CN110551200A - 一种成纤维细胞生长因子-18的生产方法及在制备促毛发制剂方面的应用 - Google Patents
一种成纤维细胞生长因子-18的生产方法及在制备促毛发制剂方面的应用 Download PDFInfo
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Abstract
本发明公开了一种成纤维细胞生长因子‑18的生产方法及在制备促毛发制剂方面的应用,该基因序列如SEQ ID NO.1所示。本发明还公开了一种用于生产FGF‑18的植物表达载体,命名为pNT3301‑FGF18;本发明还公开一种利用烟草作为生物反应器生产FGF‑18的方法;通过脱发小鼠模型鉴定了其对毛发再生的功效;利用烟草表达FGF‑18较大肠杆菌系统及动物反应器有着诸多优势,因此,因此利用植物生产要用蛋白FGF‑18具有更好的应用前景。
Description
技术领域
本发明属于基因工程领域,具体是指一种成纤维细胞生长因子-18的生产方法及在制备促毛发制剂方面的应用。
背景技术
成纤维细胞生长因子-18(FGF-18)属于FGF8亚家族的一员,1998年由日本科学家Ohbayashi首次从老鼠胚胎中分离得到。全长的FGF18基因是由207个氨基酸组成的高度保守序列,其中包含着两个潜在的N-连接糖基化位点。FGF-18主要在成年的肺和肾以及皮肤中表达,同时也在胚胎发育过程中一些离散区域表达。FGF-18具有广泛的生理功能,包括骨骼的生长和发育,毛发的生长和皮肤再生,皮质神经元活动和形态发生、血管形成。FGF-18与FGFR2c和FGFR3c具有高度亲和性,其通过自分泌和旁分泌作用活化FGFR2c受体促进成骨细胞的增殖与分化,从而影响骨的形成,FGF-18活化FGFR3c受体促进软骨细胞增殖,抑制软骨细胞分化。FGF-18能促进毛囊由休止期向生长期的转换,并促进毛囊再生,研究发现FGF-18是通过上调Wnt5α激活经典Wnt信号通路,调节毛囊的再生与毛发的生长。
尽管已有文献报道大肠杆菌和拟南芥表达系统能够实现FGF18的表达,但由于表达体系不稳定以及表达量太低使其难以达到产业化的要求。烟草作为双子叶模式植物具有生长周期短,生物量大,易于栽培等其他植物不可比拟的优势,与动物反应器相比,植物反应器产物不含热源与内毒素,也不会产生伦理方面的讨论,因此,烟草作为植物生物反应器为人类提供了一种安全和廉价的生产系统。
发明内容
本发明实施例所要解决的技术问题在于,提供一种成纤维细胞生长因子-18的生产方法及在制备促毛发制剂方面的应用。
为实现本发明的第一个目的,本发明的第一个目的是提供一种成纤维细胞生长因子-18,该基因的编码方式具有符合烟草密码子偏好性的特质,命名为FGF-18,其碱基序列如SEQ ID NO.1所示。
本发明的第二个目的是提供一种用于生产权利要求1所述成纤维细胞生长因子-18的植物叶绿体表达载体,该载体以商业化的pCAMBIA3301为骨架将GUS基因替换成烟草密码子偏好性的FGF-18基因,并在5’-UTR区加入了68bp的Ω增强子序列构建的载体,命名为pNT3301-FGF18该载体以商业化的pCAMBIA3301为骨架将GUS基因替换成烟草密码子偏好性的FGF-18基因,并在5’-UTR区加入了68bp的Ω增强子序列构建的载体,命名为pNT3301-FGF18,所述的FGF-18基因的碱基序列如SEQ ID NO.1所示。
本发明第三个目的是还提供一种携带权利要求2所述的载体,该载体为携带所述植物表达载体pNT3301-FGF18的农杆菌EHA105。
本发明第四个目的是提供一种利用烟草作为植物反应器生产成纤维细胞生长因子-18的方法,所述的成纤维细胞生长因子-18如权利要求1所示。
进一步设置是包括以下步骤:
(1)构建植物表达载体pNT3301-FGF18;
(2)利用烟草高通量遗传转化技术实现FGF18基因对烟草的遗传转化,获得大量烟草抗性植株;
(3)利用高通量筛选技术,即Taqman PCR和western点杂交对烟草抗性植株进行鉴定,筛选出拷贝数低、表达量高的转基因株系,并通过标准的southern和western杂交对其进行鉴定,ELISA进行定量。
