Gly Gly Ala Cys Cys Cys
1300 1305 1310
Ala Cys Ala Ala Thr Gly Gly Cys Gly Gly Cys Gly Ala Ala Ala Thr
1315 1320 1325
Gly Thr Thr Cys Gly Thr Gly Gly Cys Cys Cys Thr Gly Ala Ala Thr
1330 1335 1340
Cys Ala Gly Ala Ala Ala Gly Gly Cys Ala Thr Thr Cys Cys Gly Gly
1345 1350 1355 1360
Thr Gly Cys Gly Cys Gly Gly Cys Ala Ala Gly Ala Ala Gly Ala Cys
1365 1370 1375
Cys Ala Ala Gly Ala Ala Ala Gly Ala Gly Cys Ala Gly Ala Ala Gly
1380 1385 1390
Ala Cys Cys Gly Cys Cys Cys Ala Thr Thr Thr Thr Cys Thr Gly Cys
1395 1400 1405
Cys Gly Ala Thr Gly Gly Cys Cys Ala Thr Thr Ala Cys Cys Thr Ala
1410 1415 1420
Ala Gly Gly Ala Thr Cys
1425 1430
<210> 4
<211> 505
<212> PRT
<213> 人工合成
<400> 4
Cys Cys Ala Thr Gly Gly Cys Ala Thr Gly Cys Ala Ala Thr Gly Ala
5 10 15
Thr Ala Thr Gly Ala Cys Cys Cys Cys Gly Gly Ala Gly Cys Ala Gly
20 25 30
Ala Thr Gly Gly Cys Cys Ala Cys Cys Ala Ala Thr Gly Thr Gly Ala
35 40 45
Ala Thr Thr Gly Cys Ala Gly Cys Ala Gly Cys Cys Cys Gly Gly Ala
50 55 60
Ala Cys Gly Cys Cys Ala Thr Ala Cys Cys Cys Gly Cys Ala Gly Cys
65 70 75 80
Thr Ala Thr Gly Ala Cys Thr Ala Cys Ala Thr Gly Gly Ala Ala Gly
85 90 95
Gly Cys Gly Gly Thr Gly Ala Thr Ala Thr Cys Cys Gly Cys Gly Thr
100 105 110
Gly Cys Gly Cys Cys Gly Cys Cys Thr Gly Thr Thr Thr Thr Gly Cys
115 120 125
Cys Gly Thr Ala Cys Cys Cys Ala Gly Thr Gly Gly Thr Ala Thr Cys
130 135 140
Thr Gly Cys Gly Cys Ala Thr Cys Gly Ala Thr Ala Ala Gly Cys Gly
145 150 155 160
Cys Gly Gly Cys Ala Ala Ala Gly Thr Gly Ala Ala Ala Gly Gly Cys
165 170 175
Ala Cys Cys Cys Ala Gly Gly Ala Ala Ala Thr Gly Ala Ala Ala Ala
180 185 190
Ala Thr Ala Ala Thr Thr Ala Cys Ala Ala Thr Ala Thr Thr Ala Thr
195 200 205
Gly Gly Ala Ala Ala Thr Thr Cys Gly Cys Ala Cys Cys Gly Thr Gly
210 215 220
Gly Cys Cys Gly Thr Gly Gly Gly Cys Ala Thr Thr Gly Thr Gly Gly
225 230 235 240
Cys Cys Ala Thr Cys Ala Ala Ala Gly Gly Cys Gly Thr Gly Gly Ala
245 250 255
Ala Ala Gly Cys Gly Ala Gly Thr Thr Cys Thr Ala Cys Cys Thr Gly
260 265 270
Gly Cys Cys Ala Thr Gly Ala Ala Cys Ala Ala Ala Gly Ala Ala Gly
275 280 285
Gly Thr Ala Ala Ala Cys Thr Gly Thr Ala Cys Gly Cys Cys Ala Ala
290 295 300
Gly Ala Ala Gly Gly Ala Gly Thr Gly Cys Ala Ala Cys Gly Ala Ala
305 310 315 320
Gly Ala Thr Thr Gly Cys Ala Ala Thr Thr Thr Thr Ala Ala Ala Gly
325 330 335
Ala Ala Cys Thr Gly Ala Thr Thr Cys Thr Gly Gly Ala Ala Ala Ala
340 345 350
Thr Cys Ala Thr Thr Ala Cys Ala Ala Thr Ala Cys Cys Thr Ala Thr
355 360 365
Gly Cys Ala Ala Gly Cys Gly Cys Cys Ala Ala Ala Thr Gly Gly Ala
370 375 380
Cys Cys Cys Ala Cys Ala Ala Thr Gly Gly Cys Gly Gly Cys Gly Ala
385 390 395 400
Ala Ala Thr Gly Thr Thr Cys Gly Thr Gly Gly Cys Cys Cys Thr Gly
405 410 415
Ala Ala Thr Cys Ala Gly Ala Ala Ala Gly Gly Cys Ala Thr Thr Cys
420 425 430
Cys Gly Gly Thr Gly Cys Gly Cys Gly Gly Cys Ala Ala Gly Ala Ala
435 440 445
Gly Ala Cys Cys Ala Ala Gly Ala Ala Ala Gly Ala Gly Cys Ala Gly
450 455 460
Ala Ala Gly Ala Cys Cys Gly Cys Cys Cys Ala Thr Thr Thr Thr Cys
465 470 475 480
Thr Gly Cys Cys Gly Ala Thr Gly Gly Cys Cys Ala Thr Thr Ala Cys
485 490 495
Cys Thr Ala Ala Gly Gly Ala Thr Cys
500 505
Claims (6)
1.重组的成纤维细胞生长因子Halo-rhFGF7基因，其特征在于，它的碱基序列如SEQ IDNO.1所示。
2. 重组的成纤维细胞生长因子Halo-rhFGF7蛋白，其特征在于，它是碱基序列如SEQID NO.1所示的基因表达的蛋白。
3. 表达载体pET14b-Halo-rhFGF7，其特征在于，它是在表达载体pET14b中插入了碱基序列如SEQ ID NO.1所示的基因。
4.一种大肠杆菌工程菌，其特征在于，它是上述的表达载体pET14b-Halo-rhFGF7转化到大肠杆菌BL21(DE3)pLysS感受态中。
5.重组的成纤维细胞生长因子rhFGF7，其特征在于，它是由下述方法制备的：
1）将权利要求4所述的一种大肠杆菌工程菌，在30mL含氨苄青霉素和氯霉素的LB培养基中以37℃、180 rpm的条件进行培养；
当OD600达到0.8-1.0时，将培养物转移到300mL含胰蛋白胨10g/L、酵母粉10g/L，KH2PO4 1g/L，K2HPO4 3g/L，氯化钠 4g/L的改良培养基中，180rpm、37℃进行振荡培养；收集的菌体重悬于裂解缓冲液，收集上清和沉淀；
2）洗脱、纯化；
3）用rTEV蛋白酶酶解、纯化，得rhFGF7。
6.根据权利要求2所述的重组的成纤维细胞生长因子Halo-rhFGF7或权利要求5所述的重组的成纤维细胞生长因子rhFGF7制备治疗急性肝损伤药物中的应用。
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| CN114958889A (zh) * | 2022-04-13 | 2022-08-30 | 广东省科学院动物研究所 | 一种高活性fgf7蛋白的制备方法 |
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