CN110396533B - 具有噬菌体抗性的大肠杆菌菌株、筛选方法、重组工程菌表达重组蛋白的制备方法 - Google Patents
具有噬菌体抗性的大肠杆菌菌株、筛选方法、重组工程菌表达重组蛋白的制备方法 Download PDFInfo
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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Abstract
本发明公开了具有噬菌体抗性的大肠杆菌菌株、筛选方法、重组工程菌表达重组蛋白的制备方法，属于大肠杆菌菌株技术领域，筛选方法包括以下步骤：诱变过程、加压培养过程以及传代稳定性检测过程，诱变过程、加压培养过程以及传代稳定性检测过程形成一次完整的筛选过程，重复N次筛选过程得到纯化的具有噬菌体抗性的大肠杆菌菌株；其中：N为自然数，第N次筛选过程中被诱变的菌株为第N+1具有噬菌体抗性的大肠杆菌菌株且加压培养的噬菌体为第N+1噬菌体；该具有噬菌体抗性的大肠杆菌菌株N+1种噬菌体均具有抗性，不受这N+1种噬菌体的影响，因此不需要定期对设备、环境消毒或更换不同的大肠杆菌宿主，节约生产成本和提高了生产效率。
Description
技术领域
本发明属于大肠杆菌菌株技术领域，具体涉及一种具有噬菌体抗性的大肠杆菌菌株，具有抗性的大肠杆菌菌株的筛选方法以及其在形成重组工程菌种中的应用。
背景技术
自然界中，细菌培养物易受各种噬菌体的感染。特定的噬菌体会感染特定的细菌培养物，导致此培养物的完全裂解。在实验室研究阶段，细菌培养物相对规模较小，感染噬菌体后细菌培养物的处理较为容易，也较好完全清除这些噬菌体感染。近几十年来，基因工程领域技术的飞速发展，某些细菌已经被驯化并广泛用于发酵过程。通过大规模培养细菌从而生产大量的生物活性重组多肽或重组蛋白已经是非常广泛，而这类产品的大规模生产通常需要发酵大量的基因工程细菌，特别是高细胞密度发酵技术的广泛应用，使得基因工程细菌的用量和密度更大。由于每天在庞大的发酵设备中培养大量细菌，因此，大多数发酵工业中，遭遇噬菌体污染问题已经屡见不鲜。
大肠杆菌(Escherichia coli，E.coli)是用于表达重组多肽或蛋白的常用宿主。目前，为了预防噬菌体污染问题，工业生产中通常定期对设备或环境消毒，或是更换不同的大肠杆菌宿主。然而，这并不能很好地解决噬菌体污染问题，噬菌体污染现象仍然频频出现。因此，筛选具有抗噬菌体能力的大肠杆菌菌株可以从根本上有效解决噬菌体污染问题。
发明内容
为了解决现有技术存在的工业生产中通常定期对设备或环境消毒，或是更换不同的大肠杆菌宿主。然而，这并不能很好地解决噬菌体污染问题，噬菌体污染现象仍然频频出现的问题，本发明提供了一种具有噬菌体抗性的大肠杆菌菌株。
本发明所采用的技术方案为：一种具有噬菌体抗性的大肠杆菌菌株的筛选方法，包括以下步骤：
S1：将对所有噬菌体均无抗性的大肠杆菌经过化学诱变或物理诱变，得到诱变大肠杆菌菌落；
S2：将诱变大肠杆菌菌落与第一噬菌体混合加压培养，筛选出形态完整的诱变大肠杆菌菌株即为具有第一噬菌体抗性的大肠杆菌菌株；
S3：步骤S1和步骤S2形成一个诱变筛选过程，重复诱变筛选过程1-2次，得到第一具有噬菌体抗性的大肠杆菌菌株；
S4：传代稳定性检测，具体为：步骤S3得到的具有第一噬菌体抗性的纯化大肠杆菌连续传代10代，每传代1次，用双层平板法测定其噬菌体抗性，得到纯化的第一具有噬菌体抗性的大肠杆菌菌株；
S5：步骤S1、S2、S3以及S4构成一次完整的筛选过程，重复N次筛选过程得到纯化的具有噬菌体抗性的大肠杆菌菌株；其中：N为自然数，第N次筛选过程中被诱变的菌株为第N+1具有噬菌体抗性的大肠杆菌菌株且加压培养的噬菌体为第N+1噬菌体。
本发明的有益效果为：该具有噬菌体抗性的大肠杆菌菌株N+1种噬菌体均具有抗性，不受这N+1种噬菌体的影响，因此不需要定期对设备、环境消毒或是更换不同的大肠杆菌宿主，大大节约了生产成本和提高了生产效率。
具有噬菌体抗性的大肠杆菌菌株具有抗性性能稳定、生长速度以及遗传性好，与其结合形成重组菌株中的质粒基因的表达没有任何的影响，因此可普遍应用于重组多肽或蛋白类产品的生产。
将N+1种噬菌体进行单独诱变和加压培养，避免了两种及两种以上的噬菌体混合培养带来的相互竞争和拮抗作用影响筛选结果。其中，加压培养是选择性压力法培养。
