CN108410786B - 高效表达纤溶酶的枯草芽孢杆菌工程菌以及制备方法 - Google Patents
高效表达纤溶酶的枯草芽孢杆菌工程菌以及制备方法 Download PDFInfo
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Abstract
本发明公开了一种高效表达纤溶酶的枯草芽孢杆菌工程菌以及制备方法。本发明通过基因敲除技术，改造现有的枯草芽孢杆菌菌种，得到了一种高效表达纤溶酶的枯草芽孢杆菌工程菌。该方法路线清晰，简单有效，可以运用于多种枯草芽孢杆菌，具有较好的实用性。
Description
技术领域
本发明涉及生物技术领域，具体是指一种高效表达纤溶酶的枯草芽孢杆菌工程菌以及制备方法。
背景技术
纤溶酶是能专一降解纤维蛋白凝胶的蛋白水解酶，具有溶解血栓，降低血粘度，改善血液循环，软化和增加血管弹性等作用。近年来，纳豆激酶(nattokinase，简称NK)作为一种高效安全的新型纤溶酶，正在成为当今溶栓药研究的焦点。
该酶是在纳豆发酵过程中由纳豆枯草杆菌产生的一种丝氨酸蛋白酶，自1995年我国第一篇有关纳豆和纤溶酶的报道以来，至今已有多篇相关文章发表，目前纤溶酶的溶栓机制已阐明，包括①直接溶栓作用，激活交联纤维蛋白，降解为纤维蛋白降解物；②刺激血管内皮细胞产生组织型纤溶酶原激活剂(t-PA)，t-PA将纤溶酶原激活为纤溶酶，溶解纤维蛋白；③将人体内的尿激酶原激活为尿激酶，尿激酶与t-PA共同激活纤溶酶原，产生纤溶酶溶解血栓；④通过降解和失活纤溶酶原激活剂的抑制剂(PAI-1)调控纤溶作用。此外，纤溶酶在体内外溶栓的动物试验、急性毒性试验及治疗脑梗塞的初步临床试验等方面的大量研究工作已部分完成或正在进行中。
纤溶酶的主要生产菌株——纳豆芽孢杆菌，是枯草芽孢杆菌的一种，是一类好氧型、内生抗逆孢子的革兰氏阳性细菌，能分泌大量的蛋白到胞外，其研究历史已经有40多年，遗传背景清楚，其表达系统已成为继大肠杆菌表达系统之后最重要的原核表达系统，优势在于：①没有毒性；②蛋白分泌表达量高；③密码子使用偏好性不大；④基础研究、基因操作技术和发酵技术成熟。
纤溶酶传统发酵方式是固体发酵，但该方式提纯困难，回收率低，难以满足工业化放大生产的要求，因此，近年来关于纤溶酶产量的研究更倾向于液体发酵方式。目前，纤溶酶生产菌株主要以筛选方式获得，发明专利申请CN105238720A和CN101979531A等多篇专利申请和发表文章均通过筛选获得高产纤溶酶菌株，发明专利申请CN105368762A通过菌株改造得到高效分泌纤溶酶工程菌株，但是，前者只通过筛选而未进行改造，后者只经过菌株改造而所用菌株并非纤溶酶高产菌，所以在纤溶酶产量及酶活方面，两者均没有明显优势。
发明内容
本发明的目的是为了克服现有高产纤溶酶菌株在纤溶酶产量及酶活方面的缺陷，提供一种高效表达纤溶酶的枯草芽孢杆菌工程菌及其制备方法。
为实现上述目的，首先，本发明提供一种高效表达纤溶酶的枯草芽孢杆菌工程菌，其特征在于：它是通过将枯草芽孢杆菌进行基因敲除后获得；所敲除的基因包括芽孢形成编码基因spo0A，淀粉酶编码基因amyE，部分蛋白酶编码基因bpr、mpr、epr、vpr、wprA、aprX，聚谷氨酸合酶编码基因pgsB。
上述方案中，采用的枯草芽孢杆菌为枯草芽孢杆菌枯草亚种，其保藏编号为CICC20637。
本发明还提供了上述高效表达纤溶酶的枯草芽孢杆菌工程菌的制备方法，包括如下步骤：
1)根据目标基因上下游同源臂序列设计敲除引物，引物末端和载体末端具有15～20个同源碱基；
2)PCR扩增：提取枯草芽孢杆菌总DNA，以其作为模板，用各基因对应的上、下游同源臂引物进行PCR扩增，得到目的同源臂DNA片段；
3)通过SOE-PCR分别将各基因的上下游同源臂连接起来，得到9个基因敲除片段；
4)通过无缝克隆将基因敲除片段连接到敲除载体上，得到对应的9个目标基因敲除载体；
5)枯草芽孢杆菌的遗传转化，对枯草芽孢杆菌感受态的制备及利用1个目标基因敲除载体进行电转化；
6)通过抗性筛选和PCR扩增对转化子进行验证；
7)挑选阳性转化子进行传代，经过高温传代，质粒消除，点种和PCR验证后，筛选出目标基因敲除菌株；
