CN108440670B - 融合蛋白hfbi-rgd作为疏水性材料分散剂的用途 - Google Patents
融合蛋白hfbi-rgd作为疏水性材料分散剂的用途 Download PDFInfo
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Abstract
本发明公开了融合蛋白HFBI‑RGD作为疏水性材料分散剂的用途，所述融合蛋白HFBI‑RGD的氨基酸序列是SEQ ID NO.2所示。本发明的融合蛋白HFBI‑RGD具有溶解性好的特点。本发明的HFBI‑RGD融合蛋白既具有HFBI的结构和功能，又具有细胞黏附活性，这种融合蛋白能够定位到含整合素的肿瘤部位而具有较好的靶向性。HFBI‑RGD融合蛋白仍然可以作为乳化剂使小油滴在水中稳定存在，可以作为分散剂分散石墨烯、碳纳米管以及疏水性荧光探针，解决疏水性材料分散问题，并且由于该融合蛋白包含短肽RGD，这又赋予该融合蛋白整合素靶向性，可用于肿瘤诊断、药物传输等领域。
Description
技术领域
本发明属于蛋白的基因工程领域，具体地涉及一种融合蛋白HFBI-RGD的用途。
背景技术
真菌疏水蛋白(hydrophobins)是由丝状真菌分泌产生的含有丰富的疏水氨基酸的分子量大约为10kDa的两性蛋白，具有特殊的物理和化学性质，在菌丝与空气的接触中起到重要作用。真菌疏水蛋白具有疏水和亲水两部分，通过自组装可以使得在界面处形成约10nm的两性蛋白膜，这些膜可以使亲水和疏水表面发生逆转，使得亲水性的物质具有疏水性，疏水性物质具有亲水性。根据疏水蛋白形成的蛋白膜的溶解性，真菌疏水蛋白分为I型和II型。其中，II型疏水蛋白包含HFBI。真菌疏水蛋白HFBI被认为是已知的表面活性最高的蛋白之一。近些年真菌疏水蛋白在国际上得到了广泛的应用，在不同领域有许多不同的理论价值和应用价值，如个人护理产品和乳液，分离技术，生物传感器和电极，生物材料等等。
癌症已经成为威胁人类生命的重大病症之一，因此研究诊断和治疗癌症的方法已成为许多科研工作者的研究焦点。为了能更好的探测肿瘤，为了提高药物的利用率，肿瘤靶向性的研究成为主要的研究方向。RGD是含有精氨酸-甘氨酸-天冬氨酸(Arg-GlyAsp)的三肽序列。整合素是一个异源二聚体组成的膜受体蛋白家族，能特异性识别RGD。RGD肽广泛存在于生物体内，具有分子质量小、剪切力分散和有机溶剂影响较小的优点。整合素由α和β亚基组成，位于细胞表面。迄今为止已发现有18种不同的α亚基和8种不同的β亚基组成的24种异源二聚体。其中αvβ3研究较为广泛，主要在肿瘤血管生成和肿瘤细胞转移中起作用。RGD是整合素与其配体蛋白相互作用的识别位点，介导细胞与胞外基质及细胞间的黏附作用，同时具有信号传导功能，从而介导许多重要的生命活动。RGD与肿瘤细胞表面的整合素αVβ3有强的亲和性，被用于给肿瘤细胞传送药物，基因的靶向载体等。
目前，尚未有将一种真菌疏水蛋白HFBI和RGD序列制备成融合蛋白及其应用的报道。尽管对于疏水蛋白而言具有许多重要的理论和应用价值，但要将其应用于分子诊断，药物传输及靶向载体等方面仍存在不足。
发明内容
本发明的目的是克服现有技术的不足，提供一种融合蛋白HFBI-RGD作为疏水性材料分散剂的用途。
本发明的技术方案概述如下：
融合蛋白HFBI-RGD作为疏水性材料分散剂的用途。
融合蛋白HFBI-RGD的氨基酸序列是SEQ ID NO.2所示。
本发明的优点：
