CN1834250A - β-葡聚糖酶和木聚糖酶融合基因、构建法和用途 - Google Patents
β-葡聚糖酶和木聚糖酶融合基因、构建法和用途 Download PDFInfo
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- CN1834250A CN1834250A CNA2006100500756A CN200610050075A CN1834250A CN 1834250 A CN1834250 A CN 1834250A CN A2006100500756 A CNA2006100500756 A CN A2006100500756A CN 200610050075 A CN200610050075 A CN 200610050075A CN 1834250 A CN1834250 A CN 1834250A
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Abstract
本发明公开了一种β-葡聚糖酶和木聚糖酶的融合基因,其核苷酸序列是SEQ ID NO:1,该融合基因编码的氨基酸序列是SEQ ID NO:2。本发明还公开了该融合基因的构建方法,以及该融合蛋白的制备方法。本发明的融合蛋白能同时水解木聚糖和β-葡聚糖。
Description
技术领域
本发明涉及基因工程领域及蛋白质表达领域,尤其涉及β-葡聚糖酶和木聚糖酶的融合基因构建,和该融合基因的表达方法及其表达产物的用途。
背景技术
据统计,世界粮食年产量为19亿吨左右,其中植物纤维类(fibre material)加工副产品高达2.3亿吨,但这种形式的生物量很难被单胃动物有效利用。其主要原因是其中的非淀粉多糖(non-starch polysaccharides,NSP)具有抗营养作用,NSP的主要抗营养机理为增加食糜粘度和营养屏障,严重时造成肠道微生物区系紊乱。对于禾谷类饲料而言,NSP通常以阿拉伯木聚糖,β-葡聚糖,纤维素,甘露聚糖和半乳糖等几种多糖的复合形式存在,其中又以阿拉伯木聚糖和β-葡聚糖为主要组分,如小麦、黑小麦和黑麦含有较高水平的阿拉伯木聚糖,而燕麦和大麦富含β-葡聚糖。研究表明:向以小麦或黑麦为基础日粮的饲料中添加木聚糖酶,或者向以大麦为基础日粮的饲料中添加β-葡聚糖酶,均能降低NSP的抗营养作用。
β-葡聚糖酶(1,3-1,4-β-D-葡聚糖-4-葡聚糖水解酶)和木聚糖酶(1,4-β-D-木聚糖水解酶)是养殖业上使用最为广泛的两种酶。β-葡聚糖酶特异性的水解葡聚糖中与β-1,3-糖苷键毗邻的1,4-β-D-糖苷键,而木聚糖酶则特异水解阿拉伯木聚糖主链的内部β-1,4-糖苷键。实际生产中,β-葡聚糖酶和木聚糖酶单独添加均不能使NSP的抗营养作用达到显著降低的效果。因此为了达到有效降低NSP抗营养作用的目的,并较充分的利用其中的多糖,往往需要多种酶制剂的同时添加。Salobir(1998)和Mathlouthi等(2001)的研究均表明:β-葡聚糖酶和木聚糖酶的协同使用与两者的分别单独使用相比,在肉仔鸡肠道粘度的降低和营养的利用率方面,前者均显著优于后者。
发明内容
针对现有技术中存在的不足之处,本发明提供一种能翻译成具有双功能活性的β-葡聚糖酶和木聚糖酶的融合基因。
本发明为达到以上目的,是通过这样的技术方案来实现的:提供一种β-葡聚糖酶和木聚糖酶的融合基因,其核苷酸序列是SEQ ID NO:1,命名为gx-78。
本发明同时提供了上述融合基因编码的氨基酸序列是SEQ ID NO:2。
本发明还提供了上述融合基因的构建方法,包括以下步骤:
1)、设计扩增β-葡聚糖酶和木聚糖酶基因片段的PCR引物;
2)、在β-葡聚糖酶基因下游引物和木聚糖酶基因上游引物中加上一段连接肽序列,然后通过PCR反应扩增β-葡聚糖酶和木聚糖酶基因;
3)、根据基因重叠延伸技术,以步骤2)所得的两个PCR反应产物的混合物为模版,分三步进行基因重叠延伸,获得融合基因。
本发明的融合基因的构建方法:步骤2)是在β-葡聚糖酶基因和木聚糖酶基因间引入连接子序列SEQ ID NO:7。首先根据连接子的序列,设计恰当的引物,通过PCR延伸反应,使β-葡聚糖酶基因的下游含有完整的连接子序列,而木聚糖酶基因的上游含有部分的连接子序列。步骤3)根据基因重叠延伸技术,以步骤2)所得的两个PCR产物的混合物为模版,不加引物进行互为引物扩增,此时已获得融合基因。回收扩增产物作为进一步PCR反应的模板,以β-葡聚糖酶基因的上游引物和木聚糖酶基因的下游引物为引物进行扩增反应,获得更大量的融合基因产物。然后依次进行了目的片段割胶回收,并插入克隆载体或表达载体、质粒提取等步骤。
利用上述β-葡聚糖酶和木聚糖酶的融合基因所制得的融合蛋白,同时具有β-葡聚糖酶活性和木聚糖酶活性,因此该种融合蛋白能同时水解β-葡聚糖和木聚糖。
本发明还提供了上述融合蛋白的制备方法,包括以下步骤:
1)、将SEQ ID NO:1的核苷酸序列插入表达载体,形成融合基因重组表达载体;