8)在得到目标基因敲除菌株后，以其为出发菌株，重复步骤5)～7)用同样的方法对下一个目标基因进行敲除，直至得到敲除9个基因的目标菌种。
步骤4)中所述敲除载体是参考文献(张周刚，宋亚囝，姜凯等.嗜碱芽孢杆菌N16-5木寡糖结合蛋白XynE的功能表征[J].微生物学报，2015，55(1)：40-49)对质粒pE194和pHY300PLK进行改造组合后得到的枯草芽孢杆菌温敏型敲除载体pHY194。
步骤5)中所述的遗传转化方法是按照参考文献(Xue GP,Johnson JS,
Dalrymple BP.High osmolarity improves the electro-transformationefficiency of thegram-positive bacteria Bacillus subtilis and Bacilluslicheniformis.J Microbiol Method,1999,34:183-191)中描述的方法。
步骤7)中所述的质粒消除是指高温培养菌株进行传代，由于敲除载体在枯草芽孢杆菌中的复制子是温敏型复制子，所以在高温条件下质粒会丢失，再通过PCR进行验证。
步骤8)中所述为基因叠加敲除，使目标基因敲除后的效果叠加。
本发明原理：
基于纤溶酶良好的应用前景和枯草芽孢杆菌成熟的表达系统，本发明对高产纤溶酶的枯草芽孢杆菌进行了以下改造：
①除了芽孢形成编码基因spo0A，使菌体生长至稳定前期时不形成芽孢，延长有效发酵时间，提高目标蛋白的表达量；
②敲除了淀粉酶编码基因amyE，发酵条件下，枯草芽孢杆菌淀粉酶表达量较高，减低了原料的转化率，当不以淀粉作为碳源时，将其编码基因敲除，可提高原料的转化率；
③敲除了蛋白酶编码基因(bpr,mpr,epr,vpr,wprA和aprX)，纤溶酶属于丝氨酸蛋白酶，由aprN基因编码，减少菌株其他分泌蛋白酶的表达，可提高原料的利用率；
④敲除了聚谷氨酸合酶编码基因pgsB，聚谷氨酸γ-PGA是一种粘性物质，是纳豆发酵过程中的副产物之一，对其进行敲除，可以提高原料利用率，此外，由于其粘度大，液体发酵时会造成发酵液溶氧浓度不足，影响菌株生长及目标蛋白的积累，对基因pgsB进行敲除，能够提高发酵效率，整体上能够提高胞外蛋白酶的总表达量。
本发明的有益效果：通过基因敲除技术，改造现有的枯草芽孢杆菌菌种，得到了一种高效表达纤溶酶的枯草芽孢杆菌工程菌。该方法路线清晰，简单有效，可以运用于多种枯草芽孢杆菌，具有较好的实用性。
附图说明
图1为基因敲除载体的构建示意图。
图2为枯草芽孢杆菌的基因敲除流程示意图。
具体实施方式
以下结合附图和具体实施例对本发明作进一步详细描述，实施例仅为举例，不用于限制本发明。
下述实施例中所使用的培养基的配方如下：
LB培养基：蛋白胨10g/L，酵母抽提物5g/L，氯化钠10g/L，pH 7.2-7.4；固体培养基加琼脂粉15g/L。
芽孢杆菌电转感受态制备培养基：生长培养基：含0.5mol/L山梨醇的LB培养基；洗涤培养基：10％甘油，0.5mol/L山梨醇，0.5mol/L甘露醇；恢复培养基：含0.5mol/L山梨醇，0.38mol/L甘露醇的LB培养基。
液体种子培养基：大豆蛋白胨1％，牛肉膏0.5％，葡萄糖1％，NaCl 0.75％，pH7.2。
液体发酵培养基：蛋白胨2％，葡萄糖2％，CaCl2 0.02％、MgSO4 0.02％、K2HPO40.1％、KH2PO4 0.1％、KH2PO4 0.1％，pH7.2。
下述实施例中的实验材料，如无特殊说明，均可以通过常规的商业途径购得。下述实施例中所采用的方法，如无特殊说明，均为常规方法。
实施例1
9种敲除载体的构建
枯草芽孢杆菌菌种(B.subtilis)来源于中国工业微生物菌种保藏管理中心(CICC)，保藏编号No.20637。