本发明还公开了一种具有噬菌体抗性的大肠杆菌菌株的筛选方法得到的具有噬菌体抗性的大肠杆菌菌株。
本发明还公开了一种具有噬菌体抗性的大肠杆菌菌株在形成重组工程菌种中的应用。
本发明还公开了一种具有噬菌体抗性的大肠杆菌菌株作为重组工程菌宿主在重组融合蛋白生产中的应用。
本发明的有益效果为：避免了工程中使用的大肠杆菌菌株易被噬菌体感染而导致细胞破裂的作用，不用定期更换大肠杆菌和定期对生产环境进行消毒，提高了生产效率；而不会影响重组蛋白的表达。
进一步限定，重组工程菌的制备方法为：将含有编码目的多肽或蛋白质的基因连接至表达载体的启动子序列以及核糖体结合序列之后转化大肠杆菌，即可得到所述重组工程菌。
进一步限定，重组工程菌为具有噬菌体抗性的Arg34GLP-1(9-37)生产菌种；
该具有噬菌体抗性的Arg34GLP-1(9-37)生产菌种表达得到第一重组融合蛋白，第一重组融合蛋白为mTrA-Arg34GLP-1(9-37)，所述第一重组融合蛋白的结构为：辅助蛋白-连接肽-切割位点-Arg34GLP-1(9-37)。
进一步限定，重组工程菌为具有噬菌体抗性的PTH(1-34)生产菌种；
该具有噬菌体抗性的PTH(1-34)生产菌种表达得到第二重组融合蛋白，第二重组融合蛋白为P1T-PTH(1-34)，所述第二重组融合蛋白的结构为：辅助蛋白-切割位点-PTH(1-34)。
本发明还公开了一种重组工程菌表达重组蛋白的制备方法，包括以下步骤：
H1：制备种子液：将具有噬菌体抗性的大肠杆菌菌株接种于含抗生素的液体培养基，振荡培养过夜；
H2：培养表达：将步骤H1中培养过夜得到的种子液接种于新鲜的含抗生素的液体培养基，振荡培养至对数生长期，加入诱导剂开始表达，继续振荡培养数小时直至表达结束，收集菌液进行重组融合蛋白表达分析。
进一步限定，所述重组融合蛋白经过酶切后可以得到胰高血糖素样肽1类似物、胰高血糖素样肽2类似物以及甲状旁腺素类似物中的一种。
附图说明
图1是E.coli BL21(DE3)PAnr和E.coli BL21(DE3)的生长趋势，其中：E.coliBL21(DE3)PAnr是对上述六种噬菌体均具有抗性的大肠埃希氏菌的简写，E.coli BL21(DE3)为对噬菌体无抗性的大肠埃希氏菌的简写；
图2是E.coli BL21(DE3)PAnr/pET28a-mTrA/9-37表达第一重组融合蛋白mTrA-Arg34GLP-1(9-37)的电泳图，其中：E.coli BL21(DE3)PAnr/pET28a-mTrA/9-37为对上述六种噬菌体均具有抗性的大肠埃希氏菌与胰高血糖素-1类似物的核苷酸序列重组后形成的第一重组工程菌种的简写，mTrA-Arg34GLP-1(9-37)为第一重组工程菌种表达得到的第一重组融合蛋白的简写；
图3是E.coli BL21(DE3)PAnr/pET28a-P1T/PTH(1-34)表达第二重组融合蛋白P1T-PTH(1-34)的电泳图，其中：E.coli BL21(DE3)PAnr/pET28a-P1T/PTH(1-34)为对上述六种噬菌体均具有抗性的大肠埃希氏菌与人甲状旁腺激素(1-34)重组后形成的第二重组工程菌种的简写，P1T-PTH(1-34)为第二重组工程菌种表达得到的第二重组融合蛋白的简写；
图4是发酵罐生产mTrA-Arg34GLP-1(9-37)的电泳图；
图5是发酵罐生产P1T-PTH(1-34)的电泳图；
图6是切割mTrA-Arg34GLP-1(9-37)得到Arg34GLP-1(9-37)的高效液相色谱图谱，其中：Arg34GLP-1(9-37)是切割mTrA-Arg34GLP-1(9-37)获得的第一目的多肽的简写；
图7是切割P1T-PTH(1-34)得到PTH(1-34)的高效液相色谱图谱，其中：PTH(1-34)是切割P1T-PTH(1-34)获得的第二目的多肽的简写。
具体实施方式
下面结合附图及具体实施例对本发明作进一步阐述。
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
在本发明的描述中，需要理解的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本发明的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