本发明融合蛋白HFBI-RGD具有溶解性好的特点。实验表明，本发明的HFBI-RGD融合蛋白既具有HFBI的结构和功能，又具有细胞黏附活性，这种融合蛋白能够定位到含整合素的肿瘤部位而具有较好的靶向性。HFBI-RGD融合蛋白可以作为乳化剂使小油滴在水中稳定存在，可以作为分散剂分散石墨烯、碳纳米管以及疏水性荧光探针，解决疏水性材料分散问题，并且由于该融合蛋白包含短肽RGD，这又赋予该融合蛋白整合素靶向性，可用于肿瘤诊断、药物传输等领域。
附图说明
图1为BCA蛋白总量分析筛选阳性克隆。
图2为HFBI-RGD蛋白高效液相色谱层析检测图。
图3为HFBI-RGD蛋白SDS-PAGE凝胶检测图。
图4为HFBI-RGD蛋白分散荧光探针BODIPY。
图5为HFBI-RGD蛋白分散的BODIPY富集于小鼠U-87MG及HeLa肿瘤部位图。
图6为融合蛋白HFBI-RGD修饰多壁碳纳米管。
图7为HFBI-RGD/BODIPY进入U-87MG及HeLa细胞共聚焦成像。
具体实施方式
下面通过具体实施例对本发明作进一步的说明。
pPIC9k和PET-28a为市售。毕赤酵母GS115菌株市售。
实验材料：
(1)LB培养基：配制每1000mL培养基，在1000mL的一次蒸馏水中加入蛋白胨10g，酵母粉5g，NaCl 10g，溶解后，121℃、0.1MPa灭菌20min。配置固体LB培养基时向培养基内补加1.5％的琼脂粉。
(2)YPD培养基：配制每1000mL培养基，在1000mL的一次蒸馏水中加入蛋白胨20 g，酵母粉10g，NaCl 8g，葡萄糖20g。
(3)MD固体培养基：配制每100mL培养基,在100mL的一次蒸馏水中加入无氨基酸酵母氮源1.34g，葡萄糖2.0g，琼脂2.2g。
(4)MM固体培养基：配制每100mL培养基,在100mL的一次蒸馏水中加入无氨基酸酵母氮源1.34g，琼脂2.2g。灭菌后，倒入平板前，加入0.75mL无水甲醇。
(5)BMG培养基：配制每1000mL培养基,在890mL的一次蒸馏水中加入无氨基酸酵母氮源14.4g，100mL 1M pH 6.0的磷酸盐缓冲液，10mL甘油。
(6)BMM培养基：配制每1000mL培养基,在890mL的一次蒸馏水中加入无氨基酸酵母氮源14.4g，100mL 1M pH 6.0的磷酸盐缓冲液，6mL无水甲醇。
(7)SDS-PAGE(聚丙烯酰胺凝胶电泳)
30％丙烯酰胺溶液(100mL)：将30g丙烯酰胺与0.8g的甲叉双丙烯酰胺溶解在100mL 的双蒸水中，溶解后放置于4℃棕色瓶内。
1.5M Tris-HCl pH＝8.8缓冲液(1L)：称取181.71g Tris溶解在800mL纯水中，调pH至 8.8，定容至1L，室温保存。
0.5M Tris-HCl pH＝6.8缓冲液(500mL)：称取60.57g Tris溶解在400mL纯水中，调pH 至6.8，定容至500mL，室温保存。
10％十二烷基硫酸钠(10％SDS)溶液(50mL)：称取5g SDS，加纯水至50mL，室温保存。
10％过硫酸铵(10％APS)溶液(20mL)：称取2g过硫酸铵，加纯水至20mL，每管500μL分装，-20℃保存备用。
表1 12％SDS-PAGE分离胶配制(10mL)
表2 SDS-PAGE浓缩胶配制(5mL)
(8)5×Tris-甘氨酸电泳缓冲液(5L)：称取75.5g Tris，470g甘氨酸，25g SDS，加纯水溶解，定容至5L。
