WO2018099063A1 - 一种利用芽孢杆菌高效分泌表达外源蛋白的方法 - Google Patents

一种利用芽孢杆菌高效分泌表达外源蛋白的方法 Download PDF

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WO2018099063A1
WO2018099063A1 PCT/CN2017/091056 CN2017091056W WO2018099063A1 WO 2018099063 A1 WO2018099063 A1 WO 2018099063A1 CN 2017091056 W CN2017091056 W CN 2017091056W WO 2018099063 A1 WO2018099063 A1 WO 2018099063A1
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bacillus
protein
strain
expression
secreted
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French (fr)
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任钧
唐旭
雷蕾
樊超
柴进凯
曹付明
范佳
曹镜
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成都美溢德生物技术有限公司
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Priority claimed from CN201611081187.8A external-priority patent/CN106755046B/zh
Priority claimed from CN201611082702.4A external-priority patent/CN106434730B/zh
Priority claimed from CN201611085518.5A external-priority patent/CN106834161B/zh
Application filed by 成都美溢德生物技术有限公司 filed Critical 成都美溢德生物技术有限公司
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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus

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  • the invention belongs to the field of biotechnology, and particularly relates to a method for efficiently secreting and expressing a foreign protein by using Bacillus.
  • Protein expression technology is one of the core technologies of modern biology. Expression proteins can be used not only for biological research, but also for commercial protein products such as recombinant vaccines, recombinant insulin, and cytokines.
  • Currently commonly used expression systems include Escherichia coli, yeast, insect cells, and mammalian cells, but they all have distinct advantages and disadvantages.
  • the E. coli expression system is the most well-researched and has a variety of options.
  • the most commonly used is the Novagen pET expression system, which uses the phage T7 RNA polymerase to specifically transcribe the target gene after the T7 promoter, under optimal conditions.
  • the target protein can reach more than 50% of the total protein of E. coli.
  • protein is easy to form inclusion bodies, and the difficulty and cost of refolding are high; E. coli can not glycosylate the protein; cell wall contains lipopolysaccharide (endotoxin) ), not easy to completely remove; there are many intracellular proteins, and it is not easy to remove various impurity proteins during the purification of the target protein.
  • Another commonly used expression system is the yeast expression system, which has high expression, induction, protein secretion to the outside for easy purification, and has certain post-translational modification ability, but the disadvantage is that some expression products are easily degraded and expressed. Uncontrollable, proteins larger than 30KDa can hardly be secreted.
  • the animal cell and insect cell expression system is characterized by a complete modification system, the expression product has or similar natural activity, no endotoxin contamination, but low expression, long cycle, high technical requirements, and high production cost.
  • Bacillus subtilis is a Gram-positive bacterium widely present in water, air and soil. It can produce spores when the environment is not suitable. The spores can resist extreme environments such as high temperature and drought, and then germinate when the environment is suitable. Nutritional growth. The secretion capacity of Bacillus subtilis is strong, and in the high-density fermentation, the protein secretion can reach 20-25 g/L (Developments in the use of Bacillus species for industrial production, 2004). The products such as protease, amylase, hypoxanthine nucleoside and ribose glucoside produced by fermentation have already entered our daily life. Because of its biosafety, it has been rated as a GRAS additive by the FDA. Its probiotics and fermented products produced by it have been widely used in medicine and aquaculture.
  • Bacillus subtilis was used for protein expression.
  • the expression products include not only various enzymes derived from bacteria, such as amylase, protease, endoglucanase, lipase, but also hEGF and IFN-.
  • Alpha 2 Proinsulin, Streptavidin, cathelicidin-BF and other proteins from different sources.
  • Bacillus subtilis It belongs to Gram-positive bacteria and does not contain lipopolysaccharide, which is convenient for the production of injectable drugs.
  • Bacillus subtilis The nutritional requirements of Bacillus subtilis are simple, and since it has achieved scale industrial production, the large-scale culture technology is mature and the cultivation cost is low. At present, multiple strains of Bacillus subtilis and multiple species of Bacillus have completed genome sequencing work, with clear genetic background, high safety, and easy genome transformation. However, the B. subtilis expression system is not a widely used expression system, and there are still many shortcomings to be overcome.
  • the wild type strain of the 168 strain of the B. subtilis model strain has been lost, and the currently available strains are all mutants thereof. Although it satisfies the requirements of scientific research, its poor protein secretion ability is a characteristic of auxotrophic mutants and cannot meet the requirements for establishing a commercial expression system.
  • the currently established transformation systems are all based on the model strain 168, although the wild-type strains and the Bacillus subtilis species that have been produced are very numerous, and the difficulty in transformation hinders further modification of these strains.
  • the plasmids derived from Bacillus subtilis are also almost concealed plasmids, and it is difficult to construct vectors.
  • Bacillus subtilis has four known secretory pathways, the Sec pathway, the Tat pathway, the Com system, and the ABC transport pathway. The current study is more clearly the Sec pathway, and most expression systems also use the Sec pathway to secrete expressed proteins. However, the Sec pathway has an intrinsic regulatory mechanism that inhibits the secretion of incorrectly folded proteins, while foreign proteins generally fold slowly.
  • the folding process requires the participation of chaperones and is easily degraded by the Sec secretory pathway, which results in very few proteins that can utilize Sec.
  • the pathway is secreted and the expression level of the foreign protein is relatively low.
  • Bacillus subtilis also secretes up to eight proteases into the fermentation broth, which also degrade the expressed protein of interest.
  • the present invention provides a method for efficiently secreting and expressing a foreign protein by using Bacillus, which does not depend on a signal peptide or a characteristic structure, and the existing Sec pathway, Tat pathway, Com system and The ABC transport pathway is essentially different.
  • Bacillus which does not depend on a signal peptide or a characteristic structure, and the existing Sec pathway, Tat pathway, Com system and The ABC transport pathway is essentially different.