本发明的描述中，有的结构或器件未作具体描述的，理解为现有技术中有能实现的结构或器件。
加压培养为选择性压力法培养；E.coli BL21(DE3)PA1r是只对噬菌体PA-1具有抗性的大肠埃希氏菌的简写，商业化宿主E.coli BL21(DE3)是指对噬菌体没有抗性且已经投用于生产中的大肠埃希氏菌。
所述抗噬菌体的大肠杆菌菌株作为宿主用于构建重组多肽或重组蛋白的方法是指将含有编码目的多肽或蛋白质的基因连接至表达载体的启动子(Promoter)序列以及核糖体结合序列(ribosome binding site，RBS)之后，获得重组表达载体。将重组表达载体转化至所述抗噬菌体的大肠杆菌菌株，并筛选阳性转化子，获得可以表达含有目的多肽的重组融合蛋白或目的蛋白的抗噬菌体的重组大肠杆菌工程菌株。
所述抗噬菌体的重组大肠杆菌工程菌表达含有目的多肽的重组融合蛋白或蛋白质的方法，步骤如下：一、制备种子液：将抗噬菌体的重组大肠杆菌工程菌株接种于含抗生素的液体培养基，振荡培养过夜；二、培养表达：将种子液接种于新鲜的含抗生素的液体培养基，振荡培养至对数生长期，加入诱导剂开始表达，继续振荡培养数小时直至表达结束，收集菌液进行重组融合蛋白表达分析。
由本发明方法所生产的蛋白质或多肽可以是任意的可以在大肠杆菌中生产的异源蛋白质或多肽。这类蛋白质或多肽的实例可以但不仅限于组织因子、胰岛素前体、胰岛素及胰岛素类似物、生长激素、白介素、干扰素、因子VII、因子VIII、因子XIII、胰高血糖素、胰高血糖素样肽1(GLP-1)、胰高血糖素样肽1(GLP-1)类似物、胰高血糖素样肽2(GLP-2)、胰高血糖素样肽2(GLP-2)类似物、甲状旁腺素(PTH)以及甲状旁腺素(PTH)类似物；其中，胰高血糖素样肽1(GLP-1)类似物、胰高血糖素样肽2(GLP-2)类似物以及甲状旁腺素(PTH)类似物是重组融合蛋白经过酶切以后得到。
所述GLP-1是指胰高血糖素样肽1，所述GLP-2是指胰高血糖素样肽2，所述PTH是指甲状旁腺素。
所述Arg34GLP-1(9-37)是指从GLP-1第9位谷氨酸起到第37位甘氨酸止的多肽，其中34位的赖氨酸替换为精氨酸。
所述PTH(1-34)是指从PTH第1位丝氨酸起到第34位苯丙氨酸止的多肽。
所述具有噬菌体抗性的Arg34GLP-1(9-37)生产菌种，是指具有噬菌体抗性的大肠杆菌工程菌株经培养表达得到重组融合蛋白，该重组融合蛋白结构为：辅助蛋白-连接肽-切割位点-Arg34GLP-1(9-37)。该重组融合蛋白经蛋白酶切割或化学试剂切割后获得目的多肽Arg34GLP-1(9-37)。所述具有噬菌体抗性的Arg34GLP-1(9-37)生产菌种，优选为E.coliBL21(DE3)PAnr/pET28a-mTrA/9-37，所述生产菌种所生产的重组融合蛋白为mTrA-Arg34GLP-1(9-37)，其氨基酸序列如SEQ ID No.1所示，编码所述重组融合蛋白mTrA-Arg34GLP-1(9-37)的核苷酸序列如SEQ ID No.2所示。
所述具有噬菌体抗性的PTH(1-34)生产菌种，是指具有噬菌体抗性的大肠杆菌工程菌株经培养表达得到重组融合蛋白，该重组融合蛋白结构为：辅助蛋白-切割位点-PTH(1-34)。该重组融合蛋白经蛋白酶切割或化学试剂切割后获得目的多肽PTH(1-34)。所述具有噬菌体抗性的PTH(1-34)生产菌种，优选为E.coli BL21(DE3)PAnr/pET28a-P1T/PTH(1-34)，所述生产菌种所生产的重组融合蛋白为P1T-PTH(1-34)，其氨基酸序列如SEQ ID No.3所示，编码所述重组融合蛋白P1T-PTH(1-34)的核苷酸序列如SEQ ID No.4所示。
所述表达载体是指含有T7启动子序列，优选为pET系列载体(Novagen公司)。
所述大肠杆菌培养基为LB培养基，具体组成为：胰蛋白胨1％，酵母提取物0.5％，NaCl 1％。
所述TB培养基组成为：酵母提取物2.4％，酪蛋白水解物1.2％，磷酸氢二钾0.94％，磷酸二氢钾0.22％，pH7.0。
所述噬菌体缓冲液为SM buffer，具体组成为：100mM NaCl，10mM MgSO4·7H2O，50mM Tris-HCl，pH 7.5和0.01％(w/v)明胶。
