CN107217069B - 原核表达载体及rbFGF-2表达方法与工程菌和应用 - Google Patents
原核表达载体及rbFGF-2表达方法与工程菌和应用 Download PDFInfo
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Abstract
本发明提供了一种高效表达重组牛成纤维细胞生长因子‑2(rbFGF‑2)的方法。本发明首先根据大肠杆菌密码子使用偏好性,对FGF2基因序列的密码子进行替换,设计合成了rbFGF‑2核苷酸序列全长,构建得到能够表达rbFGF‑2蛋白的表达载体,将该表达载体与分子伴侣质粒先后导入到同一大肠杆菌中进行共表达。本发明解决了原核生物与真核生物在密码子使用偏好上存在差异,导致外源基因在大肠杆菌体内不能高效表达的问题;同时,通过与分子伴侣共表达,增进目标蛋白质稳定性,显著提高了rbFGF‑2蛋白的产量和活性。
Description
技术领域
本发明属于生物医药领域,涉及一种优化的生产重组牛成纤维细胞生长因子-2(rbFGF-2)的方法,以及用于该方法的源自pET-15b的原核表达载体、分子伴侣和宿主细胞(即工程菌)。
背景技术
成纤维细胞生长因子(fibroblast growth factors,FGFs)是一类生物进化上高度保守的多肽,在机体内的许多组织和器官均有分布。成纤维细胞生长因子是一个生长因子大家族,现在已经发现23个家族成员,各家族成员之间具有一定的序列同源性和结构相似性。FGFs在创伤修复、心血管系统疾病、神经系统疾病以及骨软骨再生等方面均发挥重要作用,具有重要的临床应用价值和广阔的市场前景。
成纤维细胞生长因子-2(FGF2)具有强大的促进组织修复、血管形成和细胞生长的作用。此外,大量研究已经证明,FGF2在神经发生中发挥关键作用,无论是对于发育阶段还是成人大脑中的干细胞增殖和分化。FGF2/FGFR1信号对中枢神经系统疾病的治疗和干预具有非常好的前景。
迄今为止,FGF2已通过基因重组技术利用大肠杆菌外源表达而大规模获得,但普遍存在蛋白表达量低、生物学活性差的问题,主要原因有两点:第一,大肠杆菌是原核表达系统,缺少翻译后的加工修饰如糖基化、磷酸化及酰基化等。第二,FGF2蛋白自身结构中含有4个半胱氨酸,分别位于第34、78、96和101位,其中Cys78和Cys96暴露在蛋白质的表面,极易因游离巯基氧化而形成错误的二硫键,造成多肽链折叠不正确甚至形成二聚体。
分子伴侣,是指一类多功能的蛋白质,它能够通过阻止诸如聚合作用这样的副反应,来促使其它蛋白质按正确的方式折叠,而它本身却不是最终形成的功能蛋白质的组成成分。现已证实,蛋白质转译后的有效折叠,由多肽分子组装成寡聚体结构,以及蛋白质分子在细胞内的正确定位等诸多方面的生命活动过程,都是与分子伴侣的功能作用相关联的。通过分子伴侣与克隆的外源基因在大肠杆菌宿主细胞内共表达,是增进目标蛋白质的稳定性、实现高水平表达的有用方法。
基于此,本发明根据大肠杆菌密码子使用偏好性,对牛FGF2基因序列的密码子进行替换设计合成的,主要是但不限于利用Ser78(TCC)取代Cys78(TGT),以Ser96(TCC)取代Cys96(TGT)。同时,引入分子伴侣,构建目的蛋白表达质粒pET-15b-rbFGF-2与分子伴侣groES-groEL的质粒pGro7共表达体系,增进目标蛋白质稳定性、实现高水平表达,显著提高了rbFGF-2蛋白的产量和生物学活性。
发明内容
本发明的目的是提供一种高效表达重组牛成纤维细胞生长因子-2(rbFGF-2)的方法。
本发明的另一个目的在于提供含有编码rbFGF-2基因的表达载体。
本发明的目的还在于提供用于该方法的工程菌株。
为实现上述目的,本发明首先按照大肠杆菌密码子使用偏好性对牛FGF2基因进行密码子优化,主要包括但不限于利用Ser78(TCC)取代Cys78(TGT)以及以Ser96(TCC)取代Cys96(TGT),完成重组牛成纤维细胞生长因子-2(rbFGF-2)基因核苷酸序列的设计和合成。