  • any of the proteins of interest is expressed in a large amount in the cell, it can be secreted to the outside of the cell in a large amount by this means, and the maximum secretion amount can reach the g/L level. It not only overcomes the characteristics of low secretion and poor versatility of the existing secretory pathway, but also can selectively secrete the target protein into the fermentation broth, reducing the difficulty of separation and purification in the later stage.
  • a method for efficiently secreting a foreign protein expressed by Bacillus sp. in the present invention which solves the above problems is characterized in that the target protein is accumulated in a large amount in the cell, and is secreted into the fermentation liquid in a large amount.
  • the target protein When the target protein expresses more than 2% of the total protein in the cell, it will be secreted into the fermentation broth, and the secretion amount is more than 50% of the expressed protein.
  • a method for efficiently secreting and expressing a foreign protein by using Bacillus comprises the following steps:
  • proteases are genes that express secreted proteases into the fermentation broth, which can improve the degradation of proteins in the fermentation broth.
  • the present invention expresses the target protein in a large amount in the expression system. If the target protein accumulates in the cell, it can be secreted into the fermentation broth in large quantities, and any expression system capable of expressing the target protein in a large amount can be used for the protein. Secretory expression.
  • the expression system is an expression system in which a target protein is accumulated intracellularly and is largely secreted into a fermentation broth.
  • the expression system used in the present invention is an "anti-anti-expression system", which overcomes the disadvantage that the antibiotic screening marker gene is prone to drift, the plasmid expression system is unstable, and the expression of the inducible expression is not high, which is a low background controllable induction. And highly expressed non-anti-expression system.
  • the strain construction process of the "anti-anti-expression system” includes replacing the wprA protease gene of the Z12 strain with the xylR gene, the promoter region of xylAB and the CDS fragment of the T7 RNA polymerase gene, and specifically includes the following steps:
  • the expression strain ZT7RP of the "anti-anti-expression system" used in the present invention has knocked out the wprA protease gene during the construction of the strain.
  • a method for constructing a Bacillus strain lacking eight proteases that is, constructing pBTS-aprE, pBTS-bpr, pBTS-epr, pBTS-mpr, pBTS-nprB, pBTS-nprE and pBTS-vpr vectors, respectively, is sequentially knocked out.
  • the aprE, bpr, epr, mpr, nprB, nprE and vpr genes in the ZT7RP strain were obtained to obtain a new strain Z15;
  • the intracellular expression method of the target gene of the "anti-anti-expression system" used in the present invention comprises constructing the expression plasmid pBTS-FR, synthesizing the target gene fragment X, constructing the expression vector pBTS-FR-X, and inserting the target gene by homologous recombination.
  • constructing the expression plasmid pBTS-FR synthesizing the target gene fragment X
  • constructing the expression vector pBTS-FR-X constructing the expression vector pBTS-FR-X
  • inserting the target gene by homologous recombination To the xylAB gene region, specifically including the following process:
  • the pBTS nucleotide sequence is set forth in SEQ ID NO.
  • the pBTS-T7RP nucleotide sequence is set forth in SEQ ID NO.
  • the pBTS-FR nucleotide sequence is set forth in SEQ ID NO.
  • the Bacillus is Bacillus subtilis 168, Z12 strain, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis and other Bacillus.
  • Bacillus subtilis Z12 Latin Bacillus subtilis Z12, is deposited in the Common Microbiology Center CGMCC of the China Microbial Culture Collection Management Committee, and the deposit number is 12750.
  • the secretory pathway does not require a signal peptide, a characteristic structure, or the like for transport recognition, and the secreted protein of interest does not contain an excess protein sequence.
  • the secretory pathway is not selective, and any protein that can be expressed in a large amount in the cell can be secreted into the fermentation broth, which significantly reduces the separation and purification process and cost of the subsequent target protein.
  • the invention combines Bacillus, especially Bacillus subtilis, has been recognized by the FDA as a GRAS additive, has a short fermentation cycle and low production cost, and can utilize the secretory route of the invention to produce protein polypeptide biotechnology products on a large scale and change the public. Lifestyle.
  • pBAV1K-T5-GFP http://www.addgene.org/vector-database/ ) plasmid was cleaved by EcoR I and Apa I endonuclease, and homologously recombined with the synthesized MCS fragment to obtain plasmid pBAV1K.
  • Thermo's rapid endonuclease in this experiment and subsequent experiments was developed by Thermo's rapid endonuclease.
  • the fragment was recovered using Chengdu Fuji Biotech's gel recovery kit (DE-02011).
  • the MCS fragment was synthesized by Jinweizhi Biotechnology Co., Ltd.
  • the source recombination was performed using EsayGeno Rapid Recombination Cloning Kit (VI201-02), E. coli strain was top10, KCM method was used for preparation of competent state, and plasmid extraction was carried out using the common plasmid miniprep kit (FE-01001). ).
  • the bacterial solution grows until the OD reaches between 0.85 and 0.95, the bacterial solution is placed on ice and pre-cooled for 10 min; 4 ° C, 5000g, 5min, centrifuge to remove the supernatant, resuspend the cells with an equal volume of pre-cooled EM (0.5M sorbitol + 0.5M mannitol + 10% glycerol aqueous solution), again at 4 ° C, 5000g, 5min, centrifuged Clearly, repeat the washing 4 times; add about 1/40 volume of EM to resuspend the cells to ensure the concentration of the bacteria is between 1-1.3 ⁇ 10 10 cfu/ml.
  • pre-cooled EM 0.5M sorbitol + 0.5M mannitol + 10% glycerol aqueous solution
  • the bacterial solution was centrifuged, and the supernatant was diluted to a certain ratio. 200 ul was applied to an LB plate containing 30 mg/L kanamycin, and cultured overnight at 37 ° C, while the non-electrically transformed bacterial solution was applied as a negative control. . The colony growth was observed the next morning. If the transformation plate had colony growth and the negative control did not, the transformation was successful.