本发明第五个目的是提供一种基于所述的成纤维细胞生长因子-18在用于制备出毛发再生制剂上的应用。该应用中,将提取转化烟草株系NCF-365的总可溶蛋白与cream乳剂涂抹于脱发小鼠的背部,并设置野生型烟草总可溶蛋白和cream作为阴性对照,观察小鼠毛发再生的情况,通过组织HE染色检测NCF-365表达FGF18毛发再生的效果。
在上述技术方案中,步骤1中构建植物表达载体pNT3301-FGF18具体为:
(1)利用Gene Designer将FGF18cDNA采用烟草密码子偏好性进行密码子替换,同时利用Vector NTI 11.0消除序列上第24位的BamH I和第437位的ApaL I酶切位点,使优化后的基因序列不含任何酶切位点,优化后的FGF-18基因序列如SEQ ID NO.1所示;
(2)优化后的FGF18的5’端加入Ω序列经由化学合成后连入克隆载体pUC19载体中,命名为pUC19-FGF18。利用限制性内切酶NcoI和BstEII将FGF18基因从该载体上切下,连入同样处理的植物表达载体pCAMBIA3301中,成功构建FGF18基因的植物表达载体命名为pNT3301-FGF18。
步骤2中利用烟草高通量遗传转化技术,实现FGF18基因对烟草的遗传转化。选择具有高频再生率的烟草品种“Petit Havana SR1”作为遗传转化的受体材料,将至少3000个烟草叶片用手术刀或手术剪切或剪成两半,接种到预培养培养基上,培养室光照培养7天作为遗传转化的外植体。预培养结束后将外植体置于携带植物表达载体pNT3301-FGF18的农杆菌EHA105的侵染培养基中处理10min,弃去菌液,放到共培养培养基上,暗培养3天。共培养后,把外植体置于抑菌培养基上,光照培养(16小时光照8小时黑暗)7天。随后把外植体转移到筛选/再生培养基上筛选,每两周继代一次,待胚状体长到1cm左右(通常会自己从胚状体丛上脱落),将其接种到萌发培养基萌发。约20天后,胚状体萌发获得再生苗。其中,预培养培养基具体为:MS培养基4.74g,蔗糖30g,水解酪蛋白2g/L,2,4-D 2.00mg/L,KT 0.25mg/L和8g/L琼脂,pH5.8;侵染培养基具体为:MS培养基4.74g,蔗糖30g,MES 2g/L,2,4-D2.00mg/L,KT 0.25mg/L和8g/L琼脂,pH5.8;共培养培养基具体为:MS培养基4.74g,蔗糖30g,水解酪蛋白2g/L,2,4-D 2.00mg/L,KT 0.25mg/L和8g/L琼脂,pH5.8;抑菌养培养基具体为:MS培养基4.74g,蔗糖30g,水解酪蛋白2g/L,2,4-D 2.00mg/L,KT 0.25mg/L,250mg/Lcef,250mg/L carb和8g/L琼脂,pH5.8;筛选/再生培养基具体为:MS培养基4.74g,蔗糖30g,水解酪蛋白2g/L,2,4-D 2.00mg/L,KT 0.25mg/L,125mg/L cef,125mg/L carb,1.5mg/Lbasta,pH=5.8,琼脂粉8g/L。
步骤3中利用高通量筛选平台技术对所获得的烟草植株进行检测具体为:
(1)磁珠法提取烟草总DNA为模板进行Taqman PCR检查;Taqman PCR检查使用的引物为:引物P1,碱基序列如SEQ ID NO.2所示;引物P2:碱基序列如SEQ ID NO.3所示;TaqmanPCR检查的反应体系:2×TransStart Green qPCR SuperMix 10μL,引物P1 0.4μL,引物P20.4μL,模板DNA 2μL,Passive Reference Dye(50×)0.4μL,ddH2O 6.8μL;Taqman PCR程序为:94℃预变性30sec;94℃变性5sec,54℃退火15sec,72℃退火10sec,40个循环;根据第15个循环到第40个循环之间不同样品荧光淬灭的曲线去除非转基因植株和多拷贝的转化事件。
