WO2021128766A1 - 一种具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体及其构建方法 - Google Patents

一种具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体及其构建方法 Download PDF

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WO2021128766A1
WO2021128766A1 PCT/CN2020/096714 CN2020096714W WO2021128766A1 WO 2021128766 A1 WO2021128766 A1 WO 2021128766A1 CN 2020096714 W CN2020096714 W CN 2020096714W WO 2021128766 A1 WO2021128766 A1 WO 2021128766A1
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dna fragment
plasmid
expression vector
dna
corynebacterium
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French (fr)
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陈瑞爱
黄梅
李延鹏
刘定祥
闫圆圆
方倪冉
杨小云
董楠
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华农（肇庆）生物产业技术研究院有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • 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
    • C12N15/67—General methods for enhancing the expression
    • C12N15/69—Increasing the copy number of the vector

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  • the invention belongs to the field of biotechnology, and specifically relates to a corynebacterium and E. coli dual expression vector with high copy ability and a construction method thereof.
  • genetic engineering vaccines have the advantages of simpler, easier quality control, and easier storage and transportation.
  • nucleic acid vaccines including DNA and RNA vaccines
  • subunit vaccines containing specific antigen proteins Both of them induce host cells to produce an immune response to the antigen protein through the antigen protein, so as to achieve the purpose of preventing and treating diseases.
  • genetic engineering vaccines can be prepared by biosynthesis, such as direct synthesis of polypeptides (subunit vaccines) or synthesis of DNA sequences that can be expressed in host cells (DNA vaccines), the cost of both biosynthetic peptides and synthetic nucleic acids is very high.
  • the production methods of protein subunit vaccines mainly include the preparation of expression plasmids and protein expression and purification.
  • the preparation process of antigen proteins includes transformation of plasmids into prokaryotic cells, mainly E.
  • the antigen protein is transcribed and translated to produce the antigen protein as the raw material of the protein subunit vaccine.
  • the DNA vaccine production process includes cloning the antigen gene into a vector that can be expressed in eukaryotes to obtain a plasmid expressing the antigen gene, and then transforming the plasmid into Escherichia coli, allowing the plasmid to be amplified in large quantities in the cheap factory of bacteria, and then The plasmid DNA is extracted from the bacterial factory through a series of steps, and the DNA vaccine is introduced into the host by intramuscular injection or microprojectile bombardment, and the antigen gene carried by the vaccine is expressed into antigen protein through the host cell.
  • the advantage is that the antigen protein is synthesized in the host body, can be correctly folded and modified, and has strong antigenicity. Since the main component of the vaccine is DNA, it is easier to store and transport. However, since the foreign DNA that enters the host cell cannot replicate in the host cell, the amount of DNA that can enter the host cell is another key factor that affects the efficacy of the vaccine in addition to the titer of the antigen itself.
  • both plasmids used directly as DNA vaccines or plasmids used for the production of antigen proteins are plasmids with E. coli replicons. Such plasmids can only replicate in E. coli. Because E. coli is a Gram-negative bacteria, it contains a large amount of Endotoxin, the most serious problem in preparing plasmids from this strain is endotoxin contamination. Because endotoxin is an important pyrogen and can produce serious toxic and side effects, the State Food and Drug Administration has strict requirements on it. In addition, the current host cells used to express antigen proteins are mainly Escherichia coli.
  • the most direct method is to avoid using E. coli to produce plasmids or express proteins.
  • the commercialized plasmids are basically all plasmids that can only replicate in E. coli.
  • Corynebacterium is more and more favored by scientists because it does not produce endotoxin.
  • Corynebacterium glutamicum has not only been widely used in microbial fermentation engineering to produce glutamic acid (MSG), but in recent years, people have also begun to explore and develop it in protein Production applications have received more and more attention.
  • MSG glutamic acid
  • the research on the biological metabolism and bacterial replication of coryneform bacteria is far less in-depth and comprehensive than that of E. coli. Therefore, commercial vectors that can replicate in corynebacteria are very rare on the market, and their copying ability is in the low-medium range. The copying ability directly leads to low output and push up costs, making it difficult to use for mass production.
  • the construction method is: The leaf base pyrophosphate synthase gene ERG20F96W-N127W and the valerian-derived geraniol synthase gene tVoGES with a truncated C-terminal 53 amino acid residues were linked by fusion PCR, and the ribosome binding site sequence was added to the homologous region. Insert the SacI and XbaI restriction sites of the Corynebacterium glutamicum expression plasmid pEC-XK99E to obtain plasmid 1.