  • Bacillus subtilis Z12 (((Latin is Bacillus subtilis Z12), deposited in the Common Microbiology Center (CGMCC) of China Microbial Culture Collection Management Committee, deposit number 12750, preservation date is July 11, 2016)) , white or light yellow, opaque, indicating rough, wrinkled, irregular edges.
  • the growth process of Z12 is aerobic, the optimum pH is 7.0-8.5, the optimum temperature is 30-45 °C, and the Gram stain is positive.
  • the method for cultivating Bacillus subtilis comprises the following steps:
  • Collecting wild type strains collecting soil containing Bacillus subtilis, adding LB medium according to the ratio of soil to medium: 1:100, and incubating at 37 ° C, 200 rpm for 24 hours to obtain bacteria rich in spores The suspension is obtained to obtain the original strain.
  • the soil can be collected in the original forest area, where it is rich in degrading bacteria.
  • Design primers to amplify the 602 bp fragment wprA-F of the wprA gene of Bacillus subtilis Z12 strain design primers to amplify the 561 bp fragment wprA-R downstream of wprA; the whole gene synthesis xylR promoter and the promoter region fragment of CDS and xylAB xylR; whole gene synthesis T7 RNA polymerase fragment T7RP; vector pBTS-T7RP was constructed by homologous recombination.
  • the pBTS-aprE was transformed into the ZT7RP strain by the hypertonic transformation method of Example 2 to obtain the ZT7RP-pBTS-aprE strain.
  • the whole gene synthesis green fluorescent protein (GFP) gene was inserted into the pBTS-FR vector by homologous recombination and constructed into the vector pBTS-FR-GFP.
  • Z15-GFP-45 can also be used for induction, its expression The amount is higher than Z15-GFP).
  • the sample in 10 was subjected to PAGE electrophoresis using conventional SDA-PAGE electrophoresis with a gel concentration of 12%.
  • Lane 1 is Marker. Lanes 2-8 are added with 0, 0.1 ⁇ , 0.2 ⁇ , 0.5 ⁇ , 1 ⁇ , 2 ⁇ , 5 ⁇ xylose for induction. Lanes 9 and 10 are Z15 strain, adding 0 and 2 ⁇ xylose respectively; a picture is the bacterial protein electrophoresis map, b picture is the bacterial liquid protein electrophoresis map; only Z15-GFP strain induced by adding xylose, intracellular accumulation of GFP protein amount greater than 2 Above 5%, GFP protein can be secreted into the fermentation broth, and as the xylose concentration is increased, the amount of GFP expressed is gradually increased, and the amount of GFP protein secreted into the fermentation broth is gradually increased; comparing all samples, It was found that the amount of GFP protein secreted into the fermentation broth was equal to or greater than the amount of protein remaining in the cell, and the expression and secretion amount reached 50% or more.
  • Reference implementation 7 gene synthesis of rhaM, ibpB, ribC, TEVP, groL, lacZ genes, construction of pBTS-FR-rhaM, pBTS-FR-ibpB, pBTS-FR-ribC, pBTS-FR-TEVP, pBTS- FR-groL, pBTS-FR-lacZ vector, transformed into Z15 strain, and completed the first recombination to construct expression strains Z15-rhaM-45, Z15-ibpB-45, Z15-ribC-45, Z15-TEVP-45, Z15-groL-45, Z15-lacZ-45.
  • PAGE electrophoresis was performed on the samples in 3, using gradient SDA-PAGE electrophoresis, and the gel concentration was 10%-15%.
  • Lane 1 is Marker
  • Lane 2 is RhaM
  • Lane 3 is IbpB
  • Lane 4 is RibC
  • Lane 5 is GFP
  • Lane 6 is TEVP
  • Lane 7 is GroL
  • Lane 8 For LacZ
  • Lanes 9 and 10 are Z15 strains, adding 0 and 2 ⁇ xylose respectively; a picture is bacterial protein
  • b picture is bacterial liquid protein.
  • RhaM, IbpB, RibC, GFP, GroL proteins can be expressed in a large amount in the cell, and can also be secreted into the fermentation broth in large quantities; however, the TEVP protein cannot be efficiently expressed in the cell, and it is presumed that it is degraded by intracellular proteins, so it cannot be secreted.
  • the LacZ protein may be too large (110 kDa) and cannot be expressed in a large amount in the cell, so it cannot be secreted into the fermentation broth.

