CN109576286A - 重组菌丝霉素基因的合成及其表达产物的构建方法 - Google Patents
重组菌丝霉素基因的合成及其表达产物的构建方法 Download PDFInfo
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- CN109576286A CN109576286A CN201811294825.3A CN201811294825A CN109576286A CN 109576286 A CN109576286 A CN 109576286A CN 201811294825 A CN201811294825 A CN 201811294825A CN 109576286 A CN109576286 A CN 109576286A
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Abstract
本发明提供了一种重组菌丝霉素基因的合成及其表达产物的构建方法。所述重组菌丝霉素基因序列针对毕赤酵母密码子优化表达。构建上述重组菌丝霉素基因片段的方法。同时还公开了构建表达重组菌丝霉素毕赤酵母的方法。通过针对毕赤酵母的密码子进行优化,使得由毕赤酵母表达的重组菌丝霉素的产量和纯度得到提高。
Description
技术领域
本发明涉及生物工程技术领域。
背景技术
菌丝霉素(Plectasin)是由丹麦诺维信公司从腐生子囊菌假黑盘菌(Pseudoplectania nigrella)中分离得到的首例真菌防御素。菌丝霉素基因的完整开放阅读框编码一个长为95个氨基酸的多肽,共由3部分组成。信号肽序列(1~23位氨基酸)、前肽(24~55位氨基酸)和C-末端成熟肽(56~95位氨基酸)。成熟的菌丝霉素由40个氨基酸残基组成,其二级结构属于α-β模型,由一个α-螺旋和两个反平行的β-折叠组成,其中包含三个二硫键(Cys4-Cys30,Cys15-Cys37,Cys19-Cys39),其净电荷数为+1~+3。
菌丝霉素对革兰氏阳性细菌具有明显杀伤作用,包括:链球菌属,葡萄球菌属,肠球菌属,棒状杆菌属和杆状菌属。尤其值得注意的是,菌丝霉素对肺炎链球菌的杀菌效果与万古霉素和青霉素的杀菌效果相当。菌丝霉素具有良好的盐离子耐受能力、pH稳定性和热稳定性。同时,菌丝霉素无细胞毒性、无溶血性及脑脊液渗透性好,是治疗革兰氏阳性细菌引起的疾病的潜在药物,具有广阔应用前景。
菌丝霉素的生产方式主要有三种:提取、化学合成和生物合成。腐生子囊菌中的菌丝霉素含量非常少,因此通过提取方式获得菌丝霉素是非常不经济的。化学合成得到的菌丝霉素,由于无法形成二硫键,因此菌丝霉素的空间结构不正确,抑菌活性大幅减少。还需要后续操作重新形成二硫键,步骤繁琐,价格昂贵,不适合大规模生产。生物合成主要通过基因工程手段构建工程菌株异源表达菌丝霉素,但现有技术所构建的表达系统的菌丝霉素产量都非常低。
毕赤酵母表达系统是最常用的蛋白表达系统之一,已有上千种蛋白在毕赤酵母中成功表达,并有多种蛋白实现工业化生产。
发明内容
鉴于上述现有技术的不足之处,本发明的目的在于提供一种菌丝霉素基因的合成及其表达产物的构建方法,旨在解决现有技术中菌丝霉素的表达量低的问题。
技术方案如下:
一种菌丝霉素基因片段,其序列针对毕赤酵母密码子优化表达,具体序列为:
GGTTTTGGTTGTAACGGTCCTTGGAACGAAGATGATTTGAGATGTCATAACCATTGTAAATCCATTAAGGGTTACAAGGGTGGATACTGTGCTAAAGGTGGTTTTGTTTGTAAGTGCTAC(SEQ ID No.1)。
构建上述重组菌丝霉素基因片段的方法,包括以下步骤:
(1).采用4段基因片段进行拼接重组,各基因片段的序列如下:Gene1:
GGTTTTGGTTGTAACGGTCCTTG(SEQ ID No.2);
Gene2:
TTACAATGGTTATGACATCTCAAATCATCTTCGTTCCAAGGACCGTTACAACCAAAA(SEQ IDNo.3);
Gene3:
GATTTGAGATGTCATAACCATTGTAAATCCATTAAGGGTTACAAGGGTGGATACTGT(SEQ IDNo.4);
Gene4:
TCTAGAAAGTAGCACTTACAAACAAAACCACCTTTAGCACAGTATCCACCCTTGTAACC(SEQ IDNo.5);
