CN117701410A - 介导协氧蛋白和褐藻胶裂解酶的双转录单元重组菌和应用 - Google Patents
介导协氧蛋白和褐藻胶裂解酶的双转录单元重组菌和应用 Download PDFInfo
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Abstract
本发明涉及一种介导协氧蛋白和褐藻胶裂解酶的双转录单元重组菌和应用,属于基因工程技术领域,所述重组工程菌由两个转录单元组成,一个转录单位为GAP‑α‑Vgb‑AOX1TT,另一个转录单元为AOX‑α‑102C300C‑AOX1TT,两个转录单元在pPICZαA质粒中串联表达。本发明还提供所述重组菌的制备方法及应用。本发明重组菌能够提高产褐藻胶裂解酶分泌表达效率,特别是能在低氧环境中的分泌表达。
Description
技术领域
本发明属于基因工程技术领域,具体地涉及一种介导协氧蛋白和褐藻胶裂解酶的双转录单元重组菌和应用。
背景技术
毕赤酵母目前作为基础菌株已经成功表达了用于医药、农业等领域的重组蛋白400多种,是常用的蛋白质表达系统之一。相较于大肠杆菌和枯草芽孢表达系统,其可以实现有效的分泌表达,无需后期过多的分离和纯化目标蛋白,外源基因整合到基因组中可稳定表达且容易实现高密度发酵制备目标产物。
由于毕赤酵母为高好氧微生物,在当前高密度发酵的过程中,常常受限于设备供氧能力,细胞密度和氧化降解甲醇的速率无法进一步提高,从而影响目标蛋白的表达量。透明颤菌血红蛋白(Vitreoscilla hemoglobin,VHb)是一种类似人血红蛋白的胞内可溶氧物质,分子量约为15.47kDa,其转导酵母后可以提高VHb工程菌在高密度发酵的低氧环境中时,对氧气的利用能力,进而提高目标蛋白的表达量。
当前在毕赤酵母工程菌中表达VHb蛋白的主要模式为将目标蛋白和VHb蛋白的基因进行串联,利用载体上的醇氧化酶启动子(AOX),控制外源蛋白在宿主中的表达。一个转录单元表达双基因容易导致远离启动子的目的基因无法充分表达。
发明内容
本发明针对单启动子无法充分表达双基因的问题提供一种双转录单元表达协氧蛋白VHb和产褐藻胶裂解酶重组工程菌和应用,本发明通过构建双转录单元表达模式,将克隆得到的透明颤菌血红蛋白基因Vgb利用GAP启动子将其分泌到胞外,利用AOX启动子表达产褐藻胶裂解酶的基因,提高了菌体表达VHb的能力,最终提高了褐藻胶裂解酶的产量。
本发明的目的通过下述技术方案实现:
一种介导协氧蛋白和褐藻胶裂解酶的双转录单元重组菌,所述重组工程菌由两个转录单元组成,一个转录单位为GAP-α-Vgb-AOX1TT,另一个转录单元为AOX-α-102C300C-AOX1TT,两个转录单元在pPICZαA质粒中串联表达。
所述协氧蛋白Vgb基因的核苷酸序列为SEQ ID N0.1;所述转录单元GAP-α-AOX1TT的核苷酸序列如SEQ ID N0.2,褐藻胶裂解酶的核苷酸序列为SEQ ID N0.3。
优选的实施方式之一,所述重组菌的制备方法为将协氧蛋白的基因Vgb连接到pGAPZαA质粒上,构建pGAPZαA-Vgb重组质粒,从pGAPZαA-Vgb质粒基因组中PCR扩增GAP-α-Vgb-AOX1TT片段,再将GAP-α-Vgb-AOX1TT连接到pPICZαA-102C300C的BglII位点上,使其形成双转录单元,在真核表达系统中分泌表达。
优选所述真核表达系统为毕赤酵母。更进一步优选为毕赤酵母X33。
本发明还提所述重组菌在提高褐藻胶裂解酶分泌表达中的应用。
本发明相与现有技术相比的有益效果:
1、本发明通过GAP启动子表达核苷酸序列为SEQ ID N0.1,即协氧蛋白Vgb基因,褐藻胶裂解酶的核苷酸序列为SEQ ID N0.3,构建协氧蛋白基因Vgb与褐藻胶裂解酶编码基因共表达的重组载体,提高了产褐藻胶裂解酶工程菌在低氧环境中的分泌表达。
2、本发明提供了一种协氧蛋白质粒构建模式,能在低氧的环境中提高菌体对氧气的利用率,实现褐藻胶裂解酶的高表达,特别是为毕赤酵母菌表达褐藻胶裂解酶基因提供了有效的元件。
附图说明
图1是实施例1中pPICZαA-102C300C-GAP-α-Vgb重组质粒构建示意图;
图2是实施例1中扩增Vgb基因电泳图;其中泳道M:marker DNA;泳道1为Vgb基因片段;
图3是实施例1中扩增GAP-α-Vgb-AOX1TT片段的电泳图;其中泳道M:marker DNA;泳道1为GAP-α-Vgb-AOX1TT片段;