2)、将上述融合基因重组表达载体转入表达宿主细胞,形成含融合基因表达载体的阳性细胞;
3)、在适合该融合蛋白表达的条件下,培养上述步骤中的阳性细胞;
4)、分离纯化出其中所表达的并正确加工的融合蛋白。
本发明的融合基因gx-78的序列,同时具有SEQ ID NO:3和SEQ ID NO:4的特征,基因的5’端为SEQ ID NO:3序列,来自于β-葡聚糖酶基因,全长为717bp,其中1-717位是基因开放阅读框,1-3位是起始密码子ATG,无终止密码子。3’端为SEQ ID NO:4序列,来自木聚糖酶成熟肽基因序列,全长为558bp,其中787-1344位是该基因开放阅读框,1342-1344位为终止密码子。SEQ ID NO:3和SEQ ID NO:4之间的连接肽L-78编码序列SEQ ID NO:7。
本发明的融合基因gx-78序列编码的多肽同时具有SEQ ID NO:5和SEQID NO:6中的氨基酸序列,并同时具有葡聚糖酶活性和木聚糖酶活性,将在高效低价的食品和饲料添加剂应用中具有广阔的前景。
本发明的融合基因gx-78所用的重组克隆载体,均含有SEQ ID NO:3和SEQ ID NO:4所述的DNA;克隆宿主细胞为上述重组克隆载体转化的原核细胞。
本发明的设计原理如下:与β-葡聚糖酶和木聚糖酶的单独添加相比,两者的协同使用能更为显著减轻禾谷类饲料中NSP所导致的抗营养作用。因此选择淀粉液化芽孢杆菌(Bacillus licheniformis)等来源的β-葡聚糖酶和枯草芽孢杆菌(Bacillus subtilis)等来源的木聚糖酶构建同时具有β-葡聚糖酶和木聚糖酶活性的融合蛋白。又因为当两个功能单位的蛋白(或结构域)融合时,两部分可能会因为靠的较近而影响各自的正确折叠,因此常在两融合单位间加一柔性较强的连接肽序列;同时连接肽的氨基酸组成、氨基酸性质、长短等均影响连接肽的特性,从而影响功能单位的融合。但有研究表明,α螺旋同样可以作为连接肽,用于双功能蛋白的构建研究(Ryoichi等,2001)。因此本发明设计了含有连接肽序列SEQ ID NO:8的融合蛋白,该连接肽源于天然存在的Humicola grisea的纤维素酶蛋白,其结构(PDB id:1UU6)已经解析,在该蛋白中其二级结构为一螺旋,为刚性结构,能将两个结构域独立的分开。将该融合基因在大肠杆菌表达系统中进行表达,以期获得具有双功能的重组蛋白生产菌株。在完成β-葡聚糖酶和木聚糖酶基因克隆和测序基础上,把β-葡聚糖酶和木聚糖酶基因通过SOE技术进一步连接成融合基因,并在相互间融合了连接子序列,构建成含有多糖酶活性的融合基因,融合基因成功的在大肠杆菌中获得表达,获得同时具有β-葡聚糖酶和木聚糖酶活性的双功能融合蛋白。
对本发明的序列表作如下说明:
一、SEQ ID NO:1代表gx-78融合基因序列:
序列特征如下:长度:1344碱基;类型:核酸;链性:双链;拓扑:线性。
分子型:DNA。
二、SEQ ID NO:2代表gx-78融合基因编码的氨基酸序列:
序列特征如下:长度:447个氨基酸;类型:氨基酸;拓扑结构:果冻卷状(jellyroll-like)。
分子型:多肽。
三、SEQ ID NO:3代表β-葡聚糖酶基因序列:
序列特征如下:长度:720碱基;类型:核酸;链性:双链;拓扑:线性。
分子型:DNA。
四、SEQ ID NO:4代表木聚糖酶成熟肽的编码基因序列:
序列特征如下:长度:558碱基;类型:核酸;链性:双链;拓扑:线性。
分子型:DNA。
五、SEQ ID NO:5代表β-葡聚糖酶基因编码的氨基酸序列:
序列特征如下:长度:239个氨基酸;类型:氨基酸;拓扑结构:果冻卷状(jellyroll-like)。
分子型:多肽。
六、SEQ ID NO:6代表木聚糖酶基因编码的成熟肽氨基酸序列:
序列特征如下:长度:185个氨基酸;类型:氨基酸;拓扑结构:果冻卷状(jellyroll-like)。
分子型:多肽。
七、SEQ ID NO:7代表连接肽L-78的编码序列:
序列特征如下:长度:69碱基;类型:核酸;链性:双链;拓扑:线性。
分子型:DNA。
八、SEQ ID NO:8代表连接肽L-78的氨基酸序列:
序列特征如下:长度:23个氨基酸;类型:氨基酸;拓扑结构:线性。
分子型:多肽。
具体实施方式
实施例1、PCR引物设计
在保留β-葡聚糖酶和木聚糖酶基因的功能区域基础上,设计PCR引物;本发明是由连接肽过渡连接的融合蛋白,因此在两基因的连接区引入连接肽编码序列(Linker)。
1、β-葡聚糖酶基因引物:
以淀粉液化芽孢杆菌的基因组为模版,参照Genbank上已由本申请人克隆登录β-葡聚糖酶基因,设计上下游引物,pG5和pG3,用于扩增删除终止密码子的β-葡聚糖酶基因。在上游引物中引入BamH I酶切位点序列。
上游引物序列如下,引物名称为pG5:
BamH I
下游引物序列如下,引物名称为pG3:
5′-TTGCCAGTAGTCTGTGCTAGCTTTTTTT-3′
2、木聚糖酶基因引物:
参照Genbank上所登录的木聚糖酶基因序列,进行同源性分析,根据保守序列设计上下游引物,pX5和pX3:用于扩增完整的木聚糖酶基因。在下游引物中引入Xhol I酶切位点序列。