(1)枯草芽孢杆菌基因组DNA小提：挑取B.subtilis单菌落于5ml LB液体培养基中，37℃振荡培养过夜，12000r/min离心5min收集菌体；STE洗涤菌体1次；加入200μlSolution I溶液和20μl 100mg/ml溶菌酶，重悬菌体，置于37℃下作用2-3h或置于4℃冰箱过夜；加入200μl 10％SDS，50-60℃水浴30min至澄清；加入100μl或200μl 5mol/L NaCl，混匀后12000r/min离心5min，取上清至干净离心管；等体积苯酚/氯仿抽提2次，取上清至干净离心管中；加入2倍体积的无水乙醇置于-20℃沉淀2h，12000r/min离心5min，弃上清；70％乙醇洗涤1次，乙醇晾干后溶于30-60μl的RNase(20μg/ml)，37℃作用30min后置于-20℃保存。
(2)敲除载体上下游同源臂的PCR扩增：普通PCR反应：反应条件：95℃5min；95℃30s，55℃30s，72℃2min，30个循环；72℃延伸10min；4℃保温(其中退火温度根据引物的Tm值进行调整，延伸时间根据扩增目的片段的大小进行调整)。
按下列反应体系依次加入到PCR管中，混匀并离心后置于PCR仪上进行PCR反应。
SOE-PCR反应条件：95℃5min；95℃30s，55℃30s，72℃2min，7个循环；72℃延伸10min(不加引物)；在反应体系中加入引物进行以下反应程序：95℃30s，58℃30s，72℃2min，30个循环；72℃延伸10min；4℃保温(其中退火温度根据引物的Tm值进行调整，延伸时间根据扩增目的片段的大小进行调整)。
按下列反应体系依次加入(引物暂不加入)到PCR管中，混匀并离心后置于PCR仪上进行PCR反应。
DNA体外重组方法：DNA的回收纯化、无缝克隆等操作参照相应产品供应商提供的方法或操作说明。
(3)敲除载体构建
大肠杆菌感受态细胞的制备：挑取大肠杆菌DH5α单菌落于37℃，180r/min振荡培养过夜；1％接种量接种于50ml LB，37℃，180r/min振荡培养2-4h，待细菌长至对数生长期(OD600为0.5-0.6)；将菌液在冰上预冷10min后，倒入50ml预冷的离心管中，4℃5000r/min离心5min，收集菌体；用10ml预冷的CaCl2(0.1mol/L)重悬菌体，冰上放置30min后，4℃5000r/min离心5min，用1ml预冷的CaCl2溶液重悬菌体，并于4℃保存于12-24h内可用于转化；感受态细胞也可由170μl菌悬液与30μl无菌甘油混匀，分装于无菌1.5ml离心管中，置于-70℃保存备用。
大肠杆菌感受态细胞的钙转：取100-200μl感受态细胞与一定量的无缝克隆产物混合，冰上静置15-30min；随后置于42℃水浴锅中热击90s；快速置于冰中，使细胞冷却2-5min；每管加入800μl LB液体培养基，37℃，120r/min恢复培养45min；取200μl菌液涂布于含相应抗生素的LB固体培养基上；固体培养基置于37℃培养箱，静置培养12-16h后，可长出单菌落。
转化子验证：用灭菌的牙签挑少量的转化子菌落于装有预混好的eazyTaq PCR体系中，搅拌使菌体充分混匀，进行PCR扩增反应(PCR预变性时间延长为10min，以裂解大肠杆菌细胞)。取PCR产物进行琼脂糖凝胶电泳检测。经过菌落PCR初步确定为阳性转化子后，提取转化子质粒，提取的质粒用于PCR及酶切验证。
将PCR鉴定结果为阳性的转化子进行测序分析，经过测序鉴定，得到的敲除载体序列正确，将敲除载体分别命名为pHY194Δspo0A，pHY194ΔamyE，pHY194Δbpr，pHY194Δmpr，pHY194Δepr，pHY194Δvpr，pHY194ΔwprA，pHY194ΔaprX，pHY194ΔpgsB。
实施例2
枯草芽孢杆菌的基因敲除
(1)枯草芽孢杆菌感受态的制备：挑取B.subtilis单菌落于5ml LB培养基，37℃振荡培养过夜；种子液以5％的接种量转接至50ml B.subtilis电转生长培养基中，37℃，250r/min振荡培养2-3h至OD600为0.85-0.95，将菌液冰浴10min；5000r/min离心5min收集菌体，用B.subtilis电转洗涤培养基洗涤菌体4次，最后用1ml洗涤培养基重悬细胞，分装至1.5ml离心管中，每管100μl于-80℃保存，即感受态细胞，以上步骤均在冰上操作。