为提高构建效率,可先行对步骤1制得的拼接重组片段以及由其他途径获得的MBP标签片段分别进行PCR扩增。所述MBP标签片段可以由pMAL-c5x质粒中提取获得。扩增拼接重组片段的引物序列为:
T-FP:
AGGGAAGGGGTTTTGGTTGTAACGGGTTTTGGTTGTAACGGTCCTTGG(SEQ ID No.6);
T-RP:
GGCCTCTAGAAAGTAGCACTTACAAACGTAGCACTTACAAACAAAACCACCT(SEQ ID No.7);
扩增MBP标签片的引物序列为:
M-FP:
GGCCGAATTCATGAAAATCGAAG(SEQ ID No.8);
M-RP:
ACCAAAACCCCTTCCCTCGATC(SEQ ID No.9)。
(2).取拼接重组片段和MBP标签片段,利用如SEQ ID No.7和SEQ ID No.8所示的引物搭桥PCR,纯化回收后得到合成重组有MBP标签的重组菌丝霉素基因片段。
本发明同时还公开了构建表达重组菌丝霉素毕赤酵母的方法,包括以下步骤:
(A).取上述方法制得的重组菌丝霉素基因片段,与pGAPZa A载体,用EcoRI和XbaI分别进行双酶切,分别纯化及回收;
(B).以T4连接酶将酶切后的重组菌丝霉素基因片段和pGAPZaA载体进行连接;
(C).取连接产物加到DH5α感受态细胞中混匀,转化至感受态细胞;
(D).通过PCR反应并电泳,分析载体上游和下游引物扩增的条带的理论值是否与电泳结果相符,或是直接测序鉴定;鉴定正确后扩大培养含有重组质粒的EcoliDH5α,提取重组质粒;
(E).以限制性内切酶AvrⅡ进行重组质粒的线性化处理,剩余线性化产物用有机酚/氯仿/异戊回收;
(F).以毕赤酵母GS115菌制作感受态细胞,将线性化的重组质粒电转化至毕赤酵母感受态细胞中;
(G).重组菌丝霉素经PCR鉴定产物扩增的条带的理论值与电泳结果相符,相符即为阳性单克隆,可以进行表达和纯化。
具体地,步骤(C)包括:
(C-1).取10μL连接产物加到50μL的DH5α感受态细胞中混匀,冰浴30分钟;
(C-2).将上述转化液置于42℃水浴60秒,取出后立即置于冰浴中放置5分钟;
(C-3).向其中加入500μL 37℃预热的低盐LB培养液,150rpm、37℃振荡培养45分钟;
(C-4).2500rpm离心5分钟,将上清液吸走,留100μL混匀菌液,加到Zeocin浓度25μg/mL的低盐LB固体琼脂培养基上,用无菌的玻璃珠轻轻的将细胞均匀涂开,待平板表面干燥后,倒置平板,37℃培养12-16小时。
具体地,步骤(F)包括:
(F-1).打开电转化仪,预热30min;
(F-2).设置电穿孔转化电击条件:电压1500V,电阻200Ω,电容2.5mF,5ms;
(F-3).将5-10μg纯化后的线性化质粒加入到新鲜制备的毕赤酵母感受态细胞中,轻轻旋转离心管,使质粒和感受态细胞完全混匀后,将其全部转移至冰预冷处理的0.2cm无菌电转杯中;
(F-4).将电转杯继续冰浴5min后,放入电击槽,开始电击;
(F-5).电击结束后,立即向电转杯中加入1mL冰预冷的1M山梨醇,用移液枪轻轻吹打混匀后,将其全部转移至无菌的1.5mL离心管中;
(F-6).将上述离心管置于30℃恒温培养箱中静置孵育1-2h;
(F-7).分别吸取10,25,50,100,200ul菌液涂布在含100μg/mlZeocin的YPDS固体培养基上。
(F-8).30℃恒温倒置平板2-3天,至长出单菌落
制得的重组菌丝霉素的完整氨基酸序列:
MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRGFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGGFVCKCYFLEQKLISEEDLNSAVDHHHHHH(SEQ ID No.10)。
有益效果:通过针对毕赤酵母的密码子进行优化,使得由毕赤酵母表达的重组菌丝霉素的产出浓度提高,增加6个组氨酸,使得重组菌丝霉素更容易纯化,增加BMP蛋白,使得重组菌丝霉素的生物活性增强。
附图说明
图1为实施例2的电泳结果图;
图2为实施例3的DH5α菌落PCR鉴定的电泳结果图;
图3为实施例3的重组毕赤酵母基因组PCR鉴定的电泳结果图;