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Abstract

一种利用芽孢杆菌高效分泌表达外源蛋白的方法，具体包括以下步骤：(1)构建缺失八个蛋白酶的芽孢杆菌菌株：敲除芽孢杆菌表达菌株的aprE、bpr、epr、mpr、nprB、nprE、wprA和vpr八个蛋白酶基因，避免目的蛋白分泌到发酵液中后，被枯草芽孢表达菌株分泌的蛋白酶降解掉；(2)克隆目的蛋白基因，根据表达系统的要求构建表达载体，并将表达载体转入缺失八个蛋白酶的芽孢杆菌菌株，获得表达目的蛋白的表达菌株；(3)对步骤(2)中的表达菌株进行诱导培养，即得分泌的目的蛋白。当任一目的蛋白在细胞内大量表达，就可以通过这一途径大量分泌到细胞外，最大分泌量可以达到g/L级别。

Description

一种利用芽孢杆菌高效分泌表达外源蛋白的方法 技术领域
本发明属于生物技术领域，具体涉及一种利用芽孢杆菌高效分泌表达外源蛋白的方法。
背景技术
蛋白表达技术是现代生物学的核心技术之一，表达蛋白不仅可用于生物学研究，也可以提供商业化的蛋白制品，如重组疫苗、重组胰岛素、细胞因子等产品。目前常用的表达系统有大肠杆菌、酵母、昆虫细胞和哺乳动物细胞等，但是它们都各有明显的优缺点。大肠杆菌表达系统研究最充分，有多种选择，最常用的是Novagen的pET表达系统，其利用噬菌体的T7 RNA聚合酶可专一性地转录T7启动子后的目的基因，在最佳条件下，目的蛋白可以达到大肠杆菌总蛋白的50％以上。尽管具有表达效率高，培养成本低等优点，但是缺点也非常明显：蛋白容易形成包涵体，复性难度和成本较高；大肠杆菌不能对蛋白进行糖基化修饰；细胞壁含有脂多糖(内毒素)，不易完全去除；胞内蛋白种类多，目的蛋白纯化过程中不易去除各种杂质蛋白。另外一个常用的表达系统是酵母表达系统，其具有表达量高，可诱导，蛋白分泌到胞外易于纯化，并且具有一定的翻译后修饰能力等优点，但是缺点是部分表达产物易降解，表达量不可控，大于30KDa的蛋白几乎不能分泌。动物细胞和昆虫细胞表达系统的特点是具有完整的修饰系统，表达产物具有或者类似的天然活性，没有内毒素污染，但是表达量低，周期长，技术要求高，生产成本高。
枯草芽孢杆菌是一种广泛存在于水体、空气、土壤中的革兰氏阳性菌，其可以在环境不适宜的时候产生孢子，孢子可以抵御高温、干旱等极端环境，待环境适宜时再萌发进行营养生长。枯草芽孢杆菌的分泌能力强，在进行高密度发酵时，蛋白分泌量可以达到20—25g/L(Developments in the use of Bacillus species for industrial production，2004)。其发酵生产的蛋白酶、淀粉酶、次黄嘌呤核苷、核糖甙等产品，早已经进入我们的日常生活。由于其具有生物安全性，被FDA评为GRAS类添加剂，其生产的益生菌及利用其生产的发酵产品已广泛应用于医药和养殖业中。
上世纪八十年代就开始利用枯草芽孢杆菌进行蛋白表达，其表达产物不仅包括细菌来源的各种酶类，如淀粉酶、蛋白酶、内葡聚糖酶、脂肪酶等，还包括hEGF、IFN-alpha 2、Proinsulin、Streptavidin、cathelicidin-BF等不同来源的蛋白。利用枯草芽孢杆菌的分泌途径，可将表达蛋白分泌到发酵液中，极大地降低了后期分离纯化的难度。枯草芽孢杆菌 属于革兰氏阳性菌，不含有脂多糖，便于生产注射用药品。枯草芽孢杆菌的营养要求简单，由于其已经实现规模工业化生产，大规模培养技术成熟，培养成本低。目前，枯草芽孢杆菌中的多个菌株及芽孢杆菌属中的多个种都已经完成了基因组测序工作，遗传背景清楚，安全性高，也便于进行基因组改造。但是枯草芽孢杆菌表达系统并不是一个广泛应用的表达系统，还有许多缺点待克服。
枯草芽孢杆菌的模式菌168菌的野生型菌株已经丢失，目前可以得到的菌株都是其突变体。尽管其满足于科学研究的要求，但是其蛋白分泌能力差，是营养缺陷性突变体的特性，并不能满足建立商业化表达系统的要求。目前建立的转化系统都是基于模式菌株168建立的，尽管野生型来源的菌株和已经生产应用的枯草芽孢杆菌种非常多，不易转化都阻碍了对这些菌株的进一步改造。枯草芽孢杆菌来源的质粒也几乎都是隐蔽性质粒，不易构建载体；来源于其它革兰氏阳性菌的质粒多数都是滚环复制性质粒，在枯草芽孢杆菌中复制不稳定，易丢失，拷贝数较少；这些特性都限制了直接使用质粒构建稳定表达系统的可能性。枯草芽孢杆菌有四种已知的分泌途径，Sec途径、Tat途径、Com系统和ABC转运途径，目前研究较清楚的是Sec途径，多数表达系统也利用Sec途径分泌表达蛋白。但是Sec途径具有内在调控机制，会抑制不正确折叠的蛋白分泌，而外源蛋白一般折叠较慢，折叠过程需要伴侣蛋白的参与，容易被Sec分泌途径降解，这导致只有极少数蛋白可以利用Sec途径进行分泌表达，且外源蛋白的表达量都比较低。枯草芽孢杆菌还会分泌多达八种蛋白酶到发酵液中，其同样会降解表达的目的蛋白。
发明内容
为了解决现有技术中存在的问题，本发明提供一种利用芽孢杆菌高效分泌表达外源蛋白的方法，其不依赖于信号肽或者特征结构，与现有的Sec途径、Tat途径、Com系统和ABC转运途径有本质上差异。当任一目的蛋白在细胞内大量表达，就可以通过这一途径大量分泌到细胞外，最大分泌量可以达到g/L级别。不仅克服了现有分泌途径分泌量低，通用性差的特点，还可以无选择性地将目的蛋白分泌到发酵液中，降低后期分离纯化的难度。
解决以上技术问题的本发明中的一种利用芽孢杆菌高效分泌表达外源蛋白的方法，其特征在于：所述目的蛋白在胞内大量积累，其就被大量分泌到发酵液中。