(2)将低拷贝烟草植株叶片在液氮中研磨成粉末,按1:2(W/V)比例加入PBS缓冲液,其中,PBS缓冲液为137mmol/L NaCl,2.7mmol/L KCl,10mmol/L Na2HPO4,2mmol/LKH2PO4,冰浴至完全融化;涡旋30sec;在4℃条件下15000g离心20min,取1μL上清液滴在PVDF膜上,用TBST缓冲液洗涤6次,每次5min,其中,TBST缓冲液为20mmol/L Tris-HCl,150mmol/L NaCl,0.05%Tween 20;加入封闭液100mL,37℃封闭2h,其中,封闭液为5%脱脂奶粉/TBST缓冲液;倒掉封闭液,按上述方法洗涤后加入100mL用封闭液稀释的一抗鼠抗FGF18工作液,37℃包被1h;倒掉一抗工作液,如前述方法洗涤,加入100mL用封闭液稀释的二抗碱性磷酸酶标记的羊抗鼠,包被1h;倒掉二抗工作液,如前法洗涤,加入10mL显色剂BCIP/NBT,室温暗光反应直至出现预期杂交信号后,用50ml双蒸水或TE缓冲液洗膜5min,以终止反应。选择杂交信号最强即FGF18表达量高的3个转化株系NCF-365、NCF-669、NCF-1470进行进一步检测。
(3)将野生型烟草和经点杂交筛选出的转基因烟草高表达植株的叶片剪下,在液氮中研磨成粉末用CTAB法提取总DNA。取10μg DNA用EcoR I酶切,0.8%琼脂凝胶80V电泳3个小时后转移到硝酸纤维素膜上,紫外交联两次,每次30秒,间隔2分钟。按照DIG HighPrime DNA Labeling and Detection Starter Kit I试剂盒提供的方法进行Southernblot分析,杂交探针为35S Promoter、FGF18、NOS terminater之间大小为1.4kb的DNA片段和Trans15K Marker。根据不同样品所在泳道出现杂交信号的条带数分析外源基因在基因组中的拷贝数。
(4)将野生型烟草和经点杂交筛选出的转基因烟草高表达植株的叶片剪下,在液氮中研磨成粉末,按1:2(W/V)比例加入PBS缓冲液,其中,PBS缓冲液为137mmol/L NaCl,2.7mmol/L KCl,10mmol/L Na2HPO4,2mmol/L KH2PO4,冰浴至完全融化;涡旋30sec;在4℃条件下15000g离心20min,取上清即为总可溶性蛋白;
取10μL蛋白溶液与等量2×上样缓冲液混合,沸水温浴5min,然后进行10%SDS–PAGE电泳检测,并用考马斯亮蓝R250染色,将相同条件下电泳后未进行考马斯亮蓝R250染色的凝胶,在200mA恒定电流条件下通过半干转膜仪转印到0.2μm的尼龙膜上,用TBST缓冲液洗涤6次,每次5min,其中,TBST缓冲液为20mmol/L Tris-HCl,150mmol/L NaCl,0.05%Tween 20;加入封闭液100mL,37℃封闭2h,其中,封闭液为5%脱脂奶粉/TBST缓冲液;倒掉封闭液,按上述方法洗涤后加入100mL用封闭液稀释的一抗鼠抗bFGF工作液,37℃包被1h;倒掉一抗工作液,如前述方法洗涤,加入100mL用封闭液稀释的二抗碱性磷酸酶标记的羊抗鼠,包被1h;倒掉二抗工作液,如前法洗涤,加入10mL显色剂BCIP/NBT,室温暗光反应直至出现预期杂交信号后,用50ml双蒸水或TE缓冲液洗膜5min,终止反应。
(5)按照(4)中的方式,提取蛋白。ELISA进行定量。
ELISA试剂盒使用前:预先将所需试剂取出,室温下平衡20min。配制1×WashBuffer和1×Assay Buffer(取母液2.5ml+47.5ml去离子水);确定实验所需孔数,配制Biotin-Conjugate及Streptavidin-HRP(用Assay Buffer稀释100倍);按照说明配制标准溶液,同时按一定的倍数用Sample Diluent稀释样品;用约400μl Wash Buffer润洗板子,停留10-15s后甩干,重复润洗一次,甩干后在吸水纸上轻拍扣干,去除残余的Wash Buffer;加入100μl标准溶液至标准品孔,加100μl待测样品至样品孔,各设置两复孔,用100μlSample Diluent作为空白对照。每孔加入100μl预先配置好的Biotin-Conjugate溶液,置于恒温混匀仪中,18-25℃,400rpm孵育2h;孵育完毕,甩去孔内液体,加入200μl 1×WashBuffer停留10-15s后甩干,在吸水纸上轻拍扣干,重复洗涤3次;每孔里加入100μl预先配制好的Streptavidin-HRP,18-25℃400rpm孵育1h,重复洗涤3次;每孔加入100μl TMBSubstrate Solution,避光孵育10-30min,当颜色最深的孔在620nm处吸光度值达到0.9-0.95时加入100μl Stop Buffer;用酶标仪在450nm处读吸光度值。
