CN109734782B - 一种扩张蛋白及其在几丁质胶体制备中的应用 - Google Patents
一种扩张蛋白及其在几丁质胶体制备中的应用 Download PDFInfo
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Abstract
本发明公开了一种扩张蛋白及其在几丁质胶体制备中的应用。本发明提供一种扩张蛋白氨基酸序列如SEQ ID NO.2所示,编码该扩张蛋白的核苷酸序列如SEQ ID NO.1所示。使用本发明扩张蛋白降解几丁质而制备出几丁质胶体,并且将几丁质制备成壳寡糖。采用本发明的方法,克服了传统化学法处理几丁质时产生大量的无机强酸废液,造成严重的环境污染、对设备要求高、产品质量差等现有技术中的不足。节约了成本,不造成环境污染,符合环境友好型。
Description
技术领域
本发明属于功能基因克隆表达技术领域,具体涉及一种扩张蛋白及其在几丁质胶体制备中的应用方法。
背景技术
几丁质又称甲壳素是天然的多聚物,由基本结构单元N-乙酰-D-葡萄糖胺以β-1,4-糖苷键连接构成,广泛存在于甲壳类动物的外壳和真菌的细胞壁中。未经过任何处理的甲壳素具有致密的结构,其不溶于水、稀酸、碱及其他有机溶剂。只可通过强酸破坏甲壳素的晶体结构,将几丁质制备成几丁质胶体,然后用水解酶将几丁质胶体水解为N-乙酰葡萄糖胺或壳寡糖。产物壳寡糖具备抗肿瘤、降血压、增强免疫力等独特的药理功能活性;具有抑制植物病原菌的生长,诱导高等植物体内产生相关抗体等功能,在医药、食品保健品、种植等领域有了更大的应用价值。这使得制备几丁质胶体就成为一个重要关键步骤。
目前制备几丁质胶体的主要方法是,用高浓度的无机强酸溶解粉末几丁质,再中和强酸或大量水稀释,析出沉淀,离心出沉淀,将沉淀再次溶解成胶体。此方法在生产过程中浪费大量的水资源,并产生大量的无机强酸废液,造成严重的环境污染。因此,寻找一种绿色、高效高产高品质几丁寡糖的方法越来越受重视。
扩张蛋白来自于细菌和植物,是一种非水解性的辅助蛋白,对具有多糖网状结构的底物具有破坏作用,在不水解它们的过程中断裂多糖之间的氢键,打开多糖底物的致密晶体结构。从而可以使水分子或水解酶进入多糖底物,使其形成水溶性形态。
发明内容
本发明目的在于提供一种扩张蛋白及其在几丁质胶体制备中的应用制作方法;旨在克服了传统化学法水解几丁质胶体中化学试剂污染环境、产品质量差等现有技术中的不足,提供了一种经济高效、环保型酶法制备几丁质胶体的应用。
本发明解决技术问题采用的技术方案为:
本发明所提供的扩张蛋白,其氨基酸序列为SEQ ID NO.2;
用于编码本发明的扩张蛋白的基因,其一种核苷酸序列为SEQ ID NO.1;本发明还提供一种含有扩张蛋白的重组表达菌,;
本发明的扩张蛋白用于制备几丁质胶体;
本发明提供一种制备几丁质胶体的方法,用本发明的扩张蛋白将几丁质制备成几丁质胶体,最适反应pH和温度分别为pH6.0、40℃;
本发明提供一种通过上述方法制备的几丁质胶体在几丁质酶作用下制备壳寡糖的方法,相较于未处理的几丁质,处理后的几丁质更易于水解,产生壳寡糖量更高。
本发明按真菌RNA提取试剂盒提取RNA,将提取的RNA按照RT-PCR试剂盒合成cDNA,根据目的基因的核苷酸序列设计引物,以合成的cDNA为模板进行PCR扩增,得到扩张蛋白基因,其编码区长528bp。
有益效果:
相较于已报道的几丁质胶体的制备方法,可通过生物法高效的水解几丁质中的氢键,破坏几丁质致密的网格结构来制备几丁质胶体。
附图说明:
图1:本发明的扩张蛋白基因的电泳检测图(M:蛋白Marker;22:扩张蛋白EX22基因);
图2:EX22基因同源进化树图;
图3:EX22基因保守结构域分析图;
图4:重组质粒pPICZαA-EX22序列对比图;
图5:本发明重组菌落PCR验证图(1:重组菌落;M:蛋白Marker)
图6:本发明的扩张蛋白纯化后的纯蛋白SDS-PAGE电泳图(M:蛋白Marker;1:重组菌pPICZαA-EX22/X-33表达上清;2:空菌pPICZαA/X33表达上清);
图7:本发明的扩张蛋白最适pH值的测定;
图8:本发明的扩张蛋白最适温度的测定;