所述目的蛋白在胞内表达量超过总蛋白的2％以上时，其将会被分泌到发酵液中，分泌量在占表达蛋白的50％以上。
本发明中一种利用芽孢杆菌高效分泌表达外源蛋白的方法，其特征在于：具体包括以下步骤：
(1)构建缺失八个蛋白酶的芽孢杆菌菌种：敲除芽孢杆菌表达菌株的aprE、bpr、epr、mpr、nprB、nprE、wprA和vpr八个蛋白酶基因，避免目的蛋白分泌到发酵液中后，被枯草芽孢表达菌株分泌的蛋白酶降解掉；
这八个蛋白酶为表达分泌蛋白酶到发酵液中的基因，去除后能改善发酵液中的蛋白降解情况。
(2)克隆目的蛋白基因，根据表达系统的要求构建表达载体，并将表达载体转入缺失八个蛋白酶的芽孢杆菌菌株，获得表达目的蛋白的表达菌株；
(3)对步骤(2)中的表达菌株进行诱导培养，即得分泌的目的蛋白。
本发明通过表达系统胞内大量表达目的蛋白，如果目的蛋白在胞内大量积累，其就可以被大量分泌到发酵液中，且任一可以胞内大量表达目的蛋白的表达系统都能用于蛋白分泌表达。
所述表达系统为使目的蛋白在胞内积累被大量分泌到发酵液中的表达系统。
本发明所采用的表达系统为“无抗表达系统”，其克服了抗生素筛选标记基因易发生漂移，质粒表达系统不稳定，表达不可诱导表达量不高的缺点，为一种低背景可控诱导且高效表达的无抗表达系统。
所述“无抗表达系统”的菌株构建过程，包括采用xylR基因、xylAB的启动子区域和T7 RNA聚合酶基因的CDS片段替换Z12菌株的wprA蛋白酶基因，具体包括以下步骤：
(1)PCR扩增wprA基因上游片段wprA-F和wprA基因下游片段wprA-R，全基因合成xylR操纵子的xylR基因启动子及CDS区域和xylAB的启动子区域，全基因合成T7 RNA聚合酶基因的CDS片段，通过同源重组构建载体pBTS-T7RP；
(2)转化pBTS-T7RP质粒进入Z12菌株，通过限制性培养和传代培养，分别在基因组的wprA-F和wprA-R两个片段处完成两次重组，得到不具有抗性的含有xylR-T7RP片段的菌株ZT7RP。
本发明所采用的“无抗表达系统”的表达菌株ZT7RP，在菌株构建过程中已经敲除了wprA蛋白酶基因。
本发明中构建缺失八个蛋白酶的芽孢杆菌菌株的方法，即分别构建pBTS-aprE、pBTS-bpr、pBTS-epr、pBTS-mpr、pBTS-nprB、pBTS-nprE和pBTS-vpr载体，逐次敲除ZT7RP菌株中的aprE、bpr、epr、mpr、nprB、nprE和vpr基因，获得新菌株Z15；
具体包括以下步骤：
(1)PCR扩增待敲除基因X上游500-800bp的片段X-F，插入pBTS载体中，得到质粒pBTS-X-F；扩增待敲除基因X下游500-800bp的片段X-R，插入pBTS-X-F载体中X-F片段的下游，得到质粒pBTS-X，其中，X代表为wprA、aprE、bpr、epr、mpr、nprE、nprB、vpr；
(2)转化pBTS-X质粒进入菌株，通过限制性培养和传代培养，分别在基因组的X-F和X-R两个片段处完成两次重组，得到敲除基因X的菌株；
重复以上步骤，直到菌株中aprE、bpr、epr、mpr、nprB、nprE和vpr七个蛋白酶基因均被敲除。
本发明所采用的“无抗表达系统”的目的基因胞内表达方法，包括构建表达质粒pBTS-FR、合成目的基因片段X、构建表达载体pBTS-FR-X以及通过同源重组把目的基因插入到xylAB基因区域，具体包括以下过程：
(1)PCR扩增xylAB基因上游片段xyl-F和xylAB基因下游片段xyl-R，全基因合成T7启动子、xylR结合位点、RBS位点、多克隆位点和T7终止子片段，通过同源重组，构建质粒pBTS-FR；
(2)通过PCR扩增或者全基因合成任一需要表达的基因片段，通过同源重组方式插入到pBTS-FR的多克隆位点，构建表达载体pBTS-FR-X；
(3)转化pBTS-FR-X质粒进入Z15菌株，通过限制性培养和传代培养，在xyl-F和xyl-R中任一一处发生一次重组，得到菌株Z15-X-45；或者分别在基因组的xyl-F和xyl-R两个片段处完成两次重组，得到不具有抗性的含有X片段的菌株Z15-X；
(4)接种Z15-X到培养基中，加入0.01‰-5‰浓度的木糖进行诱导，培养时间>＝20h后，即得菌体或者发酵液中的目标蛋白。培养温度为25-40℃，接种后0-8h加入木糖进行诱导。
所述pBTS核苷酸序列如SEQ ID NO.1所示。
所述pBTS-T7RP核苷酸序列如SEQ ID NO.2所示。
所述pBTS-FR核苷酸序列如SEQ ID NO.3所示。
所述芽孢杆菌为芽孢杆菌为枯草芽孢杆菌168、Z12菌株、解淀粉芽孢杆菌、短小芽孢杆菌、地衣芽孢杆菌和其它芽孢杆菌。
所述枯草芽孢杆菌Z12，拉丁文Bacillus subtilis Z12，保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏号为12750。
本发明的有益效果如下：
A、该分泌途径不需要信号肽、特征结构等用于转运识别的结构区域，分泌产生的目的蛋白不含有多余的蛋白序列。
B、该分泌途径不具有选择性，任一可以在胞内大量表达的蛋白都可以分泌到发酵液中，显著低降低后续目的蛋白的分离纯化流程和成本。
C、具有分泌量大的特点，可以达到g/L级别。
本发明结合芽孢杆菌，尤其是枯草芽孢杆菌，已被FDA认定为GRAS类添加剂，发酵周期短，生产成本低的特点，利用本发明中分泌途径可以规模化生产蛋白多肽类生物技术产品，改变大众的生活方式。
附图说明
下面结合附图及具体实施方式对本发明做更进一步详细说明：
图一不同浓度木糖诱导GFP蛋白分泌表达PAGE电泳图