图4为实施例4的Western blot检测结果图;
图5为实施例4的SDS-PAGE电泳检测结果图。
具体实施方式
下面通过具体实施方式对本发明作进一步详细说明。但本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:密码子的优化
根据菌丝霉素的氨基酸序列,进行密码子优化并确定碱基序列,设计如下8条寡核苷酸引物:
Gene1:GGTTTTGGTTGTAACGGTCCTTG(SEQ ID No.2);
Gene2:
TTACAATGGTTATGACATCTCAAATCATCTTCGTTCCAAGGACCGTTACAACCAAAA(SEQ IDNo.3);
Gene3:
GATTTGAGATGTCATAACCATTGTAAATCCATTAAGGGTTACAAGGGTGGATACTGT(SEQ IDNo.4);
Gene4:
TCTAGAAAGTAGCACTTACAAACAAAACCACCTTTAGCACAGTATCCACCCTTGTAACC(SEQ IDNo.5);
T-FP:
AGGGAAGGGGTTTTGGTTGTAACGGGTTTTGGTTGTAACGGTCCTTGG(SEQ ID No.6);
T-RP:
GGCCTCTAGAAAGTAGCACTTACAAACGTAGCACTTACAAACAAAACCACCT(SEQ ID No.7);
M-FP:
GGCCGAATTCATGAAAATCGAAG(SEQ ID No.8);
M-RP:
ACCAAAACCCCTTCCCTCGATC(SEQ ID No.9)
实施例2:拼接PCR合成重组菌丝霉素基因
2-1.编码重组菌丝霉素基因进行拼接重组。PCR反应体系设计为20μl,反应体系配制如下:
2×PfuMax HiFi PCR ProMix | 10μL |
Gene1-4引物(10uM) | 各0.4μL |
H<sub>2</sub>0 | 8.4μL |
反应条件为(降落PCR):
2-2.PCR扩增编码重组菌丝霉素基因序列,反应体系如下:
反应条件为(降落PCR):
2-3.PCR扩增编码融合标签MBP的基因序列,反应体系如下:
2×PfuMax HiFi PCR ProMix | 25μL |
M-RP、M-FP(10uM) | 各1μL |
pMAL-c5x质粒(10ng/μL) | 1μL |
H<sub>2</sub>0 | 22μL |
反应条件为(降落PCR):
2-4.PCR产物按照凝胶纯化试剂盒说明书回收PCR产物,超微量紫外分光光度计测定浓度,并根据A260/A280分析纯度。结果如下表:
样品 | 浓度(ng/ul) | A260/A280 |
拼接重组基因产物 | 4 | 1.975 |
MBP基因产物 | 16 | 2.013 |
2-5.利用搭桥PCR的方法合成重组有MBP标签的重组菌丝霉素基因序列。
2×PfuMax HiFi PCR ProMix | 25μL |
T-RP、M-FP(10uM) | 各1μL |
拼接重组基因产物(4ng/μl) | 2μL |
MBP标签片段(16ng/μl) | 4μL |
H20 | 17μL |
反应条件为(降落PCR):
PCR反应完后取PCR产物进行1.0%琼脂糖电泳分析,结果如图1所示,Marker从小到大依次为300、500、800、1500、2000、3000、5000bp,泳道1、2为拼接PCR扩增产物,扩增大小与理论大小相符。
2-6.PCR产物按照凝胶纯化试剂盒说明书回收PCR产物,超微量紫外分光光度计测定浓度,并根据A260/A280分析纯度。结果如下表:
实施例3:构建表达重组菌丝霉素毕赤酵母
3-1.用EcoRI和XbaI对重组基因和pGAPZa A载体分别进行双酶切,反应温度为37℃,反应时间为2小时。酶切反应体系如下:
组分 | 体积 |
10×FD buffer | 5μl |
模板 | 载体2μg、重组基因1μg |
EcoRI、XbaI | 各1μl |
灭菌去离子水 | 补充至50μl |
3-2.酶切结束后,纯化回收酶切产物。按照DNA纯化试剂盒说明书纯化回收酶切产物。超微量紫外分光光度计测定其浓度,并根据A260/A280分析纯度。结果如下表:
样品(双酶切后) | 浓度(ng/ul) | A260/A280 |
重组基因 | 8.5ng/ul | 2.056 |