上游引物序列如下,引物名称为pX5:
5′-ATGTTTAAGTTTAAAAAGAAT-3′
下游引物序列如下,引物名称为pX3:
Xhol I
3、融合基因gx-78构建引物:
参照β-葡聚糖酶基因和木聚糖酶基因序列,进行设计。
设计三对对引物,第一对引物(pG5和p7-1),用于克隆β-葡聚糖酶基因序列和编码连接肽L-78的部分5′-端序列,第二对引物(pG5和p7-2),用于克隆β-葡聚糖酶基因序列和编码连接肽L-78的完整序列SEQ ID NO:7。第三对引物(p8和pX3),用于克隆编码连接肽L-78的部分3′-端序列和木聚糖酶成熟肽部分的编码基因。其中第二对引物中p7-1和p7-2对连接肽的完整克隆和第三对引物中p8对连接肽的部分克隆,导致了重叠序列的存在,因此可以用SOE方法连接两个基因,融合基因构建后将在5′和3′分别引入BamH I和Xhol I酶切位点序列。
第一对引物:
上游引物名称为pG5,序列同上;
下游引物序列如下,名称为p7-1:
5′-CATATATTTGCGGCTGCCGGTTTTTTTTGTATAGCGCAC-3′
第二对引物:
上游引物名称为pG5,序列同上;
下游引物序列如下,名称为p7-2:
5′-CATGCCACGGGTCAGGGCCTCACCCATGCCCTGGGTCGCGCCCA
GTTC CATATATTTGCGGCTGCCGGT-3′
第三对引物:
上游引物序列如下,名称为p8:
5′-GAGGCCCTGACCCGTGGCATGGCTAGCACAGACTACTGG-3′
下游引物名称为p X3,序列同上。
实施例2、PCR扩增
1、扩增β-葡聚糖酶基因片段:
PCR反应体系:淀粉液化芽孢杆菌基因组DNA 0.5-1.0μl,10×Taq butter5μl,dNTP(2.5mM)2-4μl,上游引物(pG5,13pmol/μl)1-2μl,下游引物(pG3,13pmol/μl)1-2μl,Taq-plus DNA pol.(1U/μl)0.2-0.5μl,加ddH2O至总体积50μl。扩增条件为:94℃变性5min后进入循环,94℃变性45s,50℃复性1min,72℃延伸1min,35个循环后在72℃延伸10min。
2、扩增木聚糖酶基因片段:
PCR反应体系:枯草芽孢杆菌基因组DNA 0.5-1.0μl,10×Taq butter 5μl,dNTP(2.5Mm)2-4μl,上游引物(pX5,13pmol/μl)1-2μl,下游引物(pX3,13pmol/μl)1-2μl,Taq-plus DNA pol.(1U/μl)0.2-0.5μl,加ddH2O至总体积50μl。扩增条件为:94℃变性5min后进入循环,94℃变性45S,47℃复性45s,72℃延伸1min,35个循环后在72℃延伸10min。
3、β-葡聚糖酶基因和木聚糖酶基因的PCR扩增结果
β-葡聚糖酶基因和木聚糖酶基因的PCR产物长约720bp和642bp,和目的基因的大小一致。
实施例3、融合基因gx-78的构建(SOE方法)
以含β-葡聚糖酶基因的重组质粒为模板,以pG5和p7-1为引物扩增融合基因的5’端,得到剔除终止密码子的β-葡聚糖酶基因序列及连接肽L-78编码序列的5’端部分,PCR扩增条件体系同上,退火温度43℃,35个循环结束后直接结束反应,不进行72℃10分钟加碱基腺嘌呤核苷酸A;凝胶回收PCR产物作为模板,以pG5和p7-2为引物进一步扩增,得到剔除终止密码子的β-葡聚糖酶基因序列及连接肽L-78的编码全序列,PCR扩增条件不变。
以含木聚糖酶基因的重组质粒为模板,以p8和pX3为引物扩增融合基因的3’端,得到编码木聚糖酶成熟肽的全序列及连接肽编码序列的3’端部分,凝胶回收PCR产物;PCR扩增条件同上,退火温度47℃,35个循环结束后直接结束反应,不进行72℃10分钟加碱基腺嘌呤核苷酸A。
合并上述割胶回收产物,不加引物进行PCR扩增,反应体系如下(50μl):PCR反应体系:10×Taq butter 5μl,dNTP(2.5Mm)2-4μl,上游引物(pG513pmol/μl)1-2μl,下游引物(pX3,13pmol/μl)1-2μl,Taq-plus DNA pol.(1U/μl)2.5-5.0μl,加ddH2O至总体积50μl。扩增条件为94℃变性5min后进入循环,65℃复性1-1.5min,72℃延伸1.5-2.0min,30-35个循环后在72℃延伸10min。割胶回收产物加入引物pG5和pX3进一步进行PCR扩增,扩增条件同上,除Taq-plus DNA pol.(1U/μl)1.0μl。
实施例4、融合基因gx-78克隆
β-葡聚糖酶-木聚糖酶融合基因7.0μl,pUCm-T Vector 1.0μl,10×连接酶缓冲液1μl,T4DNA连接酶(1U/μl)1μl,ddH2O 4μl,总体积10μl。16℃连接过夜,将基因定向插入到质粒pUCm-T中。
实施例5、连接产物转化