图二不同蛋白的分泌表达PAGE电泳图
具体实施方式
下面结合附图，对本发明作详细的说明：
实施例1
1、通过EcoR I和Apa I内切酶切割pBAV1K-T5-GFP(http://www.addgene.org/vector-database/)质粒，与合成的MCS片段进行同源重组克隆，得到质粒pBAV1K。
本实验及后续试验中的内切酶采用Thermo公司的快速内切酶，片段回收采用成都福际生物公司的胶回收试剂盒(DE-02011)，MCS片段由金唯智生物科技有限公司合成，同源重组采用天根的EsayGeno快速重组克隆试剂盒(VI201-02)，大肠杆菌菌株为top10，制备感受态采用KCM法，质粒抽提采用福际生物的通用质粒小量提取试剂盒(DE-01001)。
2、设计引物，扩增同义突变删除Nde I酶切位点的pBAV1K片段，采用同源重组克隆的方式连接扩增片段，获得质粒命名为pBTS。
本实验及后续实验中的引物由金唯智生物科技有限公司合成。
实施例2
1、菌株转化方法参见高渗转化法(High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis，1999)。取待转化菌株的新划线单菌落，接种到约3ml GM(LB+0.5M山梨糖醇)培养基中，37℃，180rpm过夜培养。第二天早上按1:100接种到50ml GM培养基中，37℃，180rpm培养，待菌液生长到OD达到0.85-0.95之间时，将菌液放到冰上预冷10min；4℃，5000g，5min，离心去上清，用等体积预冷的EM(0.5M山梨糖醇+0.5M甘露糖醇+10％甘油水溶液)重悬菌体，再次4℃，5000g，5min，离心去上清，一共重复洗涤4次；加入约1/40体积的EM重悬菌体，保证菌液浓度在1-1.3×1010cfu/ml之间。取60ul上述菌液，加入1ul待转化质粒，质粒浓度大于100ng/ul，吹打混匀，然后加入预冷的1mm电击杯。电转化仪(EppendorfEporator)参数设定，2.1kV，实际电击时间在4.0-5.0ms时，才可能有转化菌落生长。电击后，立即加1ml RM(LB+0.5M山梨糖醇+0.38M甘露糖醇)，吹打混匀，转入5ml无菌离心管中，37℃，180rpm培养3h。将菌液离心，去上清后按一定比例稀释，取200ul涂布到含有30mg/L卡那霉素的LB平板上，37℃过夜培养，同时涂布未经电转化的菌液做阴性对照。第二天早上观察菌落生长情况，若转化板有菌落生长，阴性对照没有，则表明转化成功。
实施例3
枯草芽孢杆菌Z12(((拉丁文为Bacillus subtilis Z12)，保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号为12750，保藏日期为2016年7月11日))菌落圆形，呈白色或淡黄色，不透明，表明粗糙，有褶皱，边缘不整齐。Z12生长过程需氧，生长最适pH7.0-8.5，最适温度30-45℃，革兰氏染色呈阳性。
枯草芽孢杆菌的培育方法，包括以下步骤：
(1)采集野生型菌种：采集含有枯草芽孢杆菌的土壤，按土壤与培养基的用量比例为1：100加入LB培养基，37℃，200rpm，培养24h，得到富含芽孢杆菌孢子的菌悬液，得到原始菌种。
土壤可在原始林区，富含降解菌的地方进行采集。
(2)菌株富集与筛选：取1ml菌液，85℃加热处理30min，杀死所有菌体；然后按103和105倍稀释涂板，LB培养基，37℃过夜培养；对获得的单菌落再次划线，进行纯度鉴定。纯度鉴定是划线培养，所有的单菌落形态一致。
(3)菌株转化筛选：若需要检测转化效率，则统计生长菌落总数，并乘以稀释倍数，通过质粒添加量，计算转化效率。
菌株转化方法参见高渗转化法和电转化法,如实施例2中的内容。
实施例4
1、设计引物扩增枯草芽孢杆菌Z12菌株的wprA基因上游602bp的片段wprA-F；设计引物扩增wprA下游601bp的片段wprA-R；全基因合成xylR启动子以及CDS和xylAB的启动子区域片段xylR；全基因合成T7 RNA聚合酶片段T7RP；通过同源重组克隆构建载体pBTS-T7RP。
2、电转化pBTS-T7RP进入Z12菌株，得到菌Z12-pBTS-T7RP。
3、取菌Z12-pBTS-T7RP接种到3ml LB培养基中，45℃，180rpm，培养24h；另接菌Z12-pBTS做对照。
4、取3中的菌液200ul涂布到含有30mg/L卡那霉素的LB平板上，45℃过夜培养。若Z12-pBTS没有菌落生长，Z12-pBTS-T7RP有菌落生长，则得到的菌株完成第一次重组，命名为Z12-pBTS-T7RP-45。
5、接菌Z12-pBTS-T7RP-45到3ml LB培养基中，过夜培养，抽提基因组，PCR扩增xylR-T7RP片段进行验证。
6、取5中验证为阳性的Z12-pBTS-T7RP-45菌株，接种到3ml LB基中，37℃，180rpm培养，每隔8-16h传代一次，连续传代10次后，取菌液稀释106倍，涂布于无抗的LB平板上，得到单菌落。
7、取6中的单菌落，同时划线到LB和含有30mg/L卡那霉素的LB平板上，筛选无抗的菌落，需要得到5-10株无抗菌落。
8、取7中的无抗菌落，接种到3ml LB培养基中，37℃，180rpm过夜培养，抽提细菌基因组，通过PCR扩增xylR-T7RP片段验证同源重组结果(野生型或者插入T7RP片段)。 取阳性的片段，送金唯智生物科技有限公司进一步进行测序验证，得到阳性的菌株即为插入xylR-T7RP片段的菌株——ZT7RP(wprA::(xylR-PxylAB rpoT7))。