将rbFGF-2基因克隆至载体pMD19-T,添加BamH I酶切位点。然后,选用Nde I和BamH I酶切位点进行rbFGF-2基因与表达载体pET-15b的连接,获得在pET-15b中正确插入rbFGF-2基因的表达载体pET-15b-rbFGF-2。
构建表达质粒pET-15b-rbFGF-2与分子伴侣groES-groEL的质粒pGro7共表达体系。用分子伴侣groES-groEL的质粒pGro7(Takara)转化宿主大肠杆菌BL21(DE3),筛选得到分子伴侣质粒转化体。再将其制备成感受态细胞,用表达质粒pET-15b-rbFGF-2转化制备的感受态细胞,筛选得到成功转化表达质粒pET-15b-rbFGF-2与分子伴侣groES-groEL的质粒pGro7的转化体,即为构建得到的工程菌。
培养工程菌,IPTG诱导表达rbFGF-2蛋白,经Ni-Agrose亲和层析柱分离纯化后,制成rbFGF-2蛋白的冻干粉剂。
制成rbFGF-2蛋白的冻干粉剂时,按照纯化后1mL蛋白原液(含0.005mg·mL-1~1.5mg·mL-1rbFGF-2蛋白)中添加0.05g甘露醇和0.08g海藻糖的比例加入辅料。
本发明的优点在于:
(1)本发明根据大肠杆菌密码子使用偏好性,对FGF2基因序列的密码子进行替换,设计合成了rbFGF-2核苷酸序列全长,由此,解决了原核生物与真核生物在密码子使用偏好上存在差异,导致外源基因在大肠杆菌体内不能高效表达的问题。
(2)本发明通过分子伴侣与rbFGF-2基因在大肠杆菌宿主细胞中共表达,增进了目标蛋白质稳定性,显著提高了rbFGF-2蛋白的产量和活性。
(3)本发明所采用的大肠杆菌宿主细胞适合rbFGF-2蛋白表达载体的高效表达。
附图说明
图2:重组表达载体pET-15b-rbFGF-2的质粒图。
图3:重组表达载体pET-15b-rbFGF-2的酶切鉴定。
1:pET-15b-rbFGF-2经NdeⅠ+BamH I双酶切;2:pET-15b-rbFGF-2质粒;M:DL10,000DNA Marker。
图4:rbFGF-2蛋白的纯化。2~4:rbFGF-2蛋白;1:Premixed Protein Marker(Low)。
图5:rbFGF-2蛋白的Western blot检测图。1:可溶性蛋白中rbFGF-2蛋白;2:包涵体中rbFGF-2蛋白;3:重组牛成纤维细胞生长因子-2产品(rbbFGF)(ScienCell公司)。
图6:rbFGF-2蛋白的生物学活性检测。
图7:rbFGF-2蛋白的表达量分析。1:只含有表达质粒pET-15b-rbFGF-2的工程菌的rbFGF-2蛋白表达量;2:含有表达质粒pET-15b-rbFGF-2与分子伴侣groES-groEL的质粒pGro7共表达体系的工程菌的rbFGF-2蛋白表达量。
具体实施方式
实施例1
1.目的基因的合成
以NCBI中已公开的FGF2fibroblast growth factor 2[Bos taurus(cattle)]基因mRNA序列(No.NM_174056.3)为模板,按照大肠杆菌密码子使用偏好性对FGF2基因进行密码子优化,主要包括但不限于利用Ser78(TCC)取代Cys78(TGT)以及以Ser96(TCC)取代Cys96(TGT),完成重组牛成纤维细胞生长因子-2(rbFGF-2)基因核苷酸序列的设计和合成。rbFGF-2基因的核苷酸序列如SEQ ID NO.1所示,具体如下:
ATGGCTGCAGGTTCTATTACTACGCTGCCGGCACTGCCTGAAGACGGCGGCAGCGGTGCATTTCCGCCGGGTCACTTCAAAGACCCGAAGCGTCTGTACTGCAAGAATGGCGGCTTCTTCCTGCGCATCCACCCGGATGGTCGCGTTGATGGTGTCCGCGAGAAATCCGATCCACATATTAAACTGCAGCTGCAAGCGGAAGAACGTGGCGTGGTTTCTATCAAAGGTGTTTCCGCGAACCGTTACCTGGCGATGAAAGAGGACGGTCGTCTGCTGGCTTCTAAATCCGTGACTGACGAATGTTTCTTCTTTGAACGTCTGGAAAGCAACAACTACAACACCTATCGTAGCCGCAAATACTCCTCTTGGTATGTAGCTCTGAAACGTACCGGTCAGTACAAGCTGGGTCCGAAAACCGGCCCGGGCCAGAAAGCCATCCTGTTCCTGCCAATGTCCGCGAAATCTTGA