  • Plasmid 1 is transformed into Corynebacterium glutamicum to obtain Corynebacterium glutamicum 1 that synthesizes geraniol; the invention is The synthesis of geraniol provides more precursors, and the fermentation time is shorter than 42-48 hours. After the temperament test, no corresponding by-products are detected.
  • the expression vector is transformed into a glutamate rod.
  • the 5-aminolevulinic acid production strain obtained by the bacterium and the fermentation method of the strain is used to produce 5-aminolevulinic acid.
  • the expression vector constructed with the promoter obtained by this invention realizes the controllable expression of genes by inositol induction, which overcomes the current inducer isopropyl- ⁇ -D-thiogalactoside (Isopropyl ⁇ -D-Thiogalactoside, IPTG).
  • the promoter is integrated into the host genome or the expression vector is transformed into the host Corynebacterium glutamicum to construct a 5-aminolevulinic acid producing strain, and its 5-aminolevulinic acid production reaches 24.2g/L.
  • the above two invention patent applications are both obtained by cutting the plasmid pXMJ19 and inserting the target gene to obtain a recombinant plasmid, and introducing the recombinant plasmid into Corynebacterium glutamicum for the production of geraniol and 5-aminolevulinic acid, respectively. They are all applications in the field of microbial fermentation engineering mentioned in the above background technology, and do not involve the direct synthesis of polypeptides (subunit vaccines) or the synthesis of DNA sequences that can be expressed in host cells (DNA vaccines) mentioned in the present invention. Field of application.
  • the pXMJ19 plasmid is a plasmid with medium to low copy capacity (approximately 20).
  • the technical problem to be solved by the present invention is: how to construct a DNA replication vector with high copy ability in coryneform bacteria.
  • the purpose of the present invention is to provide a Corynebacterium and Escherichia coli dual expression vector with high copying ability and a construction method thereof, by artificially mutating the nucleotide C at position 1786 in the corynebacterium replicon regulatory part of the pXMJ19 vector , Successfully obtained the Corynebacterium/E. coli dual-use vector pXMJ19C1786T plasmid with 12.5 times higher copy capacity than pXMJ19 plasmid. Compared with pXMJ19 plasmid, the copy number of pXMJ19C1786T plasmid increased from 20 to 251.
  • a dual expression vector of Corynebacterium and Escherichia coli with high copy ability is shown in SEQ ID NO: 9.
  • the present invention also discloses a construction method of the expression vector as described above, which includes the following steps:
  • Step 1 Using plasmid pXMJ19 as a template, primers were designed to mutate the nucleotide C at position 1786 of plasmid pXMJ19 into a DNA fragment 1 with a length of 2283 bp and a DNA fragment 2 with a length of 4348 bp through PCR amplification;
  • the primers are C1786T-F, C1786T-R, V-C1786T-F, V-C1786T-R, and their nucleotide sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 4; the core of the DNA fragment 1
  • the nucleotide sequence of the nucleotide sequence is shown in SEQ ID NO: 10
  • the nucleotide sequence of the DNA fragment 2 is shown in SEQ ID NO: 11;
  • DNA fragment 2 (4348bp, SEQ ID NO: 11) sequence:
  • Step 2 Perform homologous recombination of the two DNA fragments obtained in Step 1 to obtain an expression vector pXMJ19C1786T.
  • step 1 In the above-mentioned method for constructing an expression vector, in the step 1:
  • the PCR amplification method of DNA fragment 1 and DNA fragment 2 is:
  • the homologous recombination system is: the amount of DNA fragment 1 added is the number of bases of DNA fragment 1 ⁇ 0.02 ng, and the amount of DNA fragment 2 added is that of DNA fragment 2. Base number ⁇ 0.02ng, recombinase 2uL, buffer 4uL, add nuclease-free sterile water to 20uL.
  • the present invention successfully obtains the Corynebacterium/E. coli dual-use vector pXMJ19C1786T which is 12.5 times higher than the pXMJ19 plasmid by artificially mutating the nucleotide C at position 1786 in the coryneform replicon regulatory part of the pXMJ19 vector .
  • the copy number of the pXMJ19C1786T plasmid has been increased from 20 to 251, and the mutant pXMJ19C1786T has better stability.
  • the expression plasmids prepared by the present invention can be applied to the expression of subunit antigens respectively.
  • the user can choose which plasmid to use according to the toxicity of the expressed specific antigen to the coryneform bacteria; in general, the specific antigen protein is to the host bacteria
  • Non-toxic, high-replicating vectors can be used to increase the expression yield of antigen proteins, and for those that are more toxic to host bacteria, plasmids with lower replication capacity are used to reduce the damage to host bacteria, because these proteins are in It is produced in coryneform bacteria so that endotoxin contamination can be completely avoided during the purification process, and the cost can be reasonably reduced and the quality of the product can be improved.