(6)鉴定FGF18毛发再生功效乳剂的制备:棕榈酸2g,乙醇0.875mL,烟草提取液2.5mL(FGF18约含150μg),苄索氯铵50mg。将上述材料在50℃下剧烈搅拌,得到均匀稳定的乳膏,加载值可以使得所得制剂含有1.0mg/mL至约10.0mg/mL。制好的乳剂涂于脱毛小鼠裸露的皮肤上。每次间隔24h。每3天检测一次毛发生长的情况。15天处死小鼠收集小鼠的皮肤。
(7)皮肤组织HE染色:将取下的皮肤样本组织固定与切片,切片在石蜡切片机上进行,厚度在5-10um左右。将切完后的组织切片放入盛有自来水的盆中,然后置42℃温水浸泡使组织充分伸展,大概5-10s;切片自然风干过夜,置于60℃烘箱中2-4h,用之前用烘箱烘20min,趁热加入二甲苯;组织样本脱蜡:将待测组织样本切片放于二甲苯中,充分浸泡15min,浸泡完后更换二甲苯继续浸泡15min;将浸泡过二甲苯的待测组织样本先放入无水乙醇中浸泡5min,取出再次在无水乙醇中浸泡5min,使脱蜡时用的二甲苯可以被洗脱出去,使水可以进入组织中;再依次置于95%、90%、80%、70%乙醇;移液枪吸取已经预先配制好的苏木素染色液,每个组织切片滴加100ul,充分染色7-10min。染色完毕后使用蒸馏水洗去多余的苏木素染色液。然后再使用分化液分化30s,用双蒸水将组织切片冲洗干净;向上一步驟的组织样本切片中加入伊红染液,并使组织可以充分染色几秒-3min,染色完毕后,再将组织切片进行梯度脱水。80%的乙醇脱水5s,95%乙醇脱水2min,无水乙醇脱水2min;将脱水后的组织样本切片使用二甲苯浸泡2次,每次持续15min,吸走二甲苯,并使用中性树胶封片。
本发明的有益效果是:利用烟草表达FGF-18较大肠杆菌系统及动物表达系统有独特的优势,且表达出的蛋白具有很好的促毛发再生的生物活性,相对其他表达系统植物表达系统生产成纤维细胞生长因子18具有更好的应用前景。
本发明通过农杆菌转化受体植物烟草(Petit Havana SR1)获得了大量的转化植株,通过高通量筛选出一株高表达株系。其FGF-18的表达量占总可溶蛋白的了1%,达到了产业化的要求。通过涂抹小鼠脱发模型,发现烟草表达的FGF-18具有促进脱发小鼠毛发再生的功能。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,根据这些附图获得其他的附图仍属于本发明的范畴。
图1为植物表达载体pNT3301-FGF18的结构图;
图2为烟草的遗传转化与筛选过程;
图3为烟草转基因植株的western点杂交,其中,A1:野生型烟草植株;A2-G12:不同外植体来源的烟草转基因株系;
图4为烟草转基因植株的Southern杂交结果,其中,M:DNA Marker;+:pNT3301-FGF18质粒;-:野生型烟草;1-3:分别为转基因烟草株系NCF-365、NCF-669、NCF-1470。
图5为转基因烟草的Western检测与ELISA定量结果;
图6为FGF18涂抹脱发小鼠的组织切片HE染色,其中:(A)为脱发小鼠模型给药前的组织切片HE染色(B)给药后的组织切片HE染色。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步地详细描述。
实施例1:构建植物表达载体pNT3301-FGF18
1)、在保持FGF-18序列不发生改变的前提下,利用Gene Designer将编码基因的所有密码子采用烟草密码子最适编码方式进行密码子替换,对经修饰后的基因序列再通过Vector NTI 10.0软件消除序列上第24位的BamH I和第437位的ApaL I酶切位点,优化化后的FGF-18基因序列见SEQ ID NO.1所示;