将所合成的rbFGF-2基因克隆至载体pUC57,并扩增rbFGF-2基因。利用Primer 5.0和DNAMAN设计rbFGF-2引物,具体引物核苷酸序列如SEQ ID NO.2~3所示,其中5’端引入Nde I酶切位点和保护碱基(GGAATTC)及在3’端引入Xho I酶切位点和保护碱基(CCG)。
引物核苷酸序列具体如下:
上游引物:GGAATTCCATATGGCTGCAGG(SEQ ID NO.2)
下游引物:CCGCTCGAGTCAAGATTTCG(SEQ ID NO.3)
PCR具体反应过程为:95℃3min;95℃20s,60℃30s,72℃1min,完成30个循环;72℃5min。PCR产物经1.5%琼脂糖凝胶电泳检测无非特异性条带后(图1),用DNA凝胶回收试剂盒纯化回收目的条带,即rbFGF-2基因。
2.表达质粒pET-15b-rbFGF-2的构建
表达载体为购自默克(Merck)公司的pET-15b,表达质粒pET-15b-rbFGF-2的构建结果如图2所示。
为提高连接效率,选用Nde I和BamH I酶切位点进行rbFGF-2基因与表达载体pET-15b的连接。第一步,将rbFGF-2基因克隆至载体pMD19-T,添加BamH I酶切位点。具体过程如下:利用克隆试剂盒将rbFGF-2基因与载体pMD19-T 16℃连接过夜。连接产物转化进入E.coli DH5α感受态细胞,然后,接种到含有氨苄青霉素(100μg·mL-1)的LB平板培养基,37℃培养过夜,挑取阳性克隆,利用质粒提取试剂盒提取质粒。
第二步,用NdeⅠ+BamH I分别处理阳性克隆载体pMD19-T-rbFGF-2和表达载体pET-15b,37℃过夜,回收相应的片段,用T4DNA连接酶16℃连接过夜。连接产物转化进入大肠杆菌BL21(DE3),然后,接种到含有氨苄青霉素(100μg·mL-1)的LB平板培养基,37℃培养过夜,挑取阳性克隆并进行质粒酶切鉴定。
利用质粒提取试剂盒提取质粒,将质粒DNA用NdeⅠ+BamH I双酶切,37℃过夜。酶切鉴定结果如图3所示。结果显示:经过NdeⅠ+BamH I双酶切后(泳道1),能从电泳上看到大小约500bp左右的片段,表明rbFGF-2基因已经正确插入载体pET-15b中。委托生工生物工程(上海)股份有限公司进行序列的测定,证实克隆序列与设计序列完全一致。
3.表达质粒pET-15b-rbFGF-2与分子伴侣groES-groEL的质粒pGro7共表达体系的构建
用分子伴侣groES-groEL的质粒pGro7(Takara)转化宿主大肠杆菌BL21(DE3),然后在含有氯霉素(20μg·mL-1)的平板上培养,筛选得到分子伴侣质粒转化体。
将分子伴侣质粒转化体在含有20μg·mL-1氯霉素的液体培养基中培养,再将其制备成感受态细胞。用表达质粒pET-15b-rbFGF-2转化制备的感受态细胞,在含有氯霉素(20μg·mL-1)和氨苄青霉素(50μg·mL-1)的平板上培养,筛选得到转化体。
成功转化表达质粒pET-15b-rbFGF-2与分子伴侣groES-groEL的质粒pGro7的宿主菌,即为构建得到的工程菌。该工程菌已由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏(CGMCC No.13246)。
4.工程菌表达rbFGF-2蛋白