图9:本发明的扩张蛋白作用后产物的SEM图((A)、(B)和(C):EX22作用的几丁质;(D)、(E)和(F):水作用的几丁质;(A)和(D):2000×;(B)和(E):5000×;(C)和(F):20000×);
图10:几丁质酶作用未处理几丁质及本发明的扩张蛋白制得的几丁质胶体相对活力比较。
具体实施方式:
下面通过具体实例对本发明的方法做进一步的说明。
下面通过实施例对本发明做进一步描述,以下实施例仅用于说明本发明而非用于限定本发明的范围,实施例中的实验方法,如无特殊说明,均为常规方法。如无特殊说明,本发明所使用试剂均市售可得。
实施例1扩张蛋白基因片段的克隆
按真菌RNA提取试剂盒(上海生工,SK8659)操作步骤提取线虫捕捉菌属CX1菌-Arthrobotrys sp.CX1的RNA,按TaKaRa生物公司的一步RT-PCR试剂盒(CodeNo.RR064A)的操作步骤合成cDNA。根据目的基因的核苷酸序列设计引物,上游引物5`-ATCCTAGAAATTGTTGCATTTACTT-3`,下游引物5`-TTAGACGTAATCCCAGGTGATTGAA-3`。以合成的cDNA为模板进行PCR扩增。PCR反应条件为95℃预变性5min;94℃变性1min,55℃退火30s;72℃延伸1min,30循环;72℃延伸10min。PCR产物经琼脂糖凝胶电泳检测,结果如图1所示显示在600bp附近有特异性条带,切胶将该条带回收并连接pMD18-T载体,得到重组质粒,将重组质粒热激转化E.coli DH5α,经菌落PCR鉴定重组子后进行测序,将该重组质粒命名PMD-18T-EX22。
实施例2扩张蛋白基因序列分析
测序的结果采用GenBank数据库中的Basic Local Alignment Search Tool(BLAST)和Conserved Domain Database(CDD)分析其同源性(结果如图2所示)及结构域(结果如图3所示);获得的扩张蛋白基因(命名为EX22)编码区长582bp,其核苷酸序列如SEQ IDNO.1所示。EX22编码193个氨基酸及1个终止密码子,其氨基酸序列如SEQ ID NO.2所示,保守结构域分析EX22蛋白C端序列有1个特征性的DPBB结构域,DPBB结构域是由双Ψ-β桶状结构组成的,是扩张蛋白家族特有的结构域,表明EX22为扩张蛋白家族的一名新成员。
实施例3 EX22基因在毕式酵母菌中的重组表达
实施例1中得到的重组质粒PMD-18T-EX22为模板,利用设计的上游引物5`-CTAAAGAAGGGGTATCTCTCGAGAAAAGAATGTTACAAAAGCGGGG-3`,下游引物5`-GAGTTTTTGTTCTAGAAAGCTGGCGGCCGCCACGTAATCCCAAGTG-3`,按实施例1中的程序扩增编码EX22的基因序列;PCR扩增产物和表达载体pPICZαA(Invitrogen公司,Catalog No.V195-20)通过无限制克隆法(RF克隆)连接,将连接产物转化E.coli DH5α感受态细胞,进行菌落PCR验证,结果得到大小正确的扩增产物;将正确的单克隆接入含有0.5μg/ml的Zeocin抗生素的LB培养基中培养,提取质粒,接着将该重组质粒送去测序,通过序列对比表明,在pPICZαA的α信号肽和his标签直接插入SEQ ID NO.1所示的EX22基因,且插入方向正确,结果如图4所示。所以进一步证明构建的重组质粒正确,将该重组质粒命名pPICZαA-EX22。
将重组质粒pPICZαA-EX22进行SacI线性化,按照反应体系:重组质粒40μL,SacI10μL,10×buffer 20μL,ddH2O 130μL在37℃过夜酶切,琼脂糖电泳检测,切胶回收;制备毕赤酵母Pichia pastoris X-33感受态,将重组质粒pPICZαA-EX22电转入Pichia pastorisX-33,涂布在含有0.5μg/ml Zeocin的YPD平板上筛选,挑多个单菌落于含5μLZeocin的YPD液体培养基中30℃、200rpm培养24小时,用上下游引物进行菌落PCR验证,如图5所示,结果得到大小正确的扩增产物,由于毕赤酵母基因组中自带基因AOX-1,重组质粒中带有引物AOX-3ˊ和AOX-5ˊ。因此使用通用引物AOX-3ˊ和AOX-5ˊPCR会批出两个条带,一条为基因AOX-1为2200bp,另一条正确插入毕赤酵母基因组中的EX22片段。证明正确构建的重组表达菌,将该重组菌命名pPICZαA-EX22/X-33。