2)、构建植物表达载体pNT3301-FGF18,经密码子优化后的FGF18经由化学合成。利用限制性内切酶Nco I和BstE II将FGF-18基因切下,与经相同内切酶处理过的植物表达载体pCAMBIA3301相连接,成功构建FGF-18基因的植物表达载体命名为pNT3301-FGF18(如图1所示)。
实施例2:FGF-18基因对烟草的高通量遗传转化
选择具有高频再生率的烟草品种“Petit Havana SR1”作为遗传转化的受体材料,将至少3000个烟草叶片用手术刀或手术剪切或剪成两半,接种到预培养培养基(MS培养基4.74g,蔗糖30g,水解酪蛋白2g/L,2,4-D 2.00mg/L,KT 0.25mg/L和8g/L琼脂,pH5.8)上,培养室光照培养7天作为遗传转化的外植体。预培养结束后将外植体置于携带植物表达载体pCAMBIA3301-msKGF2的农杆菌EHA105的侵染培养基(MS培养基4.74g,蔗糖30g,MES 2g/L,2,4-D 2.00mg/L,KT 0.25mg/L和8g/L琼脂,pH5.8)中处理10min,弃去菌液,放到共培养培养基(MS培养基4.74g,蔗糖30g,水解酪蛋白2g/L,2,4-D 2.00mg/L,KT0.25mg/L和8g/L琼脂,pH5.8)上,暗培养3天。共培养后,把外植体置于抑菌培养基(MS培养基4.74g,蔗糖30g,水解酪蛋白2g/L,2,4-D 2.00mg/L,KT0.25mg/L,250mg/L cef,250mg/L carb和8g/L琼脂,pH5.8)上,光照培养(16小时光照8小时黑暗)7天。随后把外植体转移到筛选/再生培养基(MS培养基4.74g,蔗糖30g,水解酪蛋白2g/L,2,4-D 2.00mg/L,KT 0.25mg/L,125mg/L cef,125mg/L carb,1.5mg/L basta,pH=5.8,琼脂粉8g/L)上筛选,每两周继代一次,待胚状体长到1cm左右(通常会自己从胚状体丛上脱落),将其接种到萌发培养基(MS培养基4.74g,蔗糖30g,125mg/L cef,125mg/L carb,1.5mg/L basta,pH=5.8,琼脂粉8g/L)萌发,约20天后,胚状体萌发获得再生苗,经统计最终获得2026株抗性再生植株。
实施例3:利用Taqman PCR技术筛选出低拷贝烟草转化植株
采用磁珠法提取全部所获得2026株烟草抗性再生植株的总DNA,由于磁珠吸附DNA的最大承载量一定,因此提取后的DNA浓度基本一致,因而可以以该方法提取的烟草总DNA为模板进行Taqman PCR检查;Taqman PCR检查使用的引物为:引物P1,碱基序列如SEQ IDNO.2所示;引物P2:,碱基序列如SEQ ID NO.3所示;Taqman PCR检查的反应体系:2×TransStart Green qPCR SuperMix 10μL,引物P1 0.4μL,引物P2 0.4μL,模板DNA 2μL,Passive Reference Dye(50×)0.4μL,ddH2O 6.8μL;Taqman PCR程序为:94℃预变性30sec;94℃变性5sec,54℃退火15sec,72℃退火10sec,40个循环;根据第15个循环到第40个循环之间不同样品荧光淬灭的曲线去除非转基因植株和多拷贝的转化事件。经检测初步从所获得的2026株抗性烟草再生植株中筛选出107株低拷贝烟草转基因植株。
实施例4:通过点杂交对转基因植株进行表达量分析
将所获得的318株低拷贝转基因植株叶片在液氮中研磨成粉末,按1:2(W/V)比例加入PBS缓冲液,其中,PBS缓冲液为137mmol/L NaCl,2.7mmol/LKCl,10mmol/L Na2HPO4,2mmol/L KH2PO4,冰浴至完全融化;涡旋30sec;在4℃条件下15000g离心20min,取1μL上清液滴在PVDF膜上,用TBST缓冲液洗涤6次,每次5min,其中,TBST缓冲液为20mmol/L Tris-HCl,150mmol/L NaCl,0.05%Tween 20;加入封闭液100mL,37℃封闭2h,其中,封闭液为5%脱脂奶粉/TBST缓冲液;倒掉封闭液,按上述方法洗涤后加入100mL用封闭液稀释的一抗鼠抗FGF18工作液,37℃包被1h;倒掉一抗工作液,如前述方法洗涤,加入100mL用封闭液稀释的二抗碱性磷酸酶标记的羊抗鼠,包被1h;倒掉二抗工作液,如前法洗涤,加入10mL显色剂BCIP/NBT,室温暗光反应直至出现预期杂交信号后,用50ml双蒸水或TE缓冲液洗膜5min,以终止反应。