(9)6×蛋白上样缓冲液：0.35M pH＝6.8Tris-HCl，10.28％W/V SDS，36％甘油，5％β- 巯基乙醇，0.012g/mL溴酚蓝，1mL每管分装，-20℃保存备用。
实施例1pET-28a-HFBI-RGD的构建：
以瑞氏木霉(Trichoderma reesei)中SEQ ID NO.3所示的HFBI核苷酸序列为模板，以 F1为正向引物，以R1为反向引物，进行第一轮PCR，得到第一种PCR产物；以第一种PCR产物为模板，以F1为正向引物，以R2为反向引物，进行第二轮PCR，得到第一种含有融合蛋白HFBI-RGD基因的序列，经酶切及T4DNA连接酶连接，将融合蛋白HFBI-RGD基因构建到PET-28a载体上，得到PET-28a-HFBI-RGD质粒。具体步骤如下：
a)酶切体系
b)条件
i.目的基因酶切1h，37℃。
ii.质粒酶切1h，37℃。
iii.酶切后80℃灭活5min。
c)以瑞氏木霉(Trichoderma reesei)中SEQ ID NO.3所示的HFBI核苷酸序列为模板，外加设计的linker及RGD序列，进行PCR以得到HFBI-RGD序列。其中linker基因序列如SEQ ID NO.4所示，RGD序列如SEQ ID NO.5所示。具体步骤如下：
根据瑞氏木霉(Trichoderma reesei)HFBI,RGD及Linker的基因序列，通过primer软件，设计出上游引物与下游引物，分别命名为F1,R1,R2。第一次PCR过程中，上游引物为F1，下游引物为R1，模板为含有HFBI基因序列的质粒。第二次PCR体系和第一次体系一样，不同的是，下游引物为R2，模板为第一次的PCR产物。在PCR仪中经过两次PCR，得到目标基因序列。PCR体系及程序设定如下表所示：
表3 PCR体系
表4 PCR仪设定程序
PCR结束，将1μL的FD Green Buffer加入到10μL的PCR产物中，用移液器吹打均匀，然后将样品加入到已配好的2％的琼脂糖凝胶中电泳分离，紫外灯下切下目的片段，使用DNA琼脂糖凝胶回收试剂盒(天根)进行回收。
将上述PCR之后的目的基因片段与载体PET-28a分别用FastDigest EcoRI和FastDigest XhoI进行双酶切，37℃水浴酶切30min，酶切产物进行琼脂糖凝胶电泳，然后进行胶回收，得到含粘性末端的目的基因片段和PET-28a载体。酶切体系50μL，各组分及含量如表5所示：
表5酶切体系
将上述的产物用胶回收试剂盒进行胶回收，得到含粘性末端的目的基因片段和PET-28a载体，并将其按照5:1摩尔比的比例加入到连接体系内，22℃连接2h，得到 PET-28a-HFBI-RGD质粒，连接体系20μL，各组分及含量如表6所示：
表6连接体系
实施例2pPIC9k-HFBI-RGD的构建：
1)选择载体为pPIC9k，酶切位点为NotI和EcoRI，设计引物进行PCR，PCR体系如表3所示。引物设计为F2、R3。将上述实施例1构建的质粒PET-28a-HFBI-RGD作为模板，PCR 可得到酶切位点为NotI和EcoRI的第二种含有融合蛋白HFBI-RGD基因的序列。
2)用限制性内切酶NotI和EcoRI对上述PCR产物和载体pPIC9k分别进行双酶切，使目的基因片段和载体出现粘性末端。具体酶切体系及方法如下所示：
3)将有粘性末端的目的基因片段和载体pPIC9k使用T4DNA连接酶进行连接，连接体系如上表6所示。
4)再经过转化及双酶切验证，将得到的阳性结果进行测序验证。最终得到pPIC9k-HFBI-RGD 构建成功质粒。