pGAPZa A载体 | 4ng/ul | 1.982 |
3-3.连接反应,将双酶切后的重组基因和双酶切后的pGAPZaA载体进行连接反应,反应体系如下:
组分 | 体积 |
10×T4ligase buffer | 2μl |
双酶切后的基因 | 50ng |
pGAPZa A载体 | 50ng |
T4连接酶 | 1μl |
灭菌去离子水 | 补充至20μl |
连接反应在22℃的温度下连接2h。
3-4.转化至感受态细胞,包括:
①取10μl连接产物加到50μl DH5α感受态细胞中混匀,冰浴30分钟。
②将上述转化液置于42℃水浴60秒,取出后立即置于冰浴中放置5分钟。
③向其中加入500μl 37℃预热的Low Salt LB(不含抗生素)培养液,150rpm、37℃振荡培养45分钟。
④2500rpm离心5分钟,将上清液吸走,留100μl混匀菌液,加到含Low Salt LB固体琼脂培养基上(Zeocin浓度25μg/ml),用无菌的玻璃珠轻轻的将细胞均匀涂开。待平板表面干燥后,倒置平板,37℃培养12-16小时。
3-5.DH5α菌落PCR鉴定,挑取上述平板上的单菌落10个,分别溶到500μl含25μg/mlZeocin的Low Salt LB培养液,37℃、180rpm/min震荡培养4h。每管取0.5μl菌液做模板,进行PCR。反应体系设计为10μl总体系:
2×Hotstart Taq PCR ProMix | 5μl |
菌液 | 0.5μl |
载体通用上游引物(10μM) | 0.5μl |
载体通用下游引物(10μM) | 0.5μl |
H20 | 3.5μl |
反应条件:
PCR反应完后取5μl PCR产物进行1.50%琼脂糖电泳分析,用载体上游和下游引物扩增的条带理论大小为1300bp左右,其实际扩增条带见图2。从电泳图可以看出,除第8、9号外,其余都为阳性克隆菌,挑取10、11号菌送往测序公司测序。
3-6.将阳性的克隆送测序公司测序进一步鉴定,详细测序结果如下:
atgaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcaaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggtgaaagatccgcgtattgccgccactatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgAacaacaacaacaataacaataacaacaacctcgggatcgagggaaggGGTTTTGGTTGTAACGGTCCTTGGAACGAAGATGATTTGAGATGTCATAACCATTGTAAATCCATTAAGGGTTACAAGGGTGGATACTGTGCTAAAGGTGGTTTTGTTTGTAAGTGCTACttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcat(SEQ IDNo.11)
大写黑色部分为SEQ ID No.1所示的重组菌丝霉素基因序列,其前面小写部分为N端融合标签MBP蛋白的基因序列,后面部分为C端融合标签的基因序列。
3-7.重组质粒的提取:
(1)从-80℃冰箱取出鉴定正确的、含有重组质粒的EcoliDH5α,冰上自融后,以1:100的比例将菌液接种于含25μg/ml Zeocin的LowSalt LB培养液中,于37℃、200r/min的摇床中培养16h;
(2)按照质粒提取试剂盒提取质粒,用微量分光光度计进行质粒浓度测定,浓度为203ng/ul,A260/A280为1.901,-20℃保存备用。
3-8.根据质粒pGAPZa A中可选择的线性化位点以及插入基因的碱基序列,最终选取限制性内切酶AvrⅡ进行重组质粒的线性化处理。线性化体系如下表所示:
将线性化溶液混匀后置于恒温水浴槽中,37℃酶切2h后,取2μL酶切后的产物用1.5%(v/v)的琼脂糖凝胶检测重组质粒是否线性化完全。剩余线性化产物用有机酚/氯仿/异戊回收,具体步骤如下:
(1)加入等体积的有机酚/氯仿/异戊醇,震荡混匀2min;
(2)加入1/10体积的3M醋酸钠及3倍体积的乙醇并混匀,置于-20℃冰箱中2h以上;
(3)13000rpm/min离心10min回收DNA沉淀,用500ul 80%乙醇洗净2遍;