图4是实施例2中构建协氧蛋白基因Vgb工程菌与改造前的对照组相比,干重的比较;
图5是实施例2中构建协氧蛋白基因Vgb工程菌与改造前的对照组相比,褐藻胶裂解酶酶活的比较。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。
以下实施例中所采用的的分子生物学实验技术包括PCR扩增、质粒提取、DNA片段连接、凝胶电泳等具体参见《分子克隆实验指南》(第三版)(Sambrook J,Russell DW,Janssen K,Argentine J.黄培堂等译,2002,北京:科学出版社)。
实施例1协氧蛋白重组工程菌的构建和筛选
通过一步克隆的方式,将协氧蛋白基因Vgb构建于pGAPZαA质粒上,协氧蛋白Vgb基因的核苷酸序列如SEQ ID NO.1所示,形成pGAPZαA-Vgb质粒;而后PCR扩增GAP-α-Vgb-AOX1TT片段,连接至褐藻胶裂解酶基因的表达载体pPICZαA-102C300C质粒上,进而构建pPICZαA-102C300C-GAP-α-Vgb质粒,结合高通量的筛选方法,从克隆中获得褐藻胶裂解酶活力最高的阳性克隆子,具体包括以下步骤:
(1)Vgb基因的调取及纯化:提取pUC57-FHb菌株的质粒(Plasmid
DNA Mini Kit D6942试剂盒),以两段人工合成片段SEQ ID N0.4(5'-agagaggctgaagctgaattcatgttggaccagcagaccatca-3')、SEQ ID N0.5(5'-tgttctagaaagctggcggccgcttactcaacagcctgagcgtaca-3')从pUC57-Vgb质粒基因组中PCR扩增钓取Vgb基因;核酸电泳验证(见图2)并纯化(Gel Extraction Kit)回收目的片段待用。
(2)酶切线性化pGAPZαA质粒:用NotI(TaKaRa)和EcoRI(TaKaRa)双酶切pGAPZαA质粒,制得线性化载体片段。线性化的载体用Fast DigestDpn I进行消化处理,处理后的片段使用纯化试剂盒(Cycle Pure Kit D6492)纯化后回收备用。
(3)pGAPZαA-Vgb质粒的构建及验证:利用DNA无缝克隆(Clon Express IIOneStep Cloning Kit试剂盒),根据重组基因和线性化的pGAPZαA片段的DNA浓度,进行适当稀释后进行重组连接,将连接后的体系热激法转化E.coli DH5α,然后涂布LB(Zeocin)平板进行阳性重组子的筛选。以pGAPZαA载体通用引物对构建的载体进行单克隆测序验证。
(4)GAP-α-Vgb-AOX1TT片段的扩增及纯化:提取pGAPZαA-Vgb菌株的质粒(Plasmid DNAMini Kit D6942试剂盒),以两段人工合成片段SEQ ID N0.6(5'-tttggtcatgagatcagatcttttttgtagaaatgtcttggtgtcct-3')、SEQ ID N0.7(5'-tcgtctttggatgttagatctgcacaaacgaaggtctcacttaatc-3')从pGAPZαA-Vgb质粒基因组中PCR扩增GAP-α-Vgb-AOX1TT片段;核酸电泳验证(见图3)并纯化(/>Gel Extraction Kit)回收目的片段待用。
(5)酶切线性化pPICZαA-102C300C质粒:用BglII(TaKaRa)单酶切pPICZαA-102C300C质粒,制得线性化载体片段。线性化的载体用Fast Digest DpnI进行消化处理,处理后的片段使用纯化试剂盒(Cycle Pure Kit D6492)纯化后回收备用。
(6)pPICZαA-102C300C-GAP-α-Vgb质粒的构建及验证:利用DNA无缝克隆(ClonExpress IIOne Step Cloning Kit试剂盒),根据GAP-α-Vgb-AOX1TT片段和线性化的pPICZαA-102C300C片段的DNA浓度,进行适当稀释后进行重组连接,将连接后的体系热激法转化E.coli DH5α,然后涂布LB(Zeocin)平板进行阳性重组子的筛选。
(7)毕赤酵母菌株X33-pPICZαA-102C300C-GAP-α-Vgb的构建:将测序正确的转化子活化后提取质粒(Plasmid DNA Mini Kit D6942试剂盒),将克隆载体线性化后电转化到毕赤酵母X33菌株中,构建毕赤酵母X33-pPICZαA-102C300C-GAP-α-Vgb菌株。
