CN111909879B - 一种高效表达外源蛋白的谷氨酸棒杆菌诱变菌株及应用 - Google Patents
一种高效表达外源蛋白的谷氨酸棒杆菌诱变菌株及应用 Download PDFInfo
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Abstract
本发明提供了一种高效表达外源蛋白的谷氨酸棒杆菌诱变菌株，其能解决现有谷氨酸棒杆菌作为外源蛋白表达宿主时，蛋白表达量低的技术问题。一种高效表达外源蛋白的谷氨酸棒杆菌诱变菌株，其特征在于：该谷氨酸棒杆菌诱变菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.20138。本发明的谷氨酸棒杆菌诱变菌株，经外源蛋白表达验证，与出发菌株进行对比，胞内蛋白的表达量、分泌蛋白的表达量均得到显著增强。
Description
技术领域
本发明涉及生物工程技术领域，具体涉及一种高效表达外源蛋白的谷氨酸棒杆菌诱变菌株及应用。
背景技术
谷氨酸棒杆菌为革兰氏阳性菌，属于放线菌目、棒状杆菌属。它是一种重要的蛋白表达宿主。包括许多优点，如无内毒素，可高密度生产，具有完善的蛋白分泌系统，目前已作为合成生物学宿主细胞来生产高附加值化合物以及重组药用蛋白，比如谷氨酸、赖氨酸等，还被应用于食品工业，动物饲料的生产。
谷氨酸棒杆菌作为外源蛋白表达宿主时，蛋白表达量低，因此，有必要对谷氨酸棒杆菌进行诱变筛选使其更有利于外源蛋白的表达。
发明内容
本发明提供了一种高效表达外源蛋白的谷氨酸棒杆菌诱变菌株，其能解决现有谷氨酸棒杆菌作为外源蛋白表达宿主时，蛋白表达量低的技术问题。
其技术方案是这样的，一种高效表达外源蛋白的谷氨酸棒杆菌诱变菌株，其特征在于：该谷氨酸棒杆菌诱变菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.20138。
进一步的，所述谷氨酸棒杆菌诱变菌株是以保藏号为CGMCC1.15647的谷氨酸棒杆菌为出发菌株经常压室温等离子体（ARTP）诱变、筛选获得。
本发明还提供了上述谷氨酸棒杆菌诱变菌株在外源蛋白表达中的应用。
进一步的，所述外源蛋白为单域重链抗体（VHH）或人I型骨胶原N端前肽（PNP）。
VHH（variable domain of heavy chin of heavy-chin antibody，VHH）是由一个重链可变区组成的单域抗体，重链可变区来源于骆驼血清中的天然缺失轻链的重链抗体。
本发明的谷氨酸棒杆菌诱变菌株，经外源蛋白表达验证，与出发菌株进行对比，胞内蛋白的表达量、分泌蛋白的表达量均得到显著增强。
生物保藏说明
拉丁学名：Corynebacterium glutamicum；
中文译名：谷氨酸棒杆菌
保藏单位全称：中国微生物菌种保藏管理委员会普通微生物中心；
保藏单位简称：CGMCC；
保藏单位地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所；
保藏日期：2020年6月24日
保藏编号：CGMCC NO.20138。
附图说明
图1为阳性突变率、致死率和诱变处理时间的关系图。
图2为流式分选诱变菌株的结果图，图2（a）为含pXMJ19-EGFP的出发菌株；图2（b）为含pXMJ19-EGFP的诱变菌株；图2（a）、2（b）中右上角矩形框选部分表示要收集的菌株。
图3为诱变菌株BZH-MLH-YB5验证的结果图，图中1为含pXMJ19-EGFP的出发菌株，2为含pXMJ19-EGFP的诱变菌株BZH-MLH-YB5，3为BZH-MLH-YB5传代4次后重新导入pXMJ19-EGFP的诱变重组菌株。
图4为重组菌株表达蛋白的WB电泳图；图4（a）中，泳道1：C. g出发菌株破碎上清，泳道2：含pXMJ19-VHH的出发菌株破碎上清，泳道3：含pXMJ19-VHH的诱变菌株BZH-MLH-YB5破碎上清；图4b中，泳道1：C. g出发菌株分泌上清；泳道2：含pXMJ19-PNP出发菌株分泌上清；泳道3：含pGX19-PINP诱变菌株BZH-MLH-YB5分泌上清。
具体实施方式
下述实施例或应用例中所用大肠杆菌DH5α购买自TAKARA。
下述实施例或应用例中所用谷氨酸棒杆菌 CGMCC1.15647是于2016年公开寄存，本领域技术人员能够在申请日前获取的现有菌种。
下述实施例或应用例中所用载体骨架pXMJ19购自Biovector，货号BiovectorpXMJ19。
下述实施例或应用例中所用限制性内切酶EcoRⅤ、XhoI、HindⅢ和BamHI购自TaKaRa公司，货号分别为1612、1635、1615、1605。
下述实施例或应用例中所用质粒提取试剂盒、胶回收和柱回收试剂盒购自Axygen公司，货号分别为AP-MD-P-10、AP-GX-250、AP-PCR-250
下述实施例或应用例中所用连接液solutionI购买自TaKaRa公司，货号 D6020A。
下述实施例或应用例中大肠杆菌的培养使用LB培养基，培养基配方为：胰蛋白胨10g、酵母提取物 5g、NaCl 10g，去离子水 1L。