实施例5
1、设计引物扩增枯草芽孢杆菌Z12菌株的aprE操纵子上游561bp的片段aprE-F，插入pBTS的EcoR I和BamHI位点间，得到质粒pBTS-aprE-F；扩增下游486bp的片段aprE-R，插入质粒pBTS-aprE-F的BamHI和Hind III之间，得到质粒pBTS-aprE质粒(敲除aprE基因)。
2、采用实施例2中的高渗转化法转化pBTS-aprE进入ZT7RP菌株，获得ZT7RP-pBTS-aprE菌株。
3、取菌ZT7RP-pBTS-aprE接种到3ml LB培养基中，45℃，180rpm，培养24h；另接菌ZT7RP-pBTS做对照。
4、取3中的菌液200ul涂布到含有30mg/L卡那霉素的LB平板上，45℃过夜培养。若ZT7RP-pBTS没有菌落生长，ZT7RP-pBTS-aprE有菌落生长，则得到的菌株完成第一次重组，命名为ZT7RP-pBTS-aprE-45。
5、接菌ZT7RP-pBTS-aprE-45到3ml LB培养基中，过夜培养，抽提基因组(成都福际生物技术有限公司，细菌基因组DNA提取试剂盒，DE-05311)，PCR扩增aprE片段进行验证(成都福际生物技术有限公司，2×快速PCR反应预混体系，DP-20041)。引物序列和条件？
6、取5中验证为阳性的ZT7RP-pBTS-aprE-45菌株，接种到3ml LB基中，37℃，180rpm培养，每隔8-16h传代一次，连续传代8次后，取菌液稀释106倍，涂布于无抗的LB平板上，得到单菌落。
7、取6中的单菌落，同时划线到LB和含有30mg/L卡那霉素的LB平板上，筛选无抗的菌落，需要得到5-10株无抗菌落。
8、取7中的无抗菌落，接种到3ml LB培养基中，37℃，180rpm过夜培养，抽提细菌基因组，通过PCR扩增aprE片段验证同源重组结果(野生型或者敲除aprE基因)。取阳性的片段，送金唯智生物科技有限公司进一步进行测序验证，得到阳性的菌株即为敲除aprE基因的菌株(△(aprE)，wprA::(xylR-PxylAB rpoT7))。
实施例6
1、参比实施例5，分别构建pBTS-bpr、pBTS-epr、pBTS-mpr、pBTS-nprB、pBTS-nprE、pBTS-vpr载体，逐次敲除ZT7RP菌株(△(aprE)，wprA::(xylR-PxylAB rpoT7))中的bpr、 epr、mpr、nprB、nprE和vpr基因，获得新菌株Z15(△(aprE),△(bpr),△(epr),△(mpr),△(nprB),△(nprE),△(vpr)，wprA::(xylR-PxylAB rpoT7))。
实施例7
1、设计引物扩增xylAB基因上游493bp片段xyl-F；设计引物扩增xylAB基因下游585bp片段xyl-R；全基因合成T7启动子、xylR结合位点、RBS位点、多克隆位点、T7终止子位点片段；通过同源重组克隆上述三个片段与pBTS载体连接，构建质粒pBTS-FR。
实施例8
1、全基因合成绿色荧光蛋白(GFP)基因，通过同源重组克隆插入到pBTS-FR载体中，构建成载体pBTS-FR-GFP。
2、电转化pBTS-FR-GFP进入Z15菌株，得到菌Z15-pBTS-FR-GFP。
3、取菌Z15-pBTS-FR-GFP接种到3ml LB培养基中，45℃，180rpm，培养24h；另接菌Z15-pBTS做对照。
4、取3中的菌液200ul涂布到含有30mg/L卡那霉素的LB平板上，45℃过夜培养。若Z15-pBTS没有菌落生长，Z15-pBTS-FR-GFP有菌落生长，则得到的菌株完成第一次重组，命名为Z15-GFP-45(Z15-GFP-45也可以用于诱导，其表达量高于Z15-GFP)。
5、接菌Z15-GFP-45到3ml LB培养基中，过夜培养，抽提基因组，PCR扩增GFP片段进行验证。
6、取5中验证为阳性的Z15-GFP-45菌株，接种到3ml LB基中，37℃，180rpm培养，每隔8-16h传代一次，连续传代10次后，取菌液稀释106倍，涂布于无抗的LB平板上，得到单菌落。
7、取6中的单菌落，同时划线到LB和含有30mg/L卡那霉素的LB平板上，筛选无抗的菌落，需要得到5-10株无抗菌落。
8、取7中的无抗菌落，接种到3ml LB培养基中，37℃，180rpm过夜培养，抽提细菌基因组，通过PCR扩增GFP片段验证同源重组结果(是否插入GFP片段)。取阳性的片段，送金唯智生物科技有限公司进一步进行测序验证，得到阳性的菌株即为插入GFP片段的菌株——Z15-GFP。
9、接菌Z15-GFP到40ml LB培养基中，添加不同浓度的木糖进行诱导(0、0.1‰、0.2‰、0.5‰、1‰、2‰、5‰)，另接种Z15到40ml LB中，添加0和2‰的木糖作对照。37℃，200rpm，培养24h。
10、取各种菌液1ml，12000g离心一分钟；取上清100ul，加入等体积2×Loading Buffer，95℃处理5min，作为菌液蛋白样品进行PAGE电泳；沉淀采用600ul Buffer A (50mM Tris，2mM EDTA，100mM NaCl，1％Triton-100)重悬，加入1ul 100mg/L的溶菌酶，37℃处理5min裂解细胞壁；取裂解液100ul，加入等体积的2×Loading Buffer，95℃处理5min，作为菌体蛋白样品进行PAGE电泳。
11、对10中的样品进行PAGE电泳，采用常规SDA-PAGE电泳，胶浓度为12％。