实施例3融合蛋白HFBI-RGD阳性克隆筛选及表达纯化：
1)重组表达载体要在酵母菌细胞中以较高拷贝插入酵母基因组中，需要在转入酵母细胞前对其进行线性化。利用引物设计软件Primer 5.0中提供的酶切位点分析功能对pPIC9k以及 HFBI-RGD基因进行酶切位点分析，结合Ivitrogen公司在毕赤酵母表达系统操作手册中提供的酶切位点信息，最终以限制性内切酶SalI对载体进行酶切线性化。酶切线性化的反应体系如表7所示。此体系在37℃水浴酶切2h。取5μL产物进行琼脂糖凝胶电泳，检测酶切是否完全，再将产物的条带进行胶回收。
表7线性化体系
2)毕赤酵母电转化感受态的制备
吸取15μL毕赤酵母GS115菌液转接至5mL YPD试管培养基中，30℃，250rpm培养过夜。第二天从试管培养物中取0.1-0.5mL过夜培养物，接种至含500mL新鲜YPD培养基的2L摇瓶，过夜生长至OD600约为1.3-1.5。从摇床上取酵母培养物，4℃，1500g离心5min以收集细胞，之后在100mL YPD-HEPES-DTT(77.5mL YPD中加入20mL pH 8.0 的HEPES缓冲液，再加入2.5mL新鲜配制的1M的DTT)培养基中重新悬浮细胞，30℃在摇床上培养15min；从摇床上取下细胞，冰浴半小时；4℃，1500g离心5min以收集细胞，再用250mL预冷的灭菌水悬浮细胞；如上离心，再用20mL预冷的1M山梨醇水溶液悬浮细胞；如上离心，用1mL预冷的1M山梨醇水溶液悬浮细胞，至终体积约1.5mL；如上离心，用500μL预冷超纯水重新悬浮细胞，冰浴中备用。
3)毕赤酵母的电转化
将5-20μg线性化的pPIC9k-HFBI-RGD加入到80μL感受态细胞中，转入预冷的0.2cm电转杯中，在冰上放置5min；1.5kV电击后，立即将1mL预冷的1M山梨醇水溶液至转化杯中，再将其转移至灭菌离心管中，在30℃培养箱中静置复苏1h，分成200-600μL 不等份，涂于MD平板上。在30℃培养箱中培养至克隆产生(约需3天)，筛选Mut+/Muts 表型。
4)毕赤酵母阳性转化子的筛选
在新的MD平板上画出四方格，并标好序号。用无菌牙签从MD平板上挑选长出的His+ 转化子至新的MD平板上，每一个小格里挑一个克隆。让其过夜生长，第二天，将40个不同的单克隆挑于40个不同的5mL YPD试管培养基中，250rpm，30℃培养过夜，然后将40 个单克隆的菌液吸600μL加入到装有400μL 50％甘油的EP管中，保存甘油菌。剩下的进行酵母基因组提取和转化子PCR鉴定。PCR体系如表8所示。
表8 PCR体系
扩增条件如下：94℃预变性5min，94℃变性1min，60℃退火45s，72℃延伸1.5 min，共30个循环，最后72℃延伸10min。PCR的产物进行琼脂糖凝胶电泳，通过跑胶结果可以检测出是否有HFBI-RGD基因插入基因组，找到阳性克隆。上述引物AOX1-3’及 AOX1-5’分别为SEQ ID NO.11及SEQ ID NO.12所示。
5)阳性克隆试表达
经过PCR验证有13个结果为阳性克隆，将这13个单克隆进行试表达。将之前保存的这13个阳性克隆的甘油菌各吸30μL分别转至于5mL的YPD培养基中，30℃，250rpm摇床过夜培养。然后吸取一定体积的菌液转到10mL的BMG培养基中，使得此时的OD值为 0.025。每过一段时间测一下OD值，当OD值为2至6时，再吸取一定量的菌液转至BMM 中，使得此时的OD值为1。转至BMM后，每隔24h加入100μL的甲醇作为诱导剂，诱导120h。诱导完成后收菌，将菌液离心取其上清液，将上清液进行BCA蛋白总量测定，选出表达量最高的阳性克隆8，如图1所示。