所述生产菌种的种子培养基及发酵培养基为：一级种子培养基为LB培养基，具体配方为：胰蛋白胨1％，酵母提取物0.5％，氯化钠1％；二级种子培养基同罐培养基，具体配方为：磷酸氢二胺0.2％、磷酸二氢钾0.675％、一水柠檬酸0.093％、七水硫酸镁0.07％、葡萄糖2％、微量盐5mL/L(微量盐组成：七水硫酸亚铁1％、五水硫酸锰0.05％、七水硫酸锌0.225％、五水硫酸铜0.1％、十水四硼酸钠0.023％、二水氯化钙0.2％、七钼酸铵0.01％、盐酸5mol/L)，pH6.8。
所述生产菌种的发酵罐培养方法具体为：一级种制备→二级种制备→罐接种及发酵培养。
实施例1
1.对噬菌体PA-1、噬菌体PA-2、噬菌体PA-3、噬菌体PA-4、噬菌体PA-5以及噬菌体PA-6均具有抗性的大肠埃希氏菌(E.coli BL21(DE3)PAnr)的高效筛选方法
1.1 E.coli BL21(DE3)的培养及第1次紫外诱变
将E.coli BL21(DE3)甘油液以1％接种量接种于LB液体培养基，37℃，220rpm培养过夜，作为种子液。将种子液以1％接种量接种于LB液体培养基，37℃，220rpm培养6-8h，离心收集菌体，然后用生理盐水洗涤3次。将菌液放置于平皿中，敞口，打开转子，于紫外下照射40s。照射完成后稀释成不同浓度梯度涂布LB固体平板，于37℃避光静置培养1-2天直至菌落长出。
1.2 E.coli BL21(DE3)与噬菌体PA-1的第1次加压培养
将长出的E.coli BL21(DE3)菌落混合接种于50mL的LB液体培养基中，37℃，220rpm振荡培养数小时，监测OD600直至0.6-1.0之间。取出0.1mL的E.coli BL21(DE3)培养液，加入0.1mL效价为108pfu/mL(plaque forming unit/mL)的噬菌体液混合，室温静置吸附5-10min，然后于37℃，100rpm培养6h。
1.3第1次加压培养得到的培养物的第2次紫外诱变
将培养6h后的E.coli BL21(DE3)和噬菌体PA-1混合物离心，然后用生理盐水洗涤3次，最后第2次紫外诱变，紫外诱变方法同步骤1.1相同。
1.4第2次加压培养
将步骤1.3所得到的混合物与噬菌体PA-1第2次加压培养，与步骤1.2相同。
1.5第2次加压培养得到的培养物的第3次紫外诱变
步骤与1.3相同。
1.6第3次加压培养
将步骤1.5所得到的混合物与噬菌体PA-1第3次加压培养，与步骤1.2相同。
1.7 E.coli BL21(DE3)PA1r的筛选
将步骤1.6所得到的混合液适当稀释，分别倒入5mL约50℃的含0.7％琼脂的LB培养基并快速混匀，再倒入已经凝固的下层LB固体培养基(1.2％琼脂)，待其凝固。37℃下静置培养1-2天，直至抗噬菌体的大肠杆菌菌落出现，划线传代3次获得纯化的单菌落E.coliBL21(DE3)PA1r；
1.8 E.coli BL21(DE3)PA1r的传代稳定性检测
将获得E.coli BL21(DE3)PA1r于LB培养基上连续传代10代，每代分别检测其对噬菌体PA-1的抗性，然后将具备抗性的单菌落进行传代，最后获得纯化的具备传代稳定性的E.coli BL21(DE3)PA1r，将新鲜的E.coli BL21(DE3)PA1r培养液保存在20％甘油中。
1.9 E.coli BL21(DE3)PAnr的筛选及保藏
将E.coli BL21(DE3)PA1r作为出发菌株，与噬菌体PA-2进行加压培养，重复步骤1.1-1.8，筛选得到对噬菌体PA-1和噬菌体PA-2均有抗性的大肠埃希氏菌，简写为E.coliBL21(DE3)PA2r，保存于20％甘油。
再以E.coli BL21(DE3)PA2r作为出发菌株，与噬菌体PA-3进行加压培养，重复步骤1.1-1.8，筛选得到对噬菌体PA-1、噬菌体PA-2以及噬菌体PA-3均有抗性的大肠埃希氏菌，简写为E.coli BL21(DE3)PA3r，保存于20％甘油。
再以E.coli BL21(DE3)PA3r作为出发菌株，与噬菌体PA-4进行加压培养，重复步骤1.1-1.8，筛选得到对噬菌体PA-1、噬菌体PA-2、噬菌体PA-3以及噬菌体PA-4均有抗性的大肠埃希氏菌，简写为E.coli BL21(DE3)PA4r，保存于20％甘油。