(4)加入30ul的Nuclease-free H2O溶解。
用微量分光光度计测定回收的线性化产物的浓度,浓度为203ng/ul,A260/A280为1.874,-20℃保存备用。
3-9.毕赤酵母GS115的电转化,包括步骤:
(1)GS115感受态细胞的制备
(1-1)将毕赤酵母GS115菌液用无菌接种环划线接种至YPD固体培养基上,用封口膜将平板封口后,于30℃恒温培养箱中倒置平板培养至有单菌落长出;
(1-2)挑取纯化后的GS115单菌落接种至5mL YPD液体培养基中,于30℃、250r/min条件下过夜培养;
(1-3)按1:1000的比例将上述酵母菌液接种至100mL YPD液体培养基中,于30℃、250r/min条件下培养至OD值达1.3-1.5;
(1-4)将上述菌液分装至两个50mL无菌离心管中,4℃条件下1500g/min离心5min后,去除培养基;
(1-5)加入40ml新配(2h内)的LDST溶液(100mM LiAc,10mM dithiothreitol,0.6Msorbitol and 10mM Tris-Hcl pH7.5,现用现配不能保存),30℃孵育30min;
(1-6)室温6000rpm离心5min。弃上清,用1ml冰浴1M山梨醇重悬菌体,转至1.5mlEP管;
(1-7)用1ml冰浴1M山梨醇洗涤菌体3次(应快速,菌脆弱)最后用400ul冰浴1M山梨醇重悬菌体,分装80ul/管(现配现用)。
(2)重组质粒电转入GS115感受态细胞
(2-1)打开电转化仪,预热30min;
(2-2)设置电穿孔转化电击条件:电压1500V,电阻200Ω,电容2.5mF,5ms;
(2-3)将5-10μg纯化后的线性化质粒加入到新鲜制备的GS115感受态细胞中,轻轻旋转离心管,使质粒和感受态细胞完全混匀后,将其全部转移至冰预冷处理的0.2cm无菌电转杯中;
(2-4)将电转杯继续冰浴5min后,放入电击槽,开始电击;
(2-5)电击结束后,立即向电转杯中加入1mL冰预冷的1M山梨醇,用移液枪轻轻吹打混匀后,将其全部转移至无菌的1.5mL离心管中;
(2-6)将上述离心管置于30℃恒温培养箱中静置孵育1-2h;
(2-7)分别吸取10,25,50,100,200ul菌液涂布在含100μg/ml Zeocin的YPDS固体培养基上;
(2-8)30℃恒温倒置平板2-3天,至长出单菌落。
3-10.重组毕赤酵母基因组PCR鉴定:
(1)将筛选出的三个耐Zeocin的转化子接种于5mL含100μg/ml Zeocin的YPD液体培养基中,于30℃、250r/min条件下培养16-18h;
(2)取酵母裂解液50ul加入1ul的过夜培养的酵母菌液,85℃孵育30min,12000r/min离心2min,取1ul上清进行PCR扩增;
(3)采用载体通用引物分别对酵母基因组DNA进行PCR扩增,PCR反应完取5μl PCR产物进行1.5%琼脂糖电泳分析,得到电泳图见图3,条带理论大小为1300bp,从图中可看出实际与理论相符合,三个单克隆菌株均为阳性单克隆。
实施例4:重组菌丝霉素的表达及检测
4-1.重组菌丝霉素在毕赤酵母中的表达:
(1)取实施例3经验证的阳性单克隆过夜培养液0.1ml接种到50ml(250ml摇瓶)的YPD培养基中,于30℃、250r/min条件下摇瓶表达;
(2)分别在培养0h、6h、12h、24h、48h、72h、96h后用15ml离心管取6ml培养液,于8000r/min离心5min,将上清转至另一15ml离心管,后将上清和菌沉淀,并放入-80℃保存,直至检测蛋白表达时取出;
4-2.重组菌丝霉素在毕赤酵母中的表达检测:
将表达时间为0h、6h、12h、18h、24h、48h、72h的培养液上清经超滤浓缩、硫酸铵沉淀并复溶后进行Western blot检测,结果如图4所示,泳道M为蛋白Marker,泳道1~7为别为0h、6h、12h、18h、24h、48h、72h培养液上清。从结果可以确定,带组氨酸标签的目的蛋白获得可溶表达,并且在48h培养后目的蛋白的表达量趋于饱和,因此确定表达培养时间为48h较好。
4-3.重组菌丝霉素纯化:
(1)硫酸氨沉淀
a、称取硫酸铵:按照每升上清加361g硫酸氨的比例称取硫酸氨粉末;
b、硫酸的添加:在0℃的条件下,并边搅拌边将硫酸粉末慢慢敲入发酵液上清;
c、静置沉淀:4℃冰箱静置4h,12000r/min离心30min,弃上清;