点杂交结果如图3所示,点A1为野生型植株,其余点为转基因烟草的杂交斑点。经杂交信号强弱的比较分析最终确定了3株杂交信号最强即FGF18表达量高的烟草转基因植株,分别为NCF-365、NCF-669、NCF-1470。
实施例5:通过southern-blot对转基因植株进行拷贝数分析
将野生型烟草植株和经点杂交筛选出的转基因烟草高表达植株的叶片剪下,在液氮中研磨成粉末用CTAB法提取总DNA。取10μg DNA用PsiI酶切,0.8%琼脂凝胶80V电泳3个小时后转移到硝酸纤维素膜上,紫外交联两次,每次30秒,间隔2分钟。按照DIG High PrimeDNA Labeling and Detection Starter Kit I试剂盒提供的方法进行Southern blot分析,杂交探针为35S Promoter、FGF18、NOS terminater之间大小为1.4kb的DNA片段和Trans15K Marker。杂交结果如图4所示,转基因株系NCF-365为单拷贝转化株系,NCF-669、NCF-1470为多拷贝转化株系。
实施例6:通过western-blot对转基因植株进行鉴定ELISA进行定量
取10μL蛋白溶液与等量2×上样缓冲液混合,沸水温浴5min,然后进行12%SDS–PAGE电泳检测,并用考马斯亮蓝R250染色,将相同条件下电泳后未进行考马斯亮蓝R250染色的凝胶,在200mA恒定电流条件下通过半干转膜仪转印到0.2μm的尼龙膜上,用TBST缓冲液洗涤6次,每次5min,其中,TBST缓冲液为20mmol/L Tris-HCl,150mmol/L NaCl,0.05%Tween 20;加入封闭液100mL,37℃封闭2h,其中,封闭液为5%脱脂奶粉/TBST缓冲液;倒掉封闭液,按上述方法洗涤后加入100mL用封闭液稀释的一抗鼠抗FGF18工作液,37℃包被1h;倒掉一抗工作液,如前述方法洗涤,加入100mL用封闭液稀释的二抗碱性磷酸酶标记的羊抗鼠,包被1h;倒掉二抗工作液,如前法洗涤,加入10mL显色剂BCIP/NBT,室温暗光反应直至出现预期杂交信号后,用50ml双蒸水或TE缓冲液洗膜5min,以终止反应,ELISA定量实验表明NCF-365的FGF18表达量占总可溶蛋白的1.19%。
实施例7:通过制备的促毛发再生乳剂检测FGF18的生物学功效
FGF18毛发再生功效乳剂的制备:棕榈酸2g,乙醇0.875mL,烟草提取液2.5mL(FGF18约含150μg),苄索氯铵50mg。将上述材料在50℃下剧烈搅拌,得到均匀稳定的乳膏,加载值可以使得所得制剂含有1.0mg/mL至约10.0mg/mL。制好的乳剂涂于脱毛小鼠裸露的皮肤上。每次间隔24h。每3天检测一次毛发生长的情况。15天处死小鼠收集小鼠的皮肤。
将取下的皮肤样本组织固定与切片,切片在石蜡切片机上进行,厚度在5-10um左右。将切完后的组织切片放入盛有自来水的盆中,然后置42℃温水浸泡使组织充分伸展,大概5-10s;切片自然风干过夜,置于60℃烘箱中2-4h,用之前用烘箱烘20min,趁热加入二甲苯;组织样本脱蜡:将待测组织样本切片放于二甲苯中,充分浸泡15min,浸泡完后更换二甲苯继续浸泡15min;将浸泡过二甲苯的待测组织样本先放入无水乙醇中浸泡5min,取出再次在无水乙醇中浸泡5min,使脱蜡时用的二甲苯可以被洗脱出去,使水可以进入组织中;再依次置于95%、90%、80%、70%乙醇;移液枪吸取已经预先配制好的苏木素染色液,每个组织切片滴加100ul,充分染色7-10min。染色完毕后使用蒸馏水洗去多余的苏木素染色液。然后再使用分化液分化30s,用双蒸水将组织切片冲洗干净;向上一步驟的组织样本切片中加入伊红染液,并使组织可以充分染色几秒-3min,染色完毕后,再将组织切片进行梯度脱水。80%的乙醇脱水5s,95%乙醇脱水2min,无水乙醇脱水2min;将脱水后的组织样本切片使用二甲苯浸泡2次,每次持续15min,吸走二甲苯,并使用中性树胶封片。