连接产物转化、感受态的制备、阳性克隆筛选、质粒DNA的提取均按文献所记载的现有技术进行。融合基因片段与质粒pUCm-T连接并导入宿主菌Top10后,经氨苄青霉素抗性及α-互补筛选后,筛选阳性重组质粒,pUCm-T/g-x。
实施例6、DNA序列分析
用碱法提取质粒,用BigDye terminator v2.0测序试剂盒(EpicentreTeclnologies)对抽提的质粒在全自动测序仪(ABI Prim 377-18,PE公司)进行测序,融合基因gx-78序列全长为1344bp,详细序列见SEQ ID No:1。
gx-78开放读框分别位于1-1344位核苷酸,1-717bp来自β-葡聚糖酶基因,序列长为717bp;718-786bp为连接肽L-78的DNA序列,共69bp;787-1344bp来自木聚糖酶基因,序列长为558bp。该融合基因全长序列具有起始和终止密码子,编码447个氨基酸残基。融合基因对应的氨基酸序列详见SEQ ID No:2。其中1-239位来自β-葡聚糖酶,240-262位为连接肽L-78的序列,263-447位来自木聚糖酶。由于翻译后加工,融合蛋白的前体所存在β-葡聚糖酶来源的信号肽(1-25)被切割掉,融合蛋白成熟肽共有422个氨基酸残基。
实施例7、融合基因gx-78在大肠杆菌BL21中的表达
重组质粒pUCm-T/g-x酶切,酶切体系如下:10×M Buffer 2.0μl,10×BSA2.0μl,BamH I限制性内切酶(10U/μl)1.0μl,Xhol I限制性内切酶(10U/μl)1.0μl,pUCm-T/g-x重组克隆载体14.0μl,反应液混匀,37℃酶切过夜。之后电泳,割胶回收目的基因。同时表达载体用相同的内切酶酶切,酶切反应体系同上,割胶回收目的片段。
目的基因和表达载体pET-30a酶切的割胶回收产物连接,连接反应体系如下:酶切载体DNA 4.0μl,目的基因4.0μl,T4 DNA连接酶1.0μl,10×T4 DNA连接酶反应缓冲液1.0μl,于12℃连接过夜。连接产物大肠杆菌BL21感受态细胞,筛选重组表达子。
实施例8、融合表达产物纯化及活性检测
将筛选到的大肠杆菌重组阳性表达子分别接种于LB液体培养基中,30℃静置培养过夜,以2.0%接种量转接于250ml LB液体培养基中,100rpm旋转式摇床,28℃培养至OD600为0.6左右,加入终浓度为0.5mMol/mL IPTG诱导6-8h,此时菌液OD600为2.5左右。
采用超声波破碎仪破碎菌悬液,条件设置为:工作时间5s,间隙时间4s,全程时间1h,工作温度0℃,一次破碎80mL左右的菌液。向菌破碎上清液加入硫酸铵至25%的饱和度,在冰上过夜,10000rpm离心30min去沉淀以去除部分分子量较大的杂蛋白,取上清,再加入硫酸铵至饱和度为65%,过夜冰浴,10000rpm离心30min去上清,取沉淀并用适量的0.2Mol/L的醋酸钠(pH6.0)溶解,0.45μm滤膜过滤液用于脱盐。取2.0mL沉淀溶解液,在KTAexplore 100蛋白纯化仪上,采用G-25脱盐柱进行脱盐,柱名为HiPrepTM 26/10desalting,洗脱缓冲液为0.2MOL/L的醋酸钠(pH6.0),流速为4.0mL/min,管收集(4mL/管)。在KTA explore 100蛋白纯化仪上,采用阳离子交换柱进行纯化,柱名HiPrep16/10SourceTM 30S,洗脱程序为:先以2个柱体积的0.2mol/L的醋酸钠(pH6.0)缓冲液平衡,样品上柱后开始以0.5mol/L NaCl进行梯度洗脱,达到100%盐浓度需要4个柱体积的混合缓冲液,然后以1.5个柱体积的0.2mol/L的醋酸钠(pH6.0)缓冲液再次平衡,管收集(2mL/管)。收集OD280和OD260的各个峰对应的试管,用于酶活力测定和SDS-PAGE电泳确定纯化效果。
酶活力测定由还原糖法进行。在试管中加入1000μl浓度为0.5%的桦木木聚糖底物,预热5min,然后加入粗酶液100μl,50℃准确反应10min,加入500μl DNS溶液终止反应,沸水浴5min,冷却后于540nm处比色,测定OD值。
用大麦β-葡聚糖底物替代上述桦木木聚糖底物,重复上述的测试方法。
结果表明,本发明的融合蛋白具有双功能酶活性,纯化后的蛋白电泳得到单一的、大小与理论分子量一致的目的条带。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
序列表
SEQ ID NO:1
1 ATGAAACGAG TGTTGCTAAT TCTTGTCACC GGATTGTTTA TGAGTTTGTG
51 TGGGATCACT TCTAGTGTTT CGGCTCAAAC AGGCGGATCG TTTTTTGAAC
101 CTTTTAACAG CTATAACTCC GGGTTATGGC AAAAAGCTGA TGGTTACTCA
151 AATGGAGATA TGTTTAACTG CACTTGGCGT GCGAATAACG TCTCTATGAC