(2)电转化：取一管感受态细胞加入5-10μl质粒DNA(单个敲除载体)，轻轻混匀后转至预冷的电转杯(0.2cm)中，冰浴1-2min，用电脉冲转化仪2.5kV电击1次，电击完后迅速加0.9ml B.subtilis电转恢复培养基，37℃，100r/min恢复培养3h，涂布含有相应抗生素的LB固体培养基上，37℃静置培养过夜，筛选转化子。
(3)转化子验证：经过菌落PCR初步确定为阳性转化子后，提取转化子质粒，提取的质粒用于PCR及酶切验证。
(4)单交换菌株的获得：挑取含有温敏型敲除载体的B.subtilis阳性转化子菌落在LB固体培养基(20μg/ml Tet)上划线，45℃培养12h；挑单菌落于5ml液体LB培养基(20μg/ml Tet)，45℃，培养12h；菌液在LB固体培养基(20μg/ml Tet)上划线，45℃培养12h，长出的单菌落进行菌落PCR验证，验证正确的即为单交换菌株。
(5)基因敲除菌株的获得和鉴定：单交换菌株在5ml无抗性液体LB培养基中传代培养6代，37℃，每代12h；每次接种前一代培养物10μl于5ml无抗性液体LB培养基。最后一代培养物稀释(10-6-10-7)，涂布LB固体培养基，37℃培养至长出单菌落，挑单菌落点种于含Tet的LB固体培养基和不含Tet的LB固体培养基上。对在不含Tet的LB固体培养基上生长而在含Tet的LB固体培养基上不长的单菌落进行菌落PCR鉴定，初步确定为双交换菌株后，提取基因组DNA，PCR验证，并将PCR产物进行测序鉴定。
(6)测序正确的菌株即为基因敲除菌株，在得到目标基因敲除菌株后，以其为出发菌株，用同样的方法对下一个目标基因进行敲除，最终得到敲除菌株B.subtilis 20637Δspo0AΔamyEΔbprΔmprΔeprΔvprΔwprAΔaprXΔpgsB(简称B.subtilis 20637Δ9)。
实施例3
工程菌株发酵及纤溶酶活性检测
(1)菌种活化：挑取甘油管保藏的B.subtilis 20637和B.subtilis 20637Δ9菌株于LB平板上划线，37℃培养12h，挑取单菌落再在LB平板上划线，37℃培养12h；
(2)种子培养：从LB固体培养基上挑取单菌落接种至50ml液体种子培养基，转速为220r/min，37℃振荡培养12h，作为发酵种子液；
(3)发酵培养：采用3L发酵罐，装液体积为1.5L，接种量为2％，发酵温度设定为37℃。搅拌转速800r/min，通气量2L/min，pH控制在6.8-7.2，发酵48h。
(4)纤溶酶酶活检测：参考专利CN103937830B，具体操作如下：①吸取发酵液1ml于离心管中，10000r/min离心10分钟，吸取上清液于试管中备用；②在另一支试管中加入50mmol/L硼砂缓冲液1.4ml及纤维蛋白原溶液0.4ml，在37℃的恒温水浴锅中加热5分钟后，加入凝血酶0.1ml，混匀。③37℃水浴中保温10分钟，再加入0.1ml样品溶液，混匀5秒钟，保温60分钟，在保温第20分钟及第40分钟时，分别取出振荡。④保温结束后，往试管中加入200mmol/L三氯乙酸2ml，再放入水浴锅保温20分钟，15000r/min离心5分钟。⑤取上清液在275nm处测定吸光值。加入相同量的灭活的酶液作空白对照，空白对照的处理方法与样品的处理方法一样。⑥每组均设3个平行样品，最终测定结果为3个测定值的平均值。
(5)检测结果如表1
表1工程菌株的纤溶酶酶活
序列表：基因敲除引物
序列表
<110> 武汉瑞法医疗器械有限公司
<120> 高效表达纤溶酶的枯草芽孢杆菌工程菌
<130> 2018
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<213> spo0A-BF
<400> 5
gtttgtgttg ctgatgataa tcggcggata agctgaggtt agagc 45
<210> 6
<211> 43
<212> DNA
<213> spo0A-BR
<400> 6
gtaaaggata aaacagcacg gtgccttata aagattaatg tgc 43
<210> 7
<211> 23
<212> DNA