下述实施例或应用例中谷氨酸棒杆菌的培养使用LBB培养基，培养基配方为：胰蛋白胨 10g、酵母提取物 5g、NaCl 10g，脑心浸液 10g、去离子水1L。
下述实施例或应用例中谷氨酸棒杆菌的转化子培养使用LBHIS培养基，培养基配方为：胰蛋白胨 5g、酵母提取物 2.5g、NaCl 5g、脑心浸液 18.5g、山梨醇 91g、去离子水1L。
1、诱变菌株基本信息
一种高效表达外源蛋白的谷氨酸棒杆菌诱变菌株，命名为BZH-MLH-YB5，该谷氨酸棒杆菌诱变菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCCNo.20138。
2、诱变菌株的制备
以谷氨酸棒杆菌
C. glutamicum CGMCC1.15647为出发菌株，导入pXMJ19-EGFP质粒，复苏后涂板于含有15μg/mL氯霉素的LBB固体平板上，30℃培养18小时，挑选单菌落于30mL的LBB液体培养基中，30℃培养10h，转接至10mL的LBB液体培养基中，使初始OD600达0.2，培养大约2h，使OD600达0.6，吸取10uL菌液于载片上，在功率为100W，气流量为10SLM，时间为120s的条件下进行ARTP诱变。诱变完成后，将其置于装有1 mL PBS的1.5mL的离心管中，振荡洗脱。利用BD FACS Aria Ⅲ流式细胞分选仪对菌液进行流式分选，以荧光强度为分选信号，筛选荧光强度较高的约102~101株菌，将得到的菌液涂布于含有15μg/mL氯霉素的LBB固体平板，30℃培养12h，挑选单菌落于48孔板中，再次筛选出50株菌，将菌株转接于含有10mL的LBB液体的100mL摇瓶中复筛，筛选出荧光强度最高的一株菌，将得到的菌株作为下一次诱变的出发菌株，重复诱变、分选，获得相较于初始菌株荧光强度提高幅度最大的诱变菌株，并进行相应保藏。
2.1 pXMJ19-EGFP质粒的制备
以pXMJ19质粒为骨架，用EcoRⅤ及XhoI酶切质粒，去掉lacI部分，胶回收骨架部分，并用上下游引物F和R进行PCR获得tac启动子片段，柱回收启动子片段，用同样的酶进行消化后将两个片段进行连接，获得组成型pXMJ19质粒。将组成型pXMJ19质粒用HindⅢ和BamHI酶切，胶回收获得酶切质粒片段，用引物EGFP-F和EGFP-R进行PCR获得绿色荧光蛋白片段，EGFP的基因序列如SEQ ID NO：1，用同样的酶进行消化，并用连接酶连接，获得组成型pXMJ19-EGFP质粒。
F: GATATCAACGTAAATGCCGCTTCGCC；
R: CTCGAGAATTAATTCTGTTTCCTGTGT；
EGFP-F: AAGCTTATGGTGAGCAAGGGC；
EGFP-R: GGATCCTTACTTGTACAGCTCGT。
2.2 确定最佳诱变时间
选择不同的时间t=0、15、30、45、60、75、90、120、150s、180s，测定阳性突变率及致死率，结果参见图1，可知诱变时间为120s时，阳性突变率达到最高约42%，此时致死率约95%，因此，选择120s作为最佳诱变时间。
2.3流式分选诱变菌株
由图2可知，经诱变后的菌株相较于出发菌株，其荧光强度明显向右偏移，所收集的高荧光菌株的占比明显增大。
2.4 筛选最佳诱变菌株
经过三轮诱变和分选后，筛选出比初始菌株荧光强度提高近1倍的诱变菌株BZH-MLH-YB5，而经过四轮、五轮或更多轮数诱变和分选所获得的诱变菌株，其荧光强度相较于诱变菌株BZH-MLH-YB5反而降低，故确定诱变菌株BZH-MLH-YB5为最佳诱变菌株。
2.5诱变菌株稳定性实验
a、将诱变菌株BZH-MLH-YB5进行稳定性验证，传代10次，测定每一代的荧光值，每一代荧光值波动范围在10%以内，基本稳定；
b、将诱变菌株BZH-MLH-YB5进行传代，在无抗性的培养基中转接4次，测得质粒丢失，重新导入pXMJ19-EGFP后培养、检测荧光强度，结果如图3所示，发现荧光值仍然提高近1倍，与诱变菌株BZH-MLH-YB5的荧光强度基本保持一致，因此，导致蛋白表达量显著提高的是诱变菌株BZH-MLH-YB5本身，而非pXMJ19-EGFP变化导致。
2.6诱变菌株外源蛋白表达实验
2.6.1胞内蛋白VHH表达实验
组成型pXMJ19-VHH质粒的制备：以组成型质粒pXMJ19为骨架，用HindⅢ和EcoRI酶切，胶回收获得酶切质粒片段，用VHH-F和VHH-R进行PCR获得VHH片段，VHH的基因序列如SEQID NO：2所示，用同样的酶进行消化，并用连接酶连接，获得组成型pXMJ19-VHH质粒。
VHH-F: AAGCTTATGCAGGTCCAACTGCAAGAAAG
VHH-R: GAATTCTCAGTGGTGGTGGTGGTGGTGTGAAGAGACGGTCACC
将pXMJ19-VHH质粒转入诱变菌株BZH-MLH-YB5和出发菌株CGMCC1.15647，将获得的转化子分别培养至48小时，超声破碎细胞，离心取上清，western blot分析，获得的条带图如图4a所示，并采用image J对条带图进行灰度分析，分析结果如表1a所示。
表1a
由图4a可知，诱变菌株与出发菌株进行对比，胞内蛋白表达量明显增强；由表1a可知，诱变菌株与出发菌株进行对比，VHH蛋白表达量增加了3.7倍。