d、蛋白复溶:按1L发酵液上清加入20ml组氨酸标签-亲和层析柱结合缓冲液(50mM三甲醇氨基甲烷pH 8.0、50mM氯化钠、5%甘油)的比例加入缓冲液,并涡旋直至所有蛋白复溶;
e、过膜转移:将复溶后所得溶液过0.22μm过滤头,转移到新的无菌瓶中。
(2)亲和层析
选用1ml组氨酸标签-亲和层析柱,纯化系统GE Healthcare AKTA pure。
a、洗泵:用蒸馏水洗A1、B1泵后,再分别用结合缓冲液Buffer A(50mM三甲醇氨基甲烷pH 8.0、50mM氯化钠、5%甘油)、洗脱缓冲液BufferB(50mM三甲醇氨基甲烷pH 8.0、50mM氯化钠、500mM咪唑、5%甘油)分别洗A1、B1泵,流速75ml/min;
b、平衡柱子:将A1泵放入BufferA(50mM三甲醇氨基甲烷pH8.0、50mM氯化钠、5%甘油)中,平衡10个柱体积结合缓冲液BufferA(50mM三甲醇氨基甲烷pH 8.0、50mM氯化钠、5%甘油),流速1ml/min;
c、上样:用A1泵上样,流速为0.5ml/min;
d、洗脱:按0%Buffer B、5%Buffer B、60%Buffer B、100%Buffer B进行梯度洗脱,每个梯度洗脱10个柱体积;
e、收集:按峰收集,并超滤浓缩;
f、电泳检测:将收集到的目的蛋白进行SDS-PAGE电泳检测;
4-4.纯化蛋白浓度、体积:
重组菌丝霉素浓度:0.5mg/ml;体积:500μL;储存缓冲液:50mM三甲醇氨基甲烷(Ph8.0)、50mM氯化钠、5%甘油。
4-5.蛋白纯度鉴定:
纯化后抗体进行SDS-PAGE电泳,考马斯亮蓝染色,电泳结果如图5所示,泳道1为0%Buffer B洗脱收集液,泳道2为5%Buffer B洗脱收集液,泳道3为60%Buffer B洗脱收集液,泳道M为蛋白Marker。可见蛋白纯化后纯度在80%以上。
SEQUENCE LISTING
<110> 佛山科学技术学院
<120> 重组菌丝霉素基因的合成及其表达产物的构建方法
<130> 2018
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 120
<212> DNA
<213> 人工序列
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ggttttggtt gtaacggtcc ttggaacgaa gatgatttga gatgtcataa ccattgtaaa 60
tccattaagg gttacaaggg tggatactgt gctaaaggtg gttttgtttg taagtgctac 120
<210> 2
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<212> DNA
<213> 人工序列
<400> 2
ggttttggtt gtaacggtcc ttg 23
<210> 3
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<212> DNA
<213> 人工序列
<400> 3
ttacaatggt tatgacatct caaatcatct tcgttccaag gaccgttaca accaaaa 57
<210> 4
<211> 57
<212> DNA
<213> 人工序列
<400> 4
gatttgagat gtcataacca ttgtaaatcc attaagggtt acaagggtgg atactgt 57
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<212> DNA
<213> 人工序列
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tctagaaagt agcacttaca aacaaaacca cctttagcac agtatccacc cttgtaacc 59
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<211> 48
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<213> 人工序列
<400> 6