实施例4扩张蛋白重组表达
将实施例3中鉴定正确的重组菌pPICZαA-EX22/X-33划线在0.5μg/ml Zeocin的YPD的平板上;挑单菌落于5mL的0.5μg/ml Zeocin YPD液体,培养24h,作为种子液;按照1:50比例接入50mL的BMGY培养中,直至OD600=2;在无菌操作台中,将上述培养基转移至50mL的离心管中;1500g离心5min,弃上清,用BMMY培养基重悬,加入3%甲醇,每24小时补加一次,培养96小时。用聚丙烯酰胺凝胶电泳检测扩张蛋白的表达情况,结果如图6所示,扩张蛋白在甲醇的诱导下有明显的表达。
实施例5 EX22协同商品几丁质酶进行协同酶学性质的研究
对实施例3获得的EX22扩张蛋白以几丁质为底物,协同商品几丁质酶(sigma公司,Catalog No.C6137)进行协同酶学性质的测定,包括最适pH和最适温度对EX22扩张蛋白的影响。
(1)最适pH测定
使用Water No.1滤纸作为底物,EX22扩张蛋白和商品几丁质酶在不同的pH条件下(3.0、4.0、5.0、6.0、7.0)30℃、160rpm、反应48小时,用DNS法测还原糖的产生量;实验组为EX22扩张蛋白和商品纤维素酶,对照组是与EX22扩张蛋白相同浓度的BSA和商品纤维素酶;结果如图7所示,EX22的最适pH为6.0。
(2)最适温度测定
在上述最适pH的条件下,同样的体系,在不同温度下(30℃、40℃、50℃、60℃、70℃)反应,用DNS法测还原糖的产生量。结果如图8所示,EX22的最适温度为40℃。
实施例6扩张蛋白重组制备几丁质胶体
取20mg实施例3获得的EX22扩张蛋白用于几丁质,在40℃、pH 6.0、160rpm下反应24h,离心除去,吸取上清,在室温下干燥不溶底物;不溶底物呈现为胶状,通过扫描电镜观察其微观变化;如图9所示未经过任何处理的甲壳素结构表面是非常致密的,看不到清晰的纤维结构,但经过EX22扩张蛋白作用后,致密的结构被打破,松散度明显增大,并且出现了多个孔洞;这些孔洞的出现正为水分子的进入几丁质提供了可行性,使几丁质形成胶状。
实施例7几丁质酶降解几丁质胶体
取1g实施例6获得的几丁质胶体,5mg商品几丁质酶加入5mL pH 6.0磷酸盐缓冲液,40℃160rpm下反应2h用DNS法测还原糖的产生量,对照为1g未处理几丁质,在5mL pH6.0磷酸盐缓冲液中加入5mg商品几丁质酶40℃160rpm下反应2h用DNS法测还原糖的产生量。如图10所示,处理后的几丁质胶体其更易于水解,产生还原糖量高于对照组。
SEQUENCE LISTING
<110> 大连工业大学
<120> 一种扩张蛋白及其在几丁质胶体制备中的应用
<130> 2019
<160> 2
<170> PatentIn version 3.5
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cctgtggtcc ctacgccgac accagagcct gttgttgctg caaatccaac tggggttcct 180
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Val
Claims (5)
1.一种基因,其核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述基因编码的扩张蛋白,其氨基酸序列如SEQ ID NO.2所示。
3.一种含有权利要求2所述扩张蛋白的重组表达菌。
4.权利要求2所述的扩张蛋白在降解几丁质制备几丁质胶体中的应用,其特征在于降解几丁质时的pH和温度分别为pH6.0、40℃。
5.权利要求2所述扩张蛋白在由几丁质制备壳寡糖中的应用。
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