<213> spo0A-yanF
<400> 7
ttctaccgaa gtcaaaaaag ggc 23
<210> 8
<211> 22
<212> DNA
<213> spo0A-yanR
<400> 8
tctagttgac cctaactgga cg 22
<210> 9
<211> 38
<212> DNA
<213> amyE-AF
<400> 9
caacgcacct ttcagcagct catgccgaga atagacac 38
<210> 10
<211> 43
<212> DNA
<213> amyE-AR
<400> 10
gccctcaatg gggaagagaa cgcagtaaag aggttttgaa tcg 43
<210> 11
<211> 43
<212> DNA
<213> amyE-BF
<400> 11
cgattcaaaa cctctttact gcgttctctt ccccattgag ggc 43
<210> 12
<211> 41
<212> DNA
<213> amyE-BR
<400> 12
gtaaaggata aaacagcacg ctcgccatga cttcactaac g 41
<210> 13
<211> 20
<212> DNA
<213> amyE-yanF
<400> 13
ttgcgctgct tgatgtgatc 20
<210> 14
<211> 23
<212> DNA
<213> amyE-yanR
<400> 14
catgttgcgt aagtcaggat atc 23
<210> 15
<211> 39
<212> DNA
<213> pgsB-AF
<400> 15
caacgcacct ttcagcattc ggactcgtat gtttagcgt 39
<210> 16
<211> 45
<212> DNA
<213> pgsB-AR
<400> 16
ctgctttacc ttgtattcgt gggatgacca gtatgacagc acagg 45
<210> 17
<211> 45
<212> DNA
<213> pgsB-BF
<400> 17
cctgtgctgt catactggtc atcccacgaa tacaaggtaa agcag 45
<210> 18
<211> 41
<212> DNA
<213> pgsB-BR
<400> 18
gtaaaggata aaacagcact tcatcgtttg acacacctta c 41
<210> 19
<211> 23
<212> DNA
<213> pgsB-yanF
<400> 19
tgacaaagtc ttcacaaata tgg 23
<210> 20
<211> 21
<212> DNA
<213> pgsB-yanR
<400> 20
gcgaacataa ggacaaaaac g 21
<210> 21
<211> 40
<212> DNA
<213> bpr-AF
<400> 21
caacgcacct ttcagcagca agcagtaaag tcacctcacc 40
<210> 22
<211> 44
<212> DNA
<213> bpr-AR
<400> 22
ggattgtcca gtgttaattc aggcattcca caccgtcagt tgct 44
<210> 23
<211> 44
<212> DNA
<213> bpr-BF
<400> 23
agcaactgac ggtgtggaat gcctgaatta acactggaca atcc 44
<210> 24
<211> 42
<212> DNA
<213> bpr-BR
<400> 24
gtaaaggata aaacagcact catctcggac gatcacttct ac 42
<210> 25
<211> 23
<212> DNA
<213> bpr-yanF
<400> 25
ggaaaaaaac gaaaaacaga ctc 23
<210> 26
<211> 23
<212> DNA