agggaagggg ttttggttgt aacgggtttt ggttgtaacg gtccttgg 48
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<213> 人工序列
<400> 7
ggcctctaga aagtagcact tacaaacgta gcacttacaa acaaaaccac ct 52
<210> 8
<211> 23
<212> DNA
<213> 人工序列
<400> 8
ggccgaattc atgaaaatcg aag 23
<210> 9
<211> 22
<212> DNA
<213> 人工序列
<400> 9
accaaaaccc cttccctcga tc 22
<210> 10
<211> 450
<212> PRT
<213> 人工序列
<400> 10
Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn
355 360 365
Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile
370 375 380
Glu Gly Arg Gly Phe Gly Cys Asn Gly Pro Trp Asn Glu Asp Asp Leu
385 390 395 400
Arg Cys His Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr
405 410 415
Cys Ala Lys Gly Gly Phe Val Cys Lys Cys Tyr Phe Leu Glu Gln Lys
420 425 430
Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His
435 440 445
His His
450
<210> 11
<211> 1349
<212> DNA
<213> 人工序列
<400> 11
atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60
ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120
ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180
atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240
accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300
aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360
gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420
aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480
ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540
gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600
aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660
ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720
gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780
ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840
ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900
ggtgccgtag cgctgaagtc ttacgaggaa gagttggtga aagatccgcg tattgccgcc 960
actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020
tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080
gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140
aacctcggga tcgagggaag gggttttggt tgtaacggtc cttggaacga agatgatttg 1200
agatgtcata accattgtaa atccattaag ggttacaagg gtggatactg tgctaaaggt 1260
ggttttgttt gtaagtgcta cttctagaac aaaaactcat ctcagaagag gatctgaata 1320
gcgccgtcga ccatcatcat catcatcat 1349
Claims (7)
1.一种菌丝霉素基因片段,其特征在于,所述基因片段的序列针对毕赤酵母密码子优化表达,如SEQ ID No.1所示。
2.一种构建重组菌丝霉素基因片段的方法,其特征在于,包括以下步骤:
(1).采用如SEQ ID No.2~5所示的基因片段进行拼接重组;
(2).取拼接重组片段和MBP标签片段,利用如SEQ ID No.7和SEQ ID No.8所示的引物搭桥PCR,纯化回收后得到合成重组有MBP标签的重组菌丝霉素基因片段。
3.根据权利要求2所述的构建重组菌丝霉素基因片段的方法,其特征在于,步骤(2)之前还包括利用如SEQ ID No.6和SEQ ID No.7所示的引物对拼接重组片段进行PCR扩增;以及,步骤(2)之前还包括利用如SEQ ID No.8和SEQ ID No.9所示的引物对MBP标签片段进行PCR扩增。
4.构建表达重组菌丝霉素毕赤酵母的方法,其特征在于,包括以下步骤:
(A).取如权利要求2或3所述方法制得的重组菌丝霉素基因片段,与pGAPZa A载体分别进行双酶切,分别纯化回收;
(B).以T4连接酶将酶切后的重组菌丝霉素基因片段和pGAPZa A载体进行连接;
(C).取连接产物加到DH5α感受态细胞中混匀,转化至感受态细胞;
(D).扩大培养含有重组质粒的EcoliDH5α,提取重组质粒;
(E).以限制性内切酶AvrⅡ进行重组质粒的线性化处理;
(F).将线性化的重组质粒电转化至毕赤酵母感受态细胞中;
(G).重组菌丝霉素的表达和纯化。
5.根据权利要求4所述的构建表达重组菌丝霉素毕赤酵母的方法,其特征在于,步骤(C)具体包括:
(C-1).取10μL连接产物加到50μL的DH5α感受态细胞中混匀,冰浴30分钟;
(C-2).将上述转化液置于42℃水浴60秒,取出后立即置于冰浴中放置5分钟;
(C-3).向其中加入500μL 37℃预热的低盐LB培养液,150rpm、37℃振荡培养45分钟;
(C-4).2500rpm离心5分钟,将上清液吸走,留100μL混匀菌液,加到Zeocin浓度25μg/mL的低盐LB固体琼脂培养基上,用无菌的玻璃珠轻轻的将细胞均匀涂开,待平板表面干燥后,倒置平板,37℃培养12-16小时。
6.根据权利要求4所述的构建表达重组菌丝霉素毕赤酵母的方法,其特征在于,步骤(F)具体包括:
(F-1).打开电转化仪,预热30min;
(F-2).设置电穿孔转化电击条件:电压1500V,电阻200Ω,电容2.5mF,5ms;
(F-3).将5-10μg纯化后的线性化质粒加入到新鲜制备的毕赤酵母感受态细胞中,轻轻旋转离心管,使质粒和感受态细胞完全混匀后,将其全部转移至冰预冷处理的0.2cm无菌电转杯中;
(F-4).将电转杯继续冰浴5min后,放入电击槽,开始电击;
(F-5).电击结束后,立即向电转杯中加入1mL冰预冷的1M山梨醇,用移液枪轻轻吹打混匀后,将其全部转移至无菌的1.5mL离心管中;
(F-6).将上述离心管置于30℃恒温培养箱中静置孵育1-2h;
(F-7).分别吸取10,25,50,100,200ul菌液涂布在含100μg/ml Zeocin的YPDS固体培养基上。
(F-8).30℃恒温倒置平板2-3天,至长出单菌落。
7.一种重组菌丝霉素,其特征在于,有权利要求4~6任一项所述方法获得的重组菌丝霉素的氨基酸序列如SEQ ID No.10所示。
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