6)毕赤酵母HFBI-RGD的大量表达纯化
经过试表达，筛选出表达量最高的阳性克隆8。于是，用该转化子进行表达纯化。吸取30 μL的甘油菌到5mL的YPD培养基中，30℃，250rpm过夜培养。然后吸取一定量的菌液分别至5个含有200mL的BMG培养基的1L培养瓶中，使得此时的OD值为0.025。每过一段时间测一下OD值，当OD值为2至6时，再吸取一定量的菌液转至BMM中，使得此时的OD值为1。转至BMM后，每隔24h加入2mL的甲醇作为诱导剂，诱导120h。诱导完成后，离心取上清，然后进行浓缩，将体积浓缩至30mL。将浓缩后的样品进行处理，用 HPLC来获取纯的目的蛋白HFBI-RGD。样品处理如下：给样品中加入乙腈至乙腈终浓度为 40％，分装到EP管中16000g，40min离心，然后取上清液抽滤。使用HPLC制备柱进行纯化。HPLC纯化如图2所示。得到的HFBI-RGD融合蛋白进行SDS-PAGE电泳，由电泳图(图 3)可知：HPLC系统纯化后，在10kDa-15kDa之间，出现一条纯净的特异性蛋白条带，其分子量与融合蛋白HFBI的分子量相吻合，表明HFBI-RGD疏水蛋白纯化成功。
实施例4
化合物(I)的制备
1)对甲氧羰基苯基-1,3,5,7-四甲基二吡咯烯氟硼化合物(BOD)(VI)的合成
取100mL单口圆底烧瓶，加入0.864g(5.2mmol)对甲氧羰基苯甲醛(III)和32mL 新蒸的二氯甲烷；加入1mL(10.0mmol)新蒸的2,4-二甲基吡咯(II)。避光环境下，氮气鼓泡30min，除去烧瓶中的氧气；常温下磁力搅拌。鼓泡结束后，氮气保护下，用注射器缓慢加入0.08mL三氟乙酸，继续避光反应。反应间隙，每隔30min取样进行TLC分析，约 4h后，原料点消失，得到化合物(IV)。2,4-二甲基吡咯反应完全后，除去氮气保护，用5mL 二氯甲烷和5mL四氢呋喃溶解1.2g(2.6mmol)2,3-二氯-5,6-二氰对苯醌(DDQ)，用恒压滴液漏斗逐滴(20min左右)加入到上述反应液中，磁力搅拌下继续反应1小时，得到化合物(V)。量取8.6mL三乙胺逐滴加入，持续约10min；之后用冰水浴冷却反应液5min，然后逐滴加入8.6mL三氟化硼乙醚，持续时间约20min。半小时后撤去冰水浴，室温下磁力搅拌避光反应。反应3h后，新生成的物质浓度不再发生变化，停止反应。用水洗反应液三次，二氯甲烷萃取，加入无水硫酸钠过夜干燥。将所得物质先用短硅胶柱初步分离，完全除去紫黑色的焦油部分；旋干所得到的深紫色粗产品溶液。柱层析分离，选用300-400目硅胶，二氯甲烷：石油醚＝2:1(v/v)淋洗，接取第二个橙黄色色带的洗脱剂，旋干，用二氯甲烷和正己烷进行重结晶，得到红色粉末状固体BOD(化合物VI)210mg(0.524mmol)，产率为 12％。1HNMR(400MHz,CDCl3):δ8.18(d,J＝8.0Hz,2H),7.41(d,J＝8.0Hz,2H),5.99(s,2H), 3.97(s,3H),2.56(s,6H),1.36(s,6H).MALDI-TOF-MS:[M]+calcd for C21H21BF2N2O2, 382.1660,found 382.30.