2.6.2分泌蛋白PNP表达实验
诱导型质粒pXMJ19-PINP以原始质粒pXMJ19为骨架，用HindⅢ和EcoRI酶切，胶回收获得酶切质粒片段，用PINP-F和PINP-R进行PCR获得PINP片段，PINP的基因序列如SEQ IDNO：3所示，用同样的酶进行消化，并用连接酶连接，获得诱导型pXMJ19-PINP质粒。
PINP-F: AAGCTTATGCAAGAAGAAGGCCAAGTGGA
PINP-R: GAATTCTTACTGGCCGCCGTGGTGATGGTG
将pXMJ19-PINP质粒转入诱变菌株BZH-MLH-YB5和出发菌株CGMCC1.15647，将获得的转化子分别培养至48小时，离心取上清，western blot分析，获得的条带图如图4b所示，并采用image J对条带图进行灰度分析，分析结果如表1b所示。
表1b
由图4b可知，诱变菌株与出发菌株进行对比，分泌蛋白表达量明显增强；由表1b可知，诱变菌株与出发菌株进行对比，PINP蛋白表达量增加了3.5倍。
序列表
<110> 江南大学
<120> 一种高效表达外源蛋白的谷氨酸棒杆菌诱变菌株及应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 720
<212> DNA
<213> 人工序列(Artificial Sequence)
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atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 2
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<212> DNA
<213> 人工序列(Artificial Sequence)
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caggtccaac tgcaagaaag cggtggtggt tccgtccaag caggcggctc cctgcgtctg 60
tcctgcaccg catccggctt caccttcgac gattccgaca tgggctggta ccaccaggct 120
ccaggcaacg agtgcgagct ggtctccacc atcggcaacg acggctccac ctactacgca 180
gactccgtga agggccgctt caccatctcc cgcgacaacg caaagaacac cgtgtacctc 240
cagatgaaca acctgaagcc agaggacacc gcaatgtact actgcgcagc agatctgcac 300
ccaaccttcc gcaagtggga ttcccgcacc tccgactgct actccggccc actggagtac 360
ggctacaact actggggcca gggtacccag gtgaccgtct cttcacacca ccaccaccac 420
cactga 426
<210> 3
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caagaagaag gccaagtgga aggtcaagat gaggacatcc caccaatcac ttgcgtgcag 60
aacggtctgc gctaccacga ccgtgatgtc tggaaaccag aaccttgccg catctgcgtg 120
tgcgacaacg gcaaggtgct gtgcgacgac gtgatctgcg atgaaaccaa gaactgcccg 180
ggcgcagaag tgccagaagg cgaatgctgc ccagtgtgcc cagatggctc cgagtcccca 240
accgatcaag aaaccaccgg cgtcgagggc cctaagggcg atactggtcc tcgtggtcca 300
cgcggtccag ccggccctcc gggccgtgac ggcatcccgg gccagccggg cctcccgggc 360
ccaccgggcc caccgggccc accgggccca ccgggtctgg gcggtaactt cgcaccagat 420
tacaaggacg acgatgataa gggccaccat catcaccacc atcaccacgg cggccagtaa 480
Claims (3)
1.一种高效表达外源蛋白的谷氨酸棒杆菌诱变菌株，其特征在于：该谷氨酸棒杆菌诱变菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCCNo.20138。
2.如权利要求1所述的谷氨酸棒杆菌诱变菌株在外源蛋白表达中的应用。
3.根据权利要求2所述的应用，其特征在于：所述外源蛋白为单域重链抗体或人I型骨胶原N端前肽。
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