<213> bpr-yanR
<400> 26
catattaagt tttccattcg cag 23
<210> 27
<211> 40
<212> DNA
<213> mpr-AF
<400> 27
caacgcacct ttcagcagct ggtgacgcaa aatttgtcag 40
<210> 28
<211> 47
<212> DNA
<213> mpr-AR
<400> 28
ccaagttata tgacgatcct ccgccaaaca caaaaccgtt aagtaag 47
<210> 29
<211> 47
<212> DNA
<213> mpr-BF
<400> 29
cttacttaac ggttttgtgt ttggcggagg atcgtcatat aacttgg 47
<210> 30
<211> 43
<212> DNA
<213> mpr-BR
<400> 30
gtaaaggata aaacagcacc cagtatatag tttggtttcc tgc 43
<210> 31
<211> 24
<212> DNA
<213> mpr-yanF
<400> 31
tgacagagca gcaaatagaa gaag 24
<210> 32
<211> 24
<212> DNA
<213> mpr-yanR
<400> 32
gtatcatctt cttctttttc ctcc 24
<210> 33
<211> 37
<212> DNA
<213> epr-AF
<400> 33
caacgcacct ttcagcacga taggcgatcc caaaagc 37
<210> 34
<211> 48
<212> DNA
<213> epr-AR
<400> 34
ggttactttt tcggtttgac ggcagagtga ctgatacaac aagtttgc 48
<210> 35
<211> 48
<212> DNA
<213> epr-BF
<400> 35
gcaaacttgt tgtatcagtc actctgccgt caaaccgaaa aagtaacc 48
<210> 36
<211> 42
<212> DNA
<213> epr-BR
<400> 36
gtaaaggata aaacagcacc ctttacatca aaacgaagac gg 42
<210> 37
<211> 21
<212> DNA
<213> epr-yanF
<400> 37
gccccctacc atgatggact c 21
<210> 38
<211> 24
<212> DNA
<213> epr-yanR
<400> 38
ggtctcccat acatgaacga cttc 24
<210> 39
<211> 38
<212> DNA
<213> vpr-AF
<400> 39
caacgcacct ttcagcacat ccctccgctt ctttttgg 38
<210> 40
<211> 46
<212> DNA
<213> vpr-AR
<400> 40
gaaaggttct tcggtcaaaa ccgatcccct ttttcaatgt gtttcc 46
<210> 41
<211> 46
<212> DNA
<213> vpr-BF
<400> 41
ggaaacacat tgaaaaaggg gatcggtttt gaccgaagaa cctttc 46
<210> 42
<211> 43
<212> DNA
<213> vpr-BR
<400> 42
gtaaaggata aaacagcacg gctatatgtt tgcccatttt atc 43
<210> 43
<211> 22
<212> DNA
<213> vpr-yanF
<400> 43
cgcctttctt tggtatgtac gc 22
<210> 44
<211> 21
<212> DNA
<213> vpr-yanR
<400> 44
gttttccgaa tctgaccgct g 21
<210> 45
<211> 42