再以E.coli BL21(DE3)PA4r作为出发菌株，与噬菌体PA-5进行加压培养，重复步骤1.1-1.8，筛选得到对噬菌体PA-1、噬菌体PA-2、噬菌体PA-3、噬菌体PA-4以及噬菌体PA-5均有抗性的大肠埃希氏菌，简写为E.coli BL21(DE3)PA5r，保存于20％甘油。
最后E.coli BL21(DE3)PA5r作为出发菌株，与噬菌体PA-6进行加压培养，重复步骤1.1-1.8，筛选得到对噬菌体PA-1、噬菌体PA-2、噬菌体PA-3、噬菌体PA-4、噬菌体PA-5以及噬菌体PA-6均有抗性的大肠埃希氏菌，简写为E.coli BL21(DE3)PAnr，保存于20％甘油，于-80℃保存，E.coli BL21(DE3)PAnr的保藏号为：CGMCC No.17916，保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心，保藏地址：北京市朝阳区北辰西路1号院3号；保藏时间为2019年06月11日。
2.E.coli BL21(DE3)PAnr的性能鉴定
2.1 E.coli BL21(DE3)PAnr的噬菌体抗性的检测
E.coli BL21(DE3)PAnr与商业化宿主E.coli BL21(DE3)分别接种于20mL的LB液体培养基，37℃，220rpm振荡过夜。将E.coli BL21(DE3)PAnr和E.coli BL21(DE3)的培养液分别分为6份(每份0.5mL)，然后于每份培养液中分别加入0.5mL效价为108pfu/mL的噬菌体PA-1液、噬菌体PA-2液、噬菌体PA-3液、噬菌体PA-4液、噬菌体PA-5液以及噬菌体PA-6液，混合均匀，室温静置吸附5-10min，然后分别倒入5mL冷却至约50℃的含0.7％琼脂的LB培养基，快速混合均匀，再倒入已凝固的下层LB固体培养基(1.2％琼脂)，待其凝固。在凝固后的上层平板再分别各自滴加5μL的噬菌体PA-1液、噬菌体PA-2液、噬菌体PA-3液、噬菌体PA-4液、噬菌体PA-5液以及噬菌体PA-6液，于37℃静置培养1-2天。结果见表1，(+)表示双层平板上出现噬菌斑，(—)则表示双层平板上无噬菌斑。
表1 E.coli BL21(DE3)PAnr的噬菌体抗性的检测结果
| E.coli BL21(DE3)PAnr | E.coli BL21(DE3) | |
| 噬菌体PA-1 | — | + |
| 噬菌体PA-2 | — | + |
| 噬菌体PA-3 | — | + |
| 噬菌体PA-4 | — | + |
| 噬菌体PA-5 | — | + |
| 噬菌体PA-6 | — | + |
由表1可知，E.coli BL21(DE3)PAnr的平板上噬菌体PA-1、噬菌体PA-2、噬菌体PA-3、噬菌体PA-4、噬菌体PA-5以及噬菌体PA-6未出现任何噬菌斑，表明其具有对噬菌体PA-1、噬菌体PA-2、噬菌体PA-3、噬菌体PA-4、噬菌体PA-5以及噬菌体PA-6的抗性，而E.coli BL21(DE3)上均出现噬菌斑，说明噬菌体PA-1、噬菌体PA-2、噬菌体PA-3、噬菌体PA-4、噬菌体PA-5以及噬菌体PA-6均可感染E.coli BL21(DE3)。
2.2 E.coli BL21(DE3)PAnr的生长测定
将E.coli BL21(DE3)PAnr和E.coli BL21(DE3)分别接种于新鲜的LB培养基，37℃，220rpm振荡培养过夜，测定其OD600，然后以相同的接种量接种于新鲜的LB培养基，37℃，220rpm培养约30-35h，过程取样测定OD600并绘制生长曲线，如图1所示，由图1可知，E.coliBL21(DE3)PAnr和E.coli BL21(DE3)的生长并无显著差异。
3.E.coli BL21(DE3)PAnr/pET28a-mTrA/9-37的构建与重组融合蛋白mTrA-Arg34GLP-1(9-37)的表达