以上所揭露的仅为本发明较佳实施例而已,当然不能以此来限定本发明之权利范围,因此依本发明权利要求所作的等同变化,仍属本发明所涵盖的范围。
序列表
<110> 温州大学
<120> 一种成纤维细胞生长因子-18的生产方法及在制备促毛发制剂方面的应用
<130> 2015
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 546
<212> DNA序列
<213> FGF-18的核酸序列
<400> 1
ATGGAGGAGAACGTGGACTTTAGAATCCACGTGGAGAACCAGACTAGAGCTAGAGACGACGTGTCTAGGAAGCAGCTTAGGCTTTACCAGCTTTACTCTAGAACTTCTGGAAAGCACATCCAGGTGCTTGGAAGGAGAATCTCTGCTAGAGGTGAGGATGGAGACAAGTACGCTCAGCTTTTGGTTGAGACAGACACTTTCGGATCTCAGGTGAGAATCAAGGGAAAGGAAACTGAATTTTATCTTTGTATGAACAGAAAGGGAAAACTGGTGGGAAAGCCTGACGGAACTTCTAAGGAGTGCGTGTTCATCGAGAAGGTGCTTGAGAACAACTACACTGCTCTTATGTCTGCTAAGTACTCTGGATGGTACGTGGGATTCACAAAGAAGGGAAGGCCTAGGAAGGGTCCAAAGACTAGGGAGAACCAGCAAGACGTCCACTTCATGAAGAGATACCCTAAGGGACAGCCTGAGCTTCAGAAGCCTTTCAAGTACACTACTGTGACTAAGAGGTCTAGGAGGATCAGGCCTACTCATCCAGCTTAA
<210> 2
<211> 19
<212> DNA引物
<213> 人工合成序列
<400> 2
ATGGAGGAGAACGTG 15
<210> 3
<211> 20
<212> DNA引物
<213> 人工合成序列
<400> 3
TTAAGCTGGATGAGT 15
Claims (8)
1.一种成纤维细胞生长因子-18,其特征在于:该基因的编码方式具有符合烟草密码子偏好性的特质,命名为FGF-18,其碱基序列如SEQ ID NO.1所示。
2.一种用于生产权利要求1所述成纤维细胞生长因子-18的植物叶绿体表达载体,其特征在于:该载体以商业化的pCAMBIA3301为骨架将GUS基因替换成烟草密码子偏好性的FGF-18基因,并在5’-UTR区加入了68bp的Ω增强子序列构建的载体,命名为pNT3301-FGF18。
3.一种携带权利要求2所述的载体,其特征在于:该载体为携带所述植物表达载体pNT3301-FGF18的农杆菌EHA105。
4.一种利用烟草作为植物反应器生产成纤维细胞生长因子-18的方法,其特征在于:所述的成纤维细胞生长因子-18如权利要求1所示。
5.根据权利要求3所示的一种方法,其特征在于包括以下步骤:
(1)构建植物表达载体pNT3301-FGF18;
(2)利用烟草高通量遗传转化技术实现FGF18基因对烟草的遗传转化,获得大量烟草抗性植株;
(3)利用高通量筛选技术,即Taqman PCR和western点杂交对烟草抗性植株进行鉴定,筛选出拷贝数低、表达量高的转基因株系,并通过标准的southern和western杂交对其进行鉴定,ELISA进行定量。
6.一种含有权利要求4所述的成纤维细胞生长因子-18在用于制备出毛发再生制剂。
7.根据权利要求6所述的制剂,其特征在于:该制剂还包括有医学或药学上可接受的载体或者添加剂。
8.根据权利要求6所述的制剂,其特征在于:还包括有棕榈酸、乙醇和苄索氯铵。
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Application publication date: 20191210 |