2)目的BODIPY衍生物(化合物I)的合成
取50mL单口圆底烧瓶，加入20mg(0.0524mmol)化合物BOD(化合物VI)和42.7 mg(0.157mmol)N-乙基-3-甲酰基二卞(化合物VII)，溶于3mL溶剂苯中，同时用注射器向溶液中加入催化剂哌啶和冰醋酸，各0.1mL。在90℃下冷凝回流，避光下磁力搅拌。反应过程中，TLC检测反应进程。1h后，反应液变为砖红色，而且颜色越来越深，2.5h后，反应液逐渐变成绿色，继续反应，若溶剂减少，则补加溶剂和催化剂哌啶、冰醋酸，反应11-12 h后，原料和产物浓度基本不再变化，停止反应，避光自然冷却。用水洗涤反应液，二氯甲烷萃取，无水硫酸钠干燥，将产物旋干，柱层析分离，洗脱剂为二氯甲烷:石油醚＝2:1(v/v)，接收蓝色色带，用二氯甲烷和正己烷重结晶，得到深绿色固体18.7mg，产物产率为50％。1HNMR(400MHz,DMSO):δ9.37(s,8.37Hz,2H),8.23(d,J＝7.7Hz,4H),7.86(4m,7.86Hz, 4H),7.52(m,J＝13.8Hz,6H),7.46(m,J＝8.2Hz,4H),7.32(t,J＝7.4Hz,2H),6.74(s,2H),4.34 (t,J＝7.1Hz,4H),4.02(s,3H),1.90(m,4H)；MALDI-TOF-MS:[M]+calcd for C55H53BF2N4O2850.4230,found 848.0673。(化合物I)
实施例5
融合蛋白HFBI-RGD修饰多壁碳纳米管
(1)准确称取1mg的融合蛋白HFBI-RGD(SEQ ID NO.2)，加双蒸水至500μL(融合蛋白HFBI-RGD的浓度2mg/mL)；
(2)吸取75μL步骤(1)配制的融合蛋白HFBI-RGD溶液，加入PBS(pH＝7.4)至1mL；
(3)用步骤(2)获得的融合蛋白HFBI-RGD溶液与1mg多壁碳纳米管(MWCNTs)混合，使用探头超声仪进行超声，功率为180W，超声1h，得到稳定分散的HFBI-RGD/MWCNTs，见图6。
实施例6
融合蛋白HFBI-RGD修饰BODIPY衍生物(化合物I)
(1)准确称取1mg的融合蛋白HFBI-RGD(SEQ ID NO.2)，加双蒸水至500μL(融合蛋白HFBI-RGD的浓度2mg/mL)；
(2)吸取75μL步骤(1)配制的融合蛋白HFBI-RGD溶液，加入PBS(pH＝7.4)至1mL；
(3)用步骤(2)获得的融合蛋白HFBI-RGD溶液与1mgBODIPY衍生物混合，使用探头超声仪进行超声，功率为180W，超声1h，得到稳定分散的HFBI-RGD/BODIPY，如图4。
实施例7
实施例6获得的稳定分散的HFBI-RGD/BODIPY用于活体成像
1)细胞荧光成像
使用DMEM培养基培养Hela细胞，使用RPMI 1640培养基培养U-87MG细胞，待细胞生长至60％左右铺板率时，使用胰酶消化细胞，分别加入到提前加入细胞爬片的48孔板内培养细胞。细胞铺板密度分别为：Hela细胞，每孔1万细胞；U-87MG细胞，每孔8000细胞。每孔加入原先各自使用的培养基200μl培养细胞。细胞培养24h后，吸弃原培养液，分别用含1μMHFBI-RGD/BODIPY的各自的培养基加入细胞中继续培养。培养4h，将细胞爬片使用 DAPI染色，封片。具体封片过程如下：
(1)将培养4h后，吸弃原含HFBI-RGD/BODIPY的培养基，使用PBS(pH＝7.4)清洗细胞三次；
(2)向细胞中加入100μl 4％多聚甲醛固定4min；
(3)加入PBS(pH＝7.4)清洗3次；
(4)向细胞中加入DAPI染色5min，使用PBS(pH＝7.4)清洗3次；
(5)将防荧光淬灭剂加入到载玻片上，将处理好的细胞爬片倒扣在其上，使用透明指甲油密封，4℃保存；
(6)使用共聚焦显微镜拍照，结果如图7所示。
2)小鼠活体成像
购买20只6周龄的雌性裸鼠(nude mouse)进行饲养，然后将U-87MG和Hela细胞接种于裸鼠右前腿腋下，让其长出肿瘤，再将HFBI-RGD/BODIPY通过尾静脉注射到老鼠体内，进行活体成像。具体步骤如下：
(1)复苏Hela和U-87MG细胞，进行培养，经过细胞传代，得到足够数量的Hela和 U-87MG细胞。
(2)将培养好的细胞用PBS(pH＝7.4)制成细胞悬浮液，U-87MG细胞体积为1mL，Hela细胞悬浮液体积为800μL。
(3)裸鼠腋下注射肿瘤细胞。每只裸鼠腋下注射50μL的肿瘤细胞，约1x107个。
(4)接种好肿瘤细胞后，饲养裸鼠大概2-3周的时间，待其肿瘤大小达到5-8mm进行测试。
(5)待肿瘤长好后，给裸鼠尾静脉注射制备好BODIPY-HFBI-RGD样品。每只老鼠尾静脉注射200μL(含17.7nmol的HFBI-RGD/BODIPY)的样品。