结果如图一，1号泳道为Marker，2-8号泳道分别添加0、0.1‰、0.2‰、0.5‰、1‰、2‰、5‰的木糖进行诱导，9号和10号泳道为Z15菌株，分别添加0和2‰的木糖；a图是菌体蛋白电泳图，b图是菌液蛋白电泳图；只有Z15-GFP菌株在添加木糖诱导，胞内积累GFP蛋白量大于2％以上时，GFP蛋白才可以分泌到发酵液中，并且随着诱导木糖浓度升高，表达的GFP量逐渐增加，分泌到发酵液中的GFP蛋白量也逐渐增加；对所有样品进行比较，发现分泌到发酵液中的GFP蛋白量等于或者大于胞内残留的蛋白量，表达分泌量达到50％以上。
实施例9
1、参比实施7，全基因合成rhaM、ibpB、ribC、TEVP、groL、lacZ基因，构建pBTS-FR-rhaM、pBTS-FR-ibpB、pBTS-FR-ribC、pBTS-FR-TEVP、pBTS-FR-groL、pBTS-FR-lacZ载体，转化进入Z15菌株，并完成第一次重组，构建表达菌株Z15-rhaM-45、Z15-ibpB-45、Z15-ribC-45、Z15-TEVP-45、Z15-groL-45、Z15-lacZ-45。
2、接菌Z15-rhaM-45、Z15-ibpB-45、Z15-ribC-45、Z15-GFP-45、Z15-TEVP-45、Z15-groL-45、Z15-lacZ-45到40ml LB中，添加2‰的木糖进行诱导，另接种Z15到40ml LB中，添加0和2‰的木糖作对照。37℃，200rpm，培养24h。
3、取各种菌液菌液1ml，12000g离心一分钟；取上清100ul，加入等体积2×Loading Buffer，95℃处理5min，作为菌液蛋白样品进行PAGE电泳；沉淀采用600ul Buffer A(50mM tris，2mM EDTA，100mM NaCl，1％Triton-100)重悬，加入1ul 100mg/L的溶菌酶，37℃处理5min裂解细胞壁；取处理液100ul，加入等体积的2×Loading Buffer，95℃处理5min，作为菌体蛋白样品进行PAGE电泳。
4、对3中的样品进行PAGE电泳，采用梯度SDA-PAGE电泳，胶浓度为10％-15％。
结果如图二，1号泳道为Marker，2号泳道为RhaM，3号泳道为IbpB，4号泳道为RibC，5号泳道为GFP，6号泳道为TEVP，7号泳道为GroL，8号泳道为LacZ，9号和10号泳道为Z15菌株，分别添加0和2‰的木糖；a图为菌体蛋白，b图为菌液蛋白。RhaM、IbpB、RibC、GFP、GroL蛋白都可以在胞内大量表达，同时也可以大量分泌到发酵液中；但是TEVP蛋白不能在胞内有效表达，推测其被胞内蛋白降解，故也不能分泌到发酵液中；LacZ蛋白可能太大(110kDa)，亦不能在胞内大量表达，故也不能分泌到发酵液中。

Claims (5)

  1. 一种利用芽孢杆菌高效分泌表达外源蛋白的方法，其特征在于：所述目的蛋白在胞内大量积累，其就被大量分泌到发酵液中。
  2. 根据权利要求1所述的一种利用芽孢杆菌高效分泌表达外源蛋白的方法，其特征在于：所述目的蛋白在胞内表达量超过总蛋白的2％以上时，其将会被分泌到发酵液中，分泌量占表达蛋白的50％以上。
  3. 根据权利要求1或2所述的一种利用芽孢杆菌高效分泌表达外源蛋白的方法，其特征在于：具体包括以下步骤：
    (1)构建缺失八个蛋白酶的芽孢杆菌菌株：敲除芽孢杆菌表达菌株的aprE、bpr、epr、mpr、nprB、nprE、wprA和vpr八个蛋白酶基因，避免目的蛋白分泌到发酵液中后，被枯草芽孢表达菌株分泌的蛋白酶降解掉；
    (2)克隆目的蛋白基因，根据表达系统的要求构建表达载体，并将表达载体转入缺失八个蛋白酶的芽孢杆菌菌株，获得表达目的蛋白的表达菌株；
    (3)对步骤(2)中的表达菌株进行诱导培养，即得分泌的目的蛋白。
  4. 根据权利要求3所述的一种利用芽孢杆菌高效分泌表达外源蛋白的方法，其特征在于：所述表达系统为使目的蛋白在胞内积累被大量分泌到发酵液中的表达系统。
  5. 根据权利要求1所述的一种利用芽孢杆菌高效分泌表达外源蛋白的方法，其特征在于：所述芽孢杆菌为枯草芽孢杆菌168、Z12菌株、解淀粉芽孢杆菌、短小芽孢杆菌、地衣芽孢杆菌和其它芽孢杆菌；所述枯草芽孢杆菌Z12，拉丁文Bacillus subtilis Z12，保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏号为12750。
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CN104073458A (zh) * 2013-03-26 2014-10-01 南京金斯瑞生物科技有限公司 一株可高效表达外源分泌蛋白酶的枯草芽孢杆菌
CN104630123A (zh) * 2013-11-12 2015-05-20 华中农业大学 地衣芽胞杆菌表达宿主

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CN104630123A (zh) * 2013-11-12 2015-05-20 华中农业大学 地衣芽胞杆菌表达宿主

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CN113957028B (zh) * 2021-10-25 2023-08-11 江南大学 一种胞外蛋白酶失活的枯草芽孢杆菌及其构建方法与应用

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