将编码重组融合蛋白的核苷酸序列分别插入载体pET28a的NcoI(CCATGG)以及XhoI(CTCGAG)多克隆位点，得到重组表达载体pET28a-mTrA/9-37，将所构建的重组表达载体的插入片段进行测序，验证插入的基因正确。该重组融合蛋白mTrA-Arg34GLP-1(9-37)的氨基酸序列如SEQ ID NO.1所示，氨基酸序列1-104为辅助蛋白硫氧还蛋白突变体，氨基酸序列105-112为连接肽-肠激酶切割位点，氨基酸序列113-141为胰高血糖素-1类似物Arg34GLP-1(9-37)对应的氨基酸序列。编码该重组融合蛋白的核苷酸序列如SEQ ID NO.2所示，核苷酸序列1-312为辅助蛋白的核苷酸序列，核苷酸序列313-336为连接肽-肠激酶切割位点所对应的核苷酸序列，核苷酸序列337-423为胰高血糖素-1类似物Arg34GLP-1(9-37)对应的核苷酸序列。将上述构建正确的表达载体pET28a-mtrA/9-37热激法分别转化具噬菌体抗性的大肠杆菌宿主E.coli BL21(DE3)PAnr中以及无抗性的商业化宿主E.coli BL21(DE3)，并涂布于含有50μg/mL硫酸卡那霉素的LB平板培养基。大肠杆菌感受态细胞的制备以及热激转化的方法参见《分子克隆实验指南(第三版)》中的第87页到99页。待转化子长出，筛选若干送出测序，测序正确的转化子分别保藏备用，测序正确的转化子即为重组工程菌E.coli BL21(DE3)PAnr/pET28a-mTrA/9-37以及E.coli BL21(DE3)/pET28a-mTrA/9-37，E.coli BL21(DE3)PAnr/pET28a-mTrA/9-37的保藏号为：CGMCC No.17918，保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心，保藏地址：北京市朝阳区北辰西路1号院3号；保藏时间为2019年06月11日。
将上述测序正确的单克隆分别接种于含有硫酸卡那霉素(50μg/mL)的LB液体培养基，37℃，220rpm培养过夜。将所得培养液作为种子液，1％(v/v)接种量接种于新鲜的含硫酸卡那霉素(50μg/mL)的LB液体培养基中，37℃培养4h，将温度调至30℃并加入IPTG(异丙基硫代半乳糖苷)至IPTG终浓度0.4mM，继续振荡培养20h。将所得培养液于室温4000rpm离心10min，收集菌体，多余菌体保存于-20℃。取一定量的菌体，向其中加入一定体积的电泳上样缓冲液，100℃煮沸5min制样。所得样品进行SDS-PAGE电泳分析，结果如图2所示，图2为E.coli BL21(DE3)PAnr/pET28a-mTrA/9-37以及E.coli BL21(DE3)/pET28a-mTrA/9-37表达结束的菌体总蛋白的SDS-PAGE结果，图2中，泳道1为E.coli BL21(DE3)PAnr/pET28a-mTrA/9-37菌体总蛋白，泳道2为对照，即无噬菌体抗性的商业化宿主所产生的第一重组菌株E.coli BL21(DE3)/pET28a-mTrA/9-37菌体总蛋白，M为蛋白分子量标准。第一重组融合蛋白mTrA-Arg34GLP-1(9-37)的理论分子量为15kD，由图2可知，约15kD(黑色箭头标注)蛋白条带量明显大于同泳道其他条带，说明其正常表达。E.coli BL21(DE3)PAnr所表达的的蛋白量(泳道1)与E.coli BL21(DE3)(泳道2)所表达的蛋白量相当，表明E.coli BL21(DE3)PAnr与E.coli BL21(DE3)相比，其表达重组蛋白mTrA-Arg34GLP-1(9-37)水平无明显差异。
4.E.coli BL21(DE3)PAnr/pET28a-P1T/PTH(1-34)的构建与重组融合蛋白P1T-PTH(1-34)的表达
将编码重组融合蛋白P1T-PTH(1-34)的核苷酸序列插入载体pET28a的多克隆位点，得到重组表达载体pET28a-P1T/PTH(1-34)，将所构建的重组表达载体的插入片段进行测序，验证插入的基因正确。该重组融合蛋白P1T-PTH(1-34)的氨基酸序列如SEQ ID NO.3所示，编码该重组融合蛋白的核苷酸序列如SEQ ID NO.4所示。将构建正确的重组表达载体pET28a-P1T/PTH(1-34)使用热激法分别转化具噬菌体抗性的大肠杆菌宿主E.coli BL21(DE3)PAnr中以及无抗性的商业化宿主E.coli BL21(DE3)，并涂布于含有50μg/mL硫酸卡那霉素的LB平板培养基。