201 GTCATCAGGT GAAATGCGTT TGGCGCTGAC AAGTCCGTCT TATAACAAGT
251 TTGACTGCGG GGAAAACCGC TCGGTTCAAA CATATGGCTA TGGACTTTAT
301 GAAGTCAGAA TGAAACCGGC TAAAAACACA GGGATTGTTT CATCGTTCTT
351 CACTTATACA GGTCCAACGG AGGGGACTCC TTGGGATGAG ATTGATATCG
401 AATTTTTGGG AAAAGACACA ACAAAGGTTC AATTTAACTA TTATACAAAT
451 GGCGCAGGAA ACCATGAGAA GTTGGCGGAT CTCGGATTTG ATGCAGCCAA
501 TGCCTATCAT ACGTATGCGT TCGATTGGCA GCCAAACTCT ATTAAATGGT
551 ATGTCGATGG GCAATTAAAA CATACTGCGA CAACCCAAAT ACCGGCAGCG
601 CCGGGGAAAA TCATGATGAA TTTGTGGAAT GGTACGGGTG TCGATGATTG
651 GCTCGGTTCC TACAATGGCG TAAATCCGCT ATACGCTCAT TACGACTGGG
701 TGCGCTATAC AAAAAAAACC GGCAGCCGCA AATATATGGA ACTGGGCGCG
751 ACCCAGGGCA TGGGTGAGGC CCTGACCCGT GGCATGGCTA GCACAGACTA
801 CTGGCAAAAT TGGACTGATG GGGGCGGTAT AGTAAACGCT GTCAATGGGT
851 CTGGCGGGAA TTACAGTGTT AATTGGTCTA ATACCGGAAA TTTTGTTGTT
901 GGTAAAGGTT GGACTACAGG TTCGCCATTT AGGACGATAA ACTATAATGC
951 CGGAGTTTGG GCGCCGAATG GCAATGGATA TTTAACTTTA TATGGTTGGA
1001 CGAGATCACC TCTCATAGAA TATTATGTAG TGGATTCATG GGGTACTTAT
1051 AGACCTACTG GAACGTATAA AGGTACTGTA AAAAGTGATG GGGGTACATA
1101 TGACATATAT ACAACTACAC GTTATAACGC ACCTTCCATT GATGGCGATC
1151 GCACTACTTT TACGCAGTAC TGGAGTGTTC GCCAGTCGAA GAGACCAACC
1201 GGAAGCAACG CTACAATCAC TTTCAGCAAT CATGTGAACG CATGGAAGAG
1251 CCATGGAATG AATCTGGGCA GTAATTGGGC TTACCAAGTC ATGGCGACAG
1301 AAGGATATCA AAGTAGTGGA AGTTCTAACG TAACAGTGTG GTAA
SEQ ID NO:2
1 Met Lys Arg Val Leu Leu Ile Leu Val Thr Gly Leu Phe Met Ser
16 Leu Cys Gly Ile Thr Ser Ser Val Ser Ala Gln Thr Gly Gly Ser
31 Phe Phe Glu Pro Phe Asn Ser Tyr Asn Ser Gly Leu Trp Gln Lys
46 Ala Asp Gly Tyr Ser Asn Gly Asp Met Phe Asn Cys Thr Trp Arg
61 Ala Asn Asn Val Ser Met Thr Ser Ser Gly Glu Met Arg Leu Ala
76 Leu Thr Ser Pro Ser Tyr Asn Lys Phe Asp Cys Gly Glu Asn Arg
91 Ser Val Gln Thr Tyr Gly Tyr Gly Leu Tyr Glu Val Arg Met Lys
106 Pro Ala Lys Asn Thr Gly Ile Val Ser Ser Phe Phe Thr Tyr Thr
121 Gly Pro Thr Glu Gly Thr Pro Trp Asp Glu Ile Asp Ile Glu Phe
136 Leu Gly Lys Asp Thr Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn
151 Gly Ala Gly Asn His Glu Lys Leu Pro Asp Leu Gly Phe Asp Ala
166 Ala Asn Ala Tyr His Thr Tyr Ala Phe Asp Trp Gln Pro Asn Ser
181 Ile Lys Trp Tyr Val Asp Gly Gln Leu Lys His Thr Ala Thr Thr