将工程菌接种到1.5mL含有20μg·mL-1氯霉素和50μg·mL-1的氨苄青霉素的LB培养基中,37℃培养3~4h,按1:50比例接种到含50mL LB培养基的250mL三角瓶中,置于180rpm的摇床,37℃振荡培养。其中,LB培养基含有20μg·mL-1氯霉素,50μg·mL-1的氨苄青霉素以及0.5mg·mL-1的L-Arabinose。当OD600达到0.4~0.8,加入1mmol·L-1的IPTG开始诱导,置于160rpm的摇床,28℃振荡培养,诱导3h结束。样品进行SDS-PAGE检测,分离胶浓度为15%,浓缩胶浓度为5%,测定rbFGF-2蛋白含量。
蛋白质提取过程具体如下:发酵液4℃下12000rpm离心5min,得菌体。按照1g菌体加入20mL细菌裂解缓冲液(含溶菌酶、DNase I和蛋白酶抑制剂)的比例,向菌体中加入裂解液,充分涡旋直至菌体完全重悬。利用超声破碎仪破碎菌体,冰浴操作,200w,20s,重复2次。然后,4℃下12000rpm离心5min,收集上清液,即为工程菌总蛋白质。
5.rbFGF-2蛋白的纯化
将工程菌总蛋白质重悬于2mL Binding Buffer中,混匀并使其充分溶解,4℃下12000rpm离心15min,收集上清液。
由于rbFGF-2蛋白含有组氨酸标签(His-Tag),采用Ni-Agrose亲和层析柱对其进行纯化。具体过程如下:将所得的上清液,采用0.45μm滤膜过滤后上柱,流速为15mL·h-1。然后,用3倍柱体积的Binding Buffer冲洗柱子,洗去杂蛋白。接下来,用2倍柱体积的ElutionBuffer洗脱目的蛋白,收集洗脱液。最后,将所得洗脱液经PD-10脱盐柱(>5000Mr)除去盐类和杂蛋白,获得纯化后的蛋白液。
纯化后的蛋白液经SDS-PAGE电泳检测(如图4所示),结果表明,rbFGF-2蛋白纯度高达95%以上。
Binding Buffer的组成如下:20mmol·L-1Tris-HCl(pH7.9),5mmol·L-1咪唑,0.5mol·L-1NaCl,8mol·L-1尿素。Elution Buffer的组成如下:20mmol·L-1Tris-HCl(pH7.9),500mmol·L-1咪唑,0.5mol·L-1NaCl,8mol·L-1尿素。
6.制备rbFGF-2蛋白的冻干粉剂
纯化后的蛋白液,经0.22μm滤膜过滤除菌后,装入无菌的样品瓶中,加入辅料,冷冻干燥12h,制成rbFGF-2蛋白的冻干粉剂。
制成rbFGF-2蛋白的冻干粉剂时,按照纯化后1mL蛋白原液(含0.2mg·mL-1~1.0mg·mL-1rbFGF-2蛋白)中添加0.05g甘露醇和0.08g海藻糖的比例加入辅料。
7.rbFGF-2蛋白的鉴定
按照电转仪操作方法进行,首先,将PVDF膜剪成适当大小,泡甲醇20s,制作“海绵-滤纸-膜-胶-滤纸-海绵”的“三明治”结构,进行转印,转印条件为70V,1.5h。将转印后的PVDF膜取出,清洗后加封闭液(5%脱脂奶粉)封闭1h。
然后,将1:750封闭液稀释的鼠抗牛的FGF2单抗制成抗体工作液,放入封闭好的PVDF膜,4℃孵育过夜。第二日,取出PVDF膜,用TTBS洗膜2次。将1:5000封闭液稀释的羊抗鼠IgG-HRP制成二抗工作液。放入PVDF膜,37℃孵育45min。取出PVDF膜并清洗后,经ECL发光液曝光,结果如图5所示。结果表明,在21KD处有条带出现,该条带为rbFGF-2蛋白。
rbFGF-2蛋白的氨基酸序列具体如下:
MAAGS ITTLP ALPED GGSGA FPPGH FKDPK RLYCK NGGFF LRIHP DGRVD GVREKSDPHI KLQLQ AEERG VVSIK GVSAN RYLAM KEDGR LLASK SVTDE CFFFE RLESN NYNTY RSRKYSSWYV ALKRT GQYKL GPKTG PGQKA ILFLP MSAKS
8.rbFGF-2蛋白的生物学活性检测
用RPMI 1640培养基培养鼠Balb/c 3T3细胞,取指数期的细胞制备单细胞悬液,继续培养,当细胞汇合率达到80%时,将细胞培养液平均分成4份。然后,将rbFGF-2蛋白冻干粉、制备冻干粉用辅料以及ScienCell公司的重组牛成纤维细胞生长因子-2(rbbFGF)产品分别用RPMI 1640培养基溶解,经0.22μm滤膜过滤除菌后,分别加入到细胞培养液中,对照组细胞培养液无任何添加,37℃孵育12h,490nm读取吸光度值。应用SPSS软件进行方差分析。