大肠杆菌感受态细胞的制备以及热激转化的方法参见《分子克隆实验指南(第三版)》中的第87页到99页。待转化子长出，筛选若干送出测序，测序正确的转化子分别保藏备用，测序正确的转化子即为重组工程菌E.coli BL21(DE3)PAnr/pET28a-P1T/PTH(1-34)以及E.coli BL21(DE3)/pET28a-P1T/PTH(1-34)，E.coli BL21(DE3)PAnr/pET28a-P1T/PTH(1-34)的保藏号：CGMCC No.17917，保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心，保藏地址：北京市朝阳区北辰西路1号院3号；保藏时间为2019年06月11日。
将上述测序正确的单克隆分别接种于含有硫酸卡那霉素(50μg/mL)的LB液体培养基，37℃，220rpm培养过夜。将所得培养液作为种子液，1％(v/v)接种量接种于新鲜的含硫酸卡那霉素(50μg/mL)的LB液体培养基中，37℃培养4h，将温度调至30℃并加入IPTG至其终浓度为0.4mM，继续振荡培养20h。将所得培养液于室温4000rpm离心10min，收集菌体，多余菌体保存于-20℃。取一定量的菌体，向其中加入一定体积的电泳缓冲液，100℃煮沸5min制样。所得样品进行SDS-PAGE电泳分析。表达结束菌体总蛋白的SDS-PAGE分析见图3，图3中泳道1为E.coli BL21(DE3)PAnr/pET28a-P1T/PTH(1-34)的菌体总蛋白，泳道2为对照，即无噬菌体抗性的商业化宿主所产生的重组工程菌E.coli BL21(DE3)/pET28a-P1T/PTH(1-34)的菌体总蛋白，泳道M为蛋白分子量标准。所述重组融合蛋白P1T-PTH(1-34)的理论分子量为20kD，图中可见约20kD(黑色箭头标注)蛋白条带量明显大于其他条带，说明其正常表达。具有噬菌体抗性的E.coli BL21(DE3)PAnr所表达的的蛋白量(泳道1)与商业化宿主E.coliBL21(DE3)所表达的蛋白量相当，表明此抗噬菌体的大肠杆菌宿主E.coli BL21(DE3)PAnr与商业化宿主E.coli BL21(DE3)相比，其表达重组蛋白水平无明显差异。
5.E.coli BL21(DE3)PAnr/pET28a-mTrA/9-37发酵生产mTrA-Arg34GLP-1(9-37)
E.coli BL21(DE3)PAnr/pET28a-mTrA/9-37发酵生产mTrA-Arg34GLP-1(9-37)的实施详见中国专利申请201910557170.2的实施例1。发酵结束后离心获得菌体沉淀并进行电泳制样，并对菌体总蛋白进行SDS-PAGE分析，结果见图4。图4中泳道1和泳道2为E.coliBL21(DE3)PAnr/pET28a-mTrA/9-37发酵结束的菌体总蛋白，泳道3为未诱导的菌体总蛋白对照，泳道M为蛋白分子量标准。重组融合蛋白mTrA-Arg34GLP-1(9-37)(氨基酸序列如SEQID No.1所示)的理论分子量为15kD，由图4可看出，发酵结束菌体总蛋白在分子量15kD处(黑色箭头标注)有明显蛋白条带，而未诱导对照则无此条带的明显表达，说明E.coli BL21(DE3)PAnr/pET28a-mTrA/9-37发酵生产时，mTrA-Arg34GLP-1(9-37)得到较好表达。
6.E.coli BL21(DE3)PAnr/pET28a-P1T/PTH(1-34)发酵生产P1T-PTH(1-34)
E.coli BL21(DE3)PAnr/pET28a-P1T/PTH(1-34)发酵生产P1T-PTH(1-34)的实施方法参见中国专利申请201910557170.2的实施例1。发酵结束后离心获得菌体沉淀并进行电泳制样，并对菌体总蛋白进行SDS-PAGE分析，结果见图5。图5中，泳道M为蛋白分子量标准，泳道1为未诱导的菌体总蛋白对照，泳道3为发酵结束的菌体总蛋白。图5可看出，20kD处(黑色箭头标准)有明显蛋白表达，而未诱导对照则无此条带的明显表达，说明E.coli BL21(DE3)PAnr/pET28a-P1T/PTH(1-34)发酵生产时，P1T-PTH(1-34)(氨基酸序列如SEQ IDNo.3所示)得到较好表达。