196 Gln Ile Pro Ala Ala Pro Gly Lys Ile Met Met Asn Leu Trp Asn
211 Gly Thr Gly Val Asp Asp Trp Leu Gly Ser Tyr Asn Gly Val Asn
226 Pro Leu Tyr Ala His Tyr Asp Trp Val Arg Tyr Thr Lys Lys Thr
241 Gly Ser Arg Lys Tyr Met Glu Leu Gly Ala Thr Gln Gly Met Gly
256 Glu Ala Leu Thr Arg Gly Met Ala Ser Thr Asp Tyr Trp Gln Asn
271 Trp Thr Asp Gly Gly Gly Ile Val Asn Ala Val Asn Gly Ser Gly
286 Gly Asn Tyr Ser Val Asn Trp Ser Asn Thr Gly Asn Phe Val Val
301 Gly Lys Gly Trp Thr Thr Gly Ser Pro Phe Arg Thr Ile Asn Tyr
316 Asn Ala Gly Val Trp Ala Pro Asn Gly Asn Gly Tyr Leu Thr Leu
331 Tyr Gly Trp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr Val Val Asp
346 Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Thr Val
361 Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr Arg Tyr
376 Asn Ala Pro Ser Ile Asp Gly Asp Arg Thr Thr Phe Thr Gln Tyr
391 Trp Ser Val Arg Gln Ser Lys Arg Pro Thr Gly Ser Asn Ala Thr
406 Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser His Gly Met
421 Asn Leu Gly Ser Asn Trp Ala Tyr Gln Val Met Ala Thr Glu Gly
436 Tyr Gln Ser Ser Gly Ser Ser Asn Val Thr Val Trp
SEQ ID NO:3
1 ATGAAACGAG TGTTGCTAAT TCTTGTCACC GGATTGTTTA TGAGTTTGTG
51 TGGGATCACT TCTAGTGTTT CGGCTCAAAC AGGCGGATCG TTTTTTGAAC
101 CTTTTAACAG CTATAACTCC GGGTTATGGC AAAAAGCTGA TGGTTACTCA
151 AATGGAGATA TGTTTAACTG CACTTGGCGT GCGAATAACG TCTCTATGAC
201 GTCATCAGGT GAAATGCGTT TGGCGCTGAC AAGTCCGTCT TATAACAAGT
251 TTGACTGCGG AGAAAACCGC TCGGTTCAAA CATATGGCTA TGGACTTTAT
301 GAAGTCAGAA TGAAACCGGC TAAAAACACA GGAATTGTTT CATCGTTCTT
351 CACTTATACA GGTCCAACGG AGGGGACTCC TTGGGATGAG ATTGATATCG
401 AATTTTTGGG AAAAGACACA ACAAAGGTTC AATTTAACTA TTATACAAAT
451 GGCGCAGGAA ACCATGAGAA GTTGCCGGAT CTCGGATTTG ATGCAGCCAA
501 TGCCTATCAT ACGTATGCGT TCGATTGGCA GCCAAACTCT ATTAAATGGT
551 ATGTCGATGG GCAATTAAAA CATACTGCGA CAACCCAAAT ACCGGCAGCG
601 CCGGGGAAAA TCATGATGAA TTTGTGGAAT GGTACGGGTG TCGATGATTG
651 GCTCGGTTCC TACAATGGCG TAAATCCGCT ATACGCTCAT TACGACTGGG
701 TGCGCTATAC AAAAAAATAA
SEQ ID NO:4
1 GCTAGCACAG ACTACTGGCA AAATTGGACT GATGGGGGCG GTATAGTAAA
51 CGCTGTCAAT GGGTCTGGCG GGAATTACAG TGTTAATTGG TCTAATACCG
101 GAAATTTTGT TGTTGGTAAA GGTTGGACTA CAGGTTCGCC ATTTAGGACG