结果表明,6.25ng·mL-1rbFGF-2蛋白作用Balb/c 3T3细胞12h时,细胞平均活力值为1.4801±0.093,与对照组相比,具有显著性差异(p<0.01);而同等浓度下的重组牛成纤维细胞生长因子-2(rbbFGF)产品(ScienCell公司)作用细胞12h时,细胞平均活力值为0.9179±0.0771,与对照组无显著性差异(图6)。
上述结果说明,按照本发明方法获得的rbFGF-2蛋白,在6.25ng·mL-1浓度时,具有显著的促细胞增殖作用,且生物学活性显著优于市售产品的生物学活性。
对比例1
所用方法同实施例1,不同之处在于,选用Nde Ⅰ+Xho I为酶切位点与载体pET-15b连接,结果发现,连接效率很低,不容易连接成功。
这可能是因为Nde I和Xho I两个酶切位点距离较近,相隔仅7bp,导致酶切不充分,且用Xho I酶所切出的突出末端的连接效率低,因此不容易连接成功,为此,选用Nde I和BamH I为酶切位点进行rbFGF-2基因与载体pET-15b的连接,以实现高效连接。
对比例2
所用方法同实施例1,不同之处在于,所构建的工程菌只含有表达质粒pET-15b-rbFGF-2,不含有分子伴侣质粒。
工程菌经过IPTG诱导表达后,采用Western blot法分析rbFGF-2蛋白相对含量。具体方法如下:采用β-actin作为内参,对ECL发光显影胶片上的rbFGF-2蛋白条带以及β-actin条带进行灰度扫描分析,以rbFGF-2蛋白条带和β-actin条带的灰度比值作为rbFGF-2蛋白相对表达量。
结果表明,含有表达质粒pET-15b-rbFGF-2与分子伴侣groES-groEL的质粒pGro7共表达体系的工程菌中rbFGF-2蛋白的表达量是只含有表达质粒pET-15b-rbFGF-2的工程菌中rbFGF-2蛋白表达量的2.28倍(图7)。上述结果说明,分子伴侣groES-groEL能够帮助rbFGF-2蛋白高效表达,采用共表达体系能够显著提高rbFGF-2蛋白的表达量。
SEQUENCE LISTING
<110> 何伟
<120> 原核表达载体及rbFGF-2表达方法与工程菌和应用
<130>
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<170> PatentIn version 3.5
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ctgcgcatcc acccggatgg tcgcgttgat ggtgtccgcg agaaatccga tccacatatt 180
aaactgcagc tgcaagcgga agaacgtggc gtggtttcta tcaaaggtgt ttccgcgaac 240
cgttacctgg cgatgaaaga ggacggtcgt ctgctggctt ctaaatccgt gactgacgaa 300
tgtttcttct ttgaacgtct ggaaagcaac aactacaaca cctatcgtag ccgcaaatac 360
tcctcttggt atgtagctct gaaacgtacc ggtcagtaca agctgggtcc gaaaaccggc 420
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Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly
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Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu
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Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg
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Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu
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Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Ser Ala Asn
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Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Ser
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Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr
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Asn Thr Tyr Arg Ser Arg Lys Tyr Ser Ser Trp Tyr Val Ala Leu Lys
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Arg Thr Gly Gln Tyr Lys Leu Gly Pro Lys Thr Gly Pro Gly Gln Lys
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Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser
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Claims (6)
1.一种表达重组牛成纤维细胞生长因子-2的方法,其特征在于:将源自pET-15b的原核表达载体pET-15b-rbFGF-2与分子伴侣质粒共同或先后导入到同一大肠杆菌宿主细胞中共表达,可获得诱导表达的rbFGF-2蛋白;
其中,所述表达载体pET-15b-rbFGF-2为含有编码rbFGF-2蛋白的重组DNA,所述编码rbFGF-2蛋白的核苷酸序列如SEQ ID NO.1所示;所述的分子伴侣质粒为pGro7,伴侣蛋白质为groES-groEL。
2.按照权利要求1所述的方法,其特征在于:所含编码rbFGF-2蛋白的核苷酸序列,是选用Nde Ⅰ+BamH I为酶切位点与载体pET-15b连接的,即编码rbFGF-2蛋白的核苷酸序列装入载体pET-15b中位于Nde Ⅰ和BamH I之间。
3.按照权利要求1所述的方法,其特征在于:所述大肠杆菌为BL21(DE3),其基因型为:F-ompT hsdS(rB -mB -)gal dcm(DE3)。
4.一种工程菌,其特征在于:其是通过权利要求3所述的方法成功转化表达质粒pET-15b-rbFGF-2与含有分子伴侣groES-groEL的质粒pGro7构建得到工程菌;该工程菌已由中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号为:CGMCC No. 13246。
5.一种权利要求4所述工程菌的应用,其特征在于:通过培养权利要求4所述的工程菌,诱导表达rbFGF-2蛋白,经分离纯化后,得蛋白原液。
6.按照权利要求5所述的应用,其特征在于:按照纯化后1 mL蛋白原液中添加0.05 g甘露醇和0.08 g海藻糖的比例加入辅料,冷冻干燥可制成rbFGF-2蛋白的冻干粉剂,其中每1mL蛋白原液含有0.005 mg·mL-1~1.5 mg·mL-1 rbFGF-2蛋白。
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