(6)分别在注射后的2h，4h，6h，8h，24h，48h和72h用活体成像仪对小鼠进行观察，结果如图5所示。
BODIPY荧光探针衍生物该荧光探针具有良好的近红外荧光特性，发射波长732nm，可应用于细胞或生物体内进行活体成像，减少细胞或组织的背景干扰。
融合蛋白HFBI-RGD基因序列SEQ ID NO.1(人工合成)
AGCAACGGCAACGGCAATGTTTGCCCTCCCGGCCTCTTCAGCAACCCCCAGTGCTGTGC CACCCAAGTCCTTGGCCTCATCGGCCTTGACTGCAAAGTCCCCTCCCAGAACGTTTACG ACGGCACCGACTTCCGCAACGTCTGCGCCAAAACCGGCGCCCAGCCTCTCTGCTGCGT GGCCCCCGTTGCCGGCCAGGCTCTTCTGTGCCAGACCGCCGTCGGTGCTGGTGGAGGA GGTTC TGGCGGTGGAGGTTCTCGTGGTGATTGA
融合蛋白HFBI-RGD氨基酸序列SEQ ID NO.2(人工合成)
SNGNGNVCPPGLFSNPQCCATQVLGLIGLDCKVPSQNVYDGTDFRNVCAKTGAQPLCCVAPVAGQALLCQTAVGAGGGGSGGGGSRGD
HFBI核苷酸序列是SEQ ID NO.3瑞氏木霉(Trichoderma reesei)
ATGAAGTTCTTCGCCATCGCCGCTCTCTTTGCCGCCGCTGCCGTTGCCCAGCCTCTCGAG GACCGCAGCAACGGCAACGGCAATGTTTGCCCTCCCGGCCTCTTCAGCAACCCCCAGT GCTGTGCCACCCAAGTCCTTGGCCTCATCGGCCTTGACTGCAAAGTCCCCTCCCAGAAC GTTTACGACGGCACCGACTTCCGCAACGTCTGCGCCAAAACCGGCGCCCAGCCTCTCTG CTGCGTGGCCCCCGTTGCCGGCCAGGCTCTTCTGTGCCAGACCGCCGTCGGTGCTTGA Linker核苷酸序列SEQ ID NO.4(人工合成)
GGTGGAGGAGGTTCTGGCGGTGGAGGTTCT
RGD核苷酸序列SEQ ID NO.5(人工合成)
CGTGGTGAT
正向引物F1核苷酸序列SEQ ID NO.6(人工合成)
GGAATTCAGCAACGGCAACGGCAATGTTTGCCCTCC
反向引物R1核苷酸序列SEQ ID NO.7(人工合成)
GAACCTCCTCCACCAGCACCGACGGCGGTCTG
反向引物R2核苷酸序列SEQ ID NO.8(人工合成)
CCGCTCGAGATCACCACGAGAACCTCCACCGCC
正向引物F2核苷酸序列SEQ ID NO.9(人工合成)
GGAATTC AGCAACGGCAACGGCAATGT
反向引物R2核苷酸序列SEQ ID NO.10(人工合成)
TTGCGGCCGC ATCACCACGAGAACCTCCACCGCCAGA
AOX1-3’核苷酸序列SEQ ID NO.11(人工合成)
GCAAATGGCATTCTGACATCC
AOX1-5’核苷酸序列SEQ ID NO.12(人工合成)
GACTGGTTCCAATTGACAAG。
序列表
<110> 天津大学
<120> 融合蛋白HFBI-RGD基因作为疏水性材料分散剂的用途
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<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gcaaatggca ttctgacatc c 21
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gactggttcc aattgacaag 20
Claims (1)
1.融合蛋白HFBI-RGD作为疏水性材料分散剂的用途；所述融合蛋白HFBI-RGD的氨基酸序列是SEQ ID NO.2所示。
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| EP3080145B1 (en) * | 2013-12-03 | 2019-07-17 | Teknologian Tutkimuskeskus VTT OY | A method for increasing product stability with hydrophobin variants |
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