151 ATAAACTATA ATGCCGGAGT TTGGGCGCCG AATGGCAATG GATATTTAAC
201 TTTATATGGT TGGACGAGAT CACCTCTCAT AGAATATTAT GTAGTGGATT
251 CATGGGGTAC TTATAGACCT ACTGGAACGT ATAAAGGTAC TGTAAAAAGT
301 GATGGGGGTA CATATGACAT ATATACAACT ACACGTTATA ACGCACCTTC
351 CATTGATGGC GATCGCACTA CTTTTACGCA GTACTGGAGT GTTCGCCAGT
401 CGAAGAGACC AACCGGAAGC AACGCTACAA TCACTTTCAG CAATCATGTG
451 AACGCATGGA AGAGCCATGG AATGAATCTG GGCAGTAATT GGGCTTACCA
501 AGTCATGGCG ACAGAAGGAT ATCAAAGTAG TGGAAGTTCT AACGTAACAG
551 TGTGGTAA
SEQ ID NO:5
1 Met Lys Arg Val Leu Leu Ile Leu Val Thr Gly Leu Phe Met Ser
16 Leu Cys Gly Ile Thr Ser Ser Val Ser Ala Gln Thr Gly Gly Ser
31 Phe Phe Glu Pro Phe Asn Ser Tyr Asn Ser Gly Leu Trp Gln Lys
46 Ala Asp Gly Tyr Ser Asn Gly Asp Met Phe Asn Cys Thr Trp Arg
61 Ala Asn Asn Val Ser Met Thr Ser Ser Gly Glu Met Arg Leu Ala
76 Leu Thr Ser Pro Ser Tyr Asn Lys Phe Asp Cys Gly Glu Asn Arg
91 Ser Val Gln Thr Tyr Gly Tyr Gly Leu Tyr Glu Val Arg Met Lys
106 Pro Ala Lys Asn Thr Gly Ile Val Ser Ser Phe Phe Thr Tyr Thr
121 Gly Pro Thr Glu Gly Thr Pro Trp Asp Glu Ile Asp Ile Glu Phe
136 Leu Gly Lys Asp Thr Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn
151 Gly Ala Gly Asn His Glu Lys Leu Pro Asp Leu Gly Phe Asp Ala
166 Ala Asn Ala Tyr His Thr Tyr Ala Phe Asp Trp Gln Pro Asn Ser
181 Ile Lys Trp Tyr Val Asp Gly Gln Leu Lys His Thr Ala Thr Thr
196 Gln Ile Pro Ala Ala Pro Gly Lys Ile Met Met Asn Leu Trp Asn
211 Gly Thr Gly Val Asp Asp Trp Leu Gly Ser Tyr Asn Gly Val Asn
226 Pro Leu Tyr Ala His Tyr Asp Trp Val Arg Tyr Thr Lys Lys
SEQ ID NO:6
1 Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Ile
16 Val Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val Asn Trp
31 Ser Asn Thr Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly
46 Ser Pro Phe Arg Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro
61 Asn Gly Asn Gly Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Ser Pro
76 Leu Ile Glu Tyr Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro
9l Thr Gly Thr Tyr Lys Gly Thr Val Lys Ser Asp Gly Gly Thr Tyr
106 Asp Ile Tyr Thr Thr Thr Arg Tyr Asn Ala Pro Ser Ile Asp Gly
121 Asp Arg Thr Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln Ser Lys