<212> DNA
<213> wprA-AF
<400> 45
caacgcacct ttcagcatac atattacgac atgggtggta gc 42
<210> 46
<211> 45
<212> DNA
<213> wprA-AR
<400> 46
gtatccacag ccgtgacgta cagctgcgtt tcatgttatc cctcc 45
<210> 47
<211> 45
<212> DNA
<213> wprA-BF
<400> 47
ggagggataa catgaaacgc agctgtacgt cacggctgtg gatac 45
<210> 48
<211> 41
<212> DNA
<213> wprA-BR
<400> 48
gtaaaggata aaacagcacg tctccaacac agcccaatct g 41
<210> 49
<211> 20
<212> DNA
<213> wprA-yanF
<400> 49
gctgaataaa actggagggc 20
<210> 50
<211> 20
<212> DNA
<213> wprA-yanR
<400> 50
cgtcttcaat gaggatggtg 20
<210> 51
<211> 40
<212> DNA
<213> aprX-AF
<400> 51
caacgcacct ttcagcagga gggttttagt aacaggctgc 40
<210> 52
<211> 43
<212> DNA
<213> aprX-AR
<400> 52
cggaattttc tgcgttgaca ggtaacggcc aatctagctt gtg 43
<210> 53
<211> 43
<212> DNA
<213> aprX-BF
<400> 53
cacaagctag attggccgtt acctgtcaac gcagaaaatt ccg 43
<210> 54
<211> 40
<212> DNA
<213> aprX-BR
<400> 54
gtaaaggata aaacagcacc ttcagcccac tcaacctgag 40
<210> 55
<211> 24
<212> DNA
<213> aprX-yanF
<400> 55
gattgtcacg gttattagcg gttc 24
<210> 56
<211> 22
<212> DNA
<213> aprX-yanR
<400> 56
ccagtcggta aaggtccagt cc 22
Claims (2)
1.一种高效表达纤溶酶的枯草芽孢杆菌工程菌，其特征在于：它是通过将枯草芽孢杆菌进行基因敲除后获得；所敲除的基因包括芽孢形成编码基因spo0A，淀粉酶编码基因amyE，部分蛋白酶编码基因bpr、mpr、epr、vpr、wprA、aprX，聚谷氨酸合酶编码基因pgsB；所述枯草芽孢杆菌为枯草芽孢杆菌枯草亚种，其保藏编号为CICC 20637。
2.权利要求1所述高效表达纤溶酶的枯草芽孢杆菌工程菌的制备方法，包括如下步骤：
1）根据目标基因上下游同源臂序列设计敲除引物，引物末端和载体末端具有15~20个同源碱基；
2）PCR扩增：提取枯草芽孢杆菌总DNA，以其作为模板，用各基因对应的上、下游同源臂引物进行PCR扩增，得到目的同源臂DNA片段；
3）通过SOE-PCR分别将各基因的上下游同源臂连接起来，得到9个基因敲除片段；
4）通过无缝克隆将基因敲除片段连接到敲除载体上，得到对应的9个目标基因敲除载体；
5）枯草芽孢杆菌的遗传转化，对枯草芽孢杆菌感受态的制备及利用1个目标基因敲除载体进行电转化；
6）通过抗性筛选和PCR扩增对转化子进行验证；
7）挑选阳性转化子进行传代，经过高温传代，质粒消除，点种和PCR验证后，筛选出目标基因敲除菌株；
8）在得到目标基因敲除菌株后，以其为出发菌株，重复步骤5）~7）用同样的方法对下一个目标基因进行敲除，直至得到敲除9个基因的目标菌种。
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