(8)重组菌株的高通量筛选:将阳性转化子挑选至48孔深孔板中进行培养,每个孔添加1mL BMGY液体培养基。30℃,200rpm,每24h添加1%甲醇,连续诱导3天。4000rpm离心5min,取上清进行褐藻胶裂解酶酶活力检测,筛选出酶活力高的菌株。
协氧蛋白基因Vgb SEQ ID NO.1
atgttggaccagcagaccatcaacatcatcaaggctaccgttccagtcttgaaagaacacggtgttactatcaccaccaccttctacaagaacctgttcgctaagcacccagaggttagaccactgttcgatatgggtagacaagagtctttggagcagcctaaggctttggctatgactgttttggctgctgctcagaacatcgagaacttgccagctattttgccagccgttaagaagatcgccgttaagcactgtcaagctggtgttgctgctgcacattacccaatcgttggtcaagagttgctgggtgccatcaaagaagttttaggcgacgctgctactgacgacattttggatgcttggggtaaagcctacggtgttatcgctgacgttttcatccaagttgaggctgacttgtacgctcaggctgttgagtaa。
转录单元GAP-α-AOX1TT的核苷酸序(SEQ ID N0.2):
tttttgtagaaatgtcttggtgtcctcgtccaatcaggtagccatctctgaaatatctggctccgttgcaactccgaacgacctgctggcaacgtaaaattctccggggtaaaacttaaatgtggagtaatggaaccagaaacgtctcttcccttctctctccttccaccgcccgttaccgtccctaggaaattttactctgctggagagcttcttctacggcccccttgcagcaatgctcttcccagcattacgttgcgggtaaaacggaggtcgtgtacccgacctagcagcccagggatggaaaagtcccggccgtcgctggcaataatagcgggcggacgcatgtcatgagattattggaaaccaccagaatcgaatataaaaggcgaacacctttcccaattttggtttctcctgacccaaagactttaaatttaatttatttgtccctatttcaatcaattgaacaactatttcgaaacgatgagatttccttcaatttttactgctgttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagagaggctgaagctgaattcacgtggcccagccggccgtctcggatcggtacctcgagccgcggcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagttttagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgaga。
褐藻胶裂解酶的核苷酸序列(SEQ ID N0.3):
actgaatctggttctggttcttcttctggtggttcttcctccggatcttcttcttcctcatcctcttccggtggatcatcctctggtggatcaggtggtagtagttcaggtggatctttggacccaaacttgccaccatcttccaacttcgatttgtccgcttggtacttgtccgttccaactgataacaacggtgacggtaaggccgactccatcaaagaaaacgatttgaacgctggttacgccgacggtacttacttttacactgctgctgatggtggtatggtgttcagatgtccaatctgtggttacaagacttctaccaacacctcctacaccagaaccgagttgagagaaatgctgagaagaggtgacacctccattgctactcaaggtgtcaacggtaacaactgggttttcggttctgctccagcttccgctagagaagctgctggtggtgtagatggtgttttgagagctactttggccgttaaccacgttactactactggtgactctggtcaggttggtagagttatcgttggtcagatccacgctaacaacgacgaaccattgagactgtactacagaaagttgccaggtcactccaagggttccgtttacattgctcatgaacctaacggtggttccgactcttggtacgacatgattggttctagatcctcctctgcttctgacccatctgacggtattgctttggacgaagtttggtcctacgaggttaaggttgtcggtaacactttgaccgtgaccatcttcagagctggtaaggacgacgttgttcaggttgttgacatgggtaactccggttacgatgttgctgaccagtaccagtacttcaaggccggtgtttacaaccagaacaatactggtaactgttccgactacgttcaggttactttctacgctttggagcaatctcacgatcatcatcaccatcaccactaa。
实施例2含协氧蛋白菌株的生长和产酶水平评价
(1)将构建前的菌株X33-102C300C和构建后的菌株X33-102C300C-GAP-α-Vgb-AOX1TT分别接种20mL液体YPD培养基中,37℃、200r/min震荡培养24h,作为种子液。
(2)吸取1mL种子液至20mL BMGY培养基,每24h添加1%甲醇进行诱导产酶,共诱导5次,前四天30℃、200r/min振荡培养,后两天30℃,50r/min振荡培养。每24h取2mL菌液5000r/min离心5min,沉淀测定干重,上清测定酶活。本实施例在发酵过程中通过降低摇床的转速从而营造了低氧环境。
(3)取洁净玻璃制的扁形称量瓶,置于105℃干燥箱中,瓶盖斜支于瓶边,加热2h,取出盖好,置干燥器内冷却0.5h,称量,并重复干燥至前后两次质量差不超过2mg,即为恒重。
(4)褐藻胶裂解酶的酶活测定采用DNS法,酶活力单位定义:1个酶活力单位(U)定义为每1min产生1μmol还原糖所需的酶量。
(5)毕赤酵母菌体干重和褐藻胶裂解酶酶活的测定:与对照组(15.575g/L)相比,由双启动子表达系统介导协氧蛋白的产褐藻胶裂解酶毕赤酵母工程菌在发酵144h时干重为19.2g/L,随着96h转速的降低,改造菌株的菌体量较原始菌呈上升趋势(见图4)。协氧蛋白VHb可以使毕赤酵母在低氧环境中产褐藻胶裂解酶由12.3015U/mL增加至14.5736U/mL,增长了15.6%(见图5)。
Claims (6)
1.一种介导协氧蛋白和褐藻胶裂解酶的双转录单元重组菌,其特征在于,所述重组工程菌由两个转录单元组成,一个转录单位为GAP-α-Vgb-AOX1TT,另一个转录单元为AOX-α-102C300C-AOX1TT,两个转录单元在pPICZαA质粒中串联表达。
2.根据权利要求1所述的一种介导协氧蛋白和褐藻胶裂解酶的双转录单元重组菌,其特征在于,所述协氧蛋白Vgb基因的核苷酸序列为SEQ ID N0.1;所述转录单元GAP-α-AOX1TT的核苷酸序列如SEQ ID N0.2,褐藻胶裂解酶的核苷酸序列为SEQ ID N0.3。
3.权利要求1所述的一种介导协氧蛋白和褐藻胶裂解酶的双转录单元重组菌的制备方法,其特征在于,所述重组菌的制备方法为将协氧蛋白的基因Vgb连接到pGAPZαA质粒上,构建pGAPZαA-Vgb重组质粒,从pGAPZαA-Vgb质粒基因组中PCR扩增GAP-α-Vgb-AOX1TT片段,再将GAP-α-Vgb-AOX1TT连接到pPICZαA-102C300C的BglII位点上,使其形成双转录单元,在真核表达系统中分泌表达。
4.根据权利要求3所述的一种介导协氧蛋白和褐藻胶裂解酶的双转录单元重组菌的制备方法,其特征在于,所述真核表达系统为毕赤酵母。
5.根据权利要求3所述的一种介导协氧蛋白和褐藻胶裂解酶的双转录单元重组菌的制备方法,其特征在于,所述真核表达系统为毕赤酵母X33。
6.权利要求1所述的重组菌在提高褐藻胶裂解酶分泌表达中的应用。
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