136 Arg Pro Thr Gly Ser Asn Ala Thr Ile Thr Phe Ser Asn His Val
151 Asn Ala Trp Lys Ser His Gly Met Asn Leu Gly Ser Asn Trp Ala
166 Tyr Gln Val Met Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser
181 Asn Val Thr Val Trp
SEQ ID NO:7
1 ACCGGCAGCC GCAAATATAT GGAACTGGGC GCGACCCAGG GCATGGGTGA
51 GGCCCTGACC CGTGGCATG
SEQ ID NO:8
1 Thr Gly Ser Arg Lys Tyr Met Glu Leu Gly Ala Thr Gln Gly Met
16 Gly Glu Ala Leu Thr Arg Gly Met
Claims (5)
1、一种β-葡聚糖酶和木聚糖酶的融合基因,其核苷酸序列是SEQ ID NO:1。
2、如权利要求1所述的β-葡聚糖酶和木聚糖酶的融合基因编码的氨基酸序列是SEQ ID NO:2。
3、如权利要求1所述的β-葡聚糖酶和木聚糖酶的融合基因的构建方法,其特征在于包括以下步骤:
1)、设计扩增β-葡聚糖酶和木聚糖酶基因片段的PCR引物;
2)、在β-葡聚糖酶基因下游引物和木聚糖酶基因上游引物中加上一段连接肽序列,然后通过PCR反应扩增β-葡聚糖酶和木聚糖酶基因;
3)、根据基因重叠延伸技术,以步骤2)所得的两个PCR反应产物的混合物为模版,分三步进行基因重叠延伸,获得融合基因。
4、如权利要求1所述的β-葡聚糖酶和木聚糖酶的融合基因制得的融合蛋白的用途,其特征在于:该融合蛋白能同时水解木聚糖和β-葡聚糖。
5、如权利要求4所述的融合蛋白的制备方法,其特征在于包括以下步骤:
1)、将SEQ ID NO:1的核苷酸序列插入表达载体,形成融合基因重组表达载体;
2)、将上述融合基因重组表达载体转入表达宿主细胞,形成含融合基因表达载体的阳性细胞;
3)、在适合该融合蛋白表达的条件下,培养上述步骤中的阳性细胞;
4)、分离纯化出其中所表达的并正确加工的融合蛋白。
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US20160002616A1 (en) * | 2013-03-12 | 2016-01-07 | Basf Enzymes Llc | Genes with codon mutations encoding xylanase |
CN108570107A (zh) * | 2017-03-08 | 2018-09-25 | 南京工业大学 | 一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用 |
CN113564092A (zh) * | 2021-08-16 | 2021-10-29 | 合肥工业大学 | 一种定向合成右旋糖酐的融合酶及其构建方法与应用 |
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US20160002616A1 (en) * | 2013-03-12 | 2016-01-07 | Basf Enzymes Llc | Genes with codon mutations encoding xylanase |
US10131895B2 (en) * | 2013-03-12 | 2018-11-20 | Basf Enzymes Llc | Genes with codon mutations encoding xylanase |
US10619142B2 (en) | 2013-03-12 | 2020-04-14 | Basf Enzymes Llc | Genes with codon mutations encoding xylanase |
CN108570107A (zh) * | 2017-03-08 | 2018-09-25 | 南京工业大学 | 一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用 |
CN108570107B (zh) * | 2017-03-08 | 2021-12-28 | 南京工业大学 | 一种扩张蛋白和木聚糖酶融合蛋白、其编码基因和应用 |
CN113564092A (zh) * | 2021-08-16 | 2021-10-29 | 合肥工业大学 | 一种定向合成右旋糖酐的融合酶及其构建方法与应用 |
CN113564092B (zh) * | 2021-08-16 | 2024-02-27 | 合肥工业大学 | 一种定向合成右旋糖酐的融合酶及其构建方法与应用 |
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