7.mTrA-Arg34GLP-1(9-37)切割获得第一目的多肽Arg34GLP-1(9-37)
mTrA-Arg34GLP-1(9-37)(氨基酸序列如SEQ ID No.1所示)切割获得目的多肽Arg34GLP-1(9-37)的方法以及高效液相色谱分析方法均参见中国专利申请201910557170.2的实施例1，切割反应前以及反应后的液相图谱结果见图6。
8.P1T-PTH(1-34)切割获得第二目的多肽PTH(1-34)
P1T-PTH(1-34)(氨基酸序列如SEQ ID No.3所示)切割获得目的多肽PTH(1-34)的方法以及高效液相色谱分析方法均参见中国专利申请201910557170.2的实施例1，所切割反应前以及反应后的液相图谱结果见图7。
本发明不局限于上述可选实施方式，任何人在本发明的启示下都可得出其他各种形式的产品，但不论在其形状或结构上作任何变化，凡是落入本发明权利要求界定范围内的技术方案，均落在本发明的保护范围之内。
序列表
<110> 成都英普博集生物科技有限公司
<120> 具有噬菌体抗性的大肠杆菌菌株、筛选方法、重组工程菌表达重组蛋白的制备方法
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Claims (6)
1. 一种具有噬菌体抗性的大肠杆菌菌株作为重组工程菌宿主在重组融合蛋白生产中的应用，其特征在于，所述具有噬菌体抗性的大肠杆菌菌株为：E.coli BL21(DE3)PAnr，保藏号为：CGMCC No.17916，保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心；
所述重组工程菌的制备方法为：将含有编码目的多肽或蛋白质的基因连接至表达载体的启动子序列以及核糖体结合序列之后转化大肠杆菌，即可得到所述重组工程菌。
2.根据权利要求1所述的应用，其特征在于，所述重组工程菌表达重组融合蛋白的制备方法，包括以下步骤：
H1：制备种子液：将具有噬菌体抗性的大肠杆菌菌株接种于含抗生素的液体培养基，振荡培养过夜；
H2：培养表达：将步骤H1中培养过夜得到的种子液接种于新鲜的含抗生素的液体培养基，振荡培养至对数生长期，加入诱导剂开始表达，继续振荡培养数小时直至表达结束，收集菌液进行重组融合蛋白表达分析。
3. 一种具有噬菌体抗性的大肠杆菌菌株作为重组工程菌宿主在重组融合蛋白生产中的应用，其特征在于，所述重组工程菌为具有噬菌体抗性的Arg34GLP-1(9-37)生产菌，所述具有噬菌体抗性的Arg34GLP-1(9-37)大肠杆菌生产菌株为：E.coli BL21(DE3)PAnr/pET28a-mTrA/9-37，保藏号为：CGMCC No.17918，保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心；
该具有噬菌体抗性的Arg34GLP-1(9-37)生产菌表达得到重组融合蛋白，所述重组融合蛋白为mTrA-Arg34GLP-1(9-37)，所述重组融合蛋白的结构为：辅助蛋白-连接肽-切割位点- Arg34GLP-1(9-37)；
所述重组融合蛋白的氨基酸序列如SEQ ID No.1所示。
4. 一种具有噬菌体抗性的大肠杆菌菌株作为重组工程菌宿主在重组融合蛋白生产中的应用，其特征在于，所述重组工程菌为具有噬菌体抗性的PTH(1-34)生产菌，所述具有噬菌体抗性的PTH(1-34)大肠杆菌生产菌株为：E.coli BL21(DE3)PAnr/pET28a-P1T/PTH(1-34)，保藏号为：CGMCC No.17917，保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心；
该具有噬菌体抗性的PTH(1-34)生产菌表达得到重组融合蛋白，所述重组融合蛋白为P1T-PTH(1-34)，所述重组融合蛋白的结构为：辅助蛋白-切割位点-PTH(1-34)；
所述重组融合蛋白的氨基酸序列如SEQ ID No.3所示。
5.根据权利要求3所述的应用，其特征在于，所述重组融合蛋白经过酶切后得到胰高血糖素样肽1。
6.根据权利要求4所述的应用，其特征在于，所述重组融合蛋白经过酶切后得到甲状旁腺素。
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