  • the Corynebacterium replicon with high replication ability in the expression plasmid prepared by the present invention can replace the E. coli replicon in the commonly used eukaryotic cell expression vector, and a high replication ability can be obtained that can be expressed in eukaryotic cells. Plasmids. These plasmids can be used to produce DNA vaccines. Because these plasmids replicate in coryneform bacteria, they can completely avoid endotoxin contamination.
  • Figure 1 is a diagram of the plasmid structure of pXMJ19
  • Figure 2 is an electrophoresis diagram of the product after PCR amplification in Example 1, wherein the left L1 and the right L1 are respectively derived from two fragments generated after PCR amplification;
  • Figure 3 is an electrophoresis diagram of the double digestion product of the expression plasmid prepared in Example 1;
  • Figure 4 is a structural diagram of the expression plasmid pXMJ19C1786T prepared in Example 1;
  • Figure 5 is a diagram of the sequencing verification result of the expression plasmid prepared in Example 1;
  • Figure 6 is an electrophoresis diagram of PCR amplified products in the copy ability test, where L1, L2 are from pXMJ19, and L3, L4 are from pXMJ19C1786T;
  • Fig. 7 is a histogram of the qPCR results using software Grappd prism 5 in the copy ability test;
  • Figure 8 is the electrophoresis diagram of the plasmid digestion product in the stability test, where L1 and L3 represent the plasmid pXMJ19; L2 and L4 represent the plasmid pXMJ19C1786T; L1 ⁇ L2 are the products of MluI digestion; L3 ⁇ L4 are the products of BsmB1 digestion ⁇ The product.
  • a method for constructing a dual expression vector of Corynebacterium and Escherichia coli with high copy ability including the following steps:
  • the nucleotide sequence of the primer is as follows:
  • V-C1786T-F tgcgagctactaactcatatgcacg (SEQ ID NO: 3)
  • V-C1786T-R gaattcgagctcggtacccgg (SEQ ID NO: 4)
  • the PCR reaction conditions are:
  • the electrophoresis diagram of the PCR amplified product is shown in Figure 2, where the left L1 and The right L1 comes from two fragments generated after PCR amplification. As can be seen from Figure 2, the left L1 fragment is 2283 bp; the right L1 fragment is 4348 bp.
  • the plasmid prepared in Example 1 was sent to biosequencing for verification. The results are shown in Figure 5, which proves that the plasmid prepared in Example 1 has a mutation of the nucleotide C at position 1786 to T.
  • Plasmid pXMJ19 or pXMJ19C1786T plasmid pXMJ19 or pXMJ19C1786T
  • Competent Cell Recovery Medium LBHIS is as follows: (g/L)
  • Yeast powder 2.5 tryptone 5.0, sodium chloride 5.0, brain heart infusion 18.5, sorbitol 90.0, pH 6.8-7.0 deionized water dilute to 1L and autoclave at 121°C.
  • TG buffer (1M Tris-HCL, pH 7.5+10% glycerol) pre-cooled on ice, centrifuge at 4000 rpm 4°C for 10 min, and discard the supernatant.
  • BHI medium (g/L) (Huankai Microbial Product No. 024053)
  • Peptone 10.0, dehydrated calf brain extract powder 12.5, dehydrated beef heart extract powder 5.0, sodium chloride 5.0, glucose 2.0, disodium hydrogen phosphate 2.5, PH 7.4 ⁇ 0.2.
  • Yeast powder 5.0, tryptone 10.0, sodium chloride 10.0, glycine 25.0, isoniazid 5.0, Tween80 1mL,
  • step (10) will Put the DNA Mini binding column on a 2mL collection tube, transfer all the liquid obtained in step (9) to the column, centrifuge at 10000 ⁇ g at room temperature for 1 min, discard the collection tube and filtrate;
  • (11) will Put the DNA Mini binding column on a new 2mL collection tube, add 500uL of HBC Buffer, centrifuge at 10000 ⁇ g at room temperature for 1 min, discard the filtrate;
  • primers were selected for fluorescence quantitative PCR to determine the relative replication capacity of plasmids pXMJ19 and pXMJ19C1786T.
  • the nucleotide sequences of the primers are as follows:
  • the amplified PCR product was subjected to agarose gel electrophoresis, the result is shown in Figure 6, the product is 140bp; L1, L2 are from pXMJ19, L3, and L4 are from pXMJ19C1786T.
  • the plasmids pXMJ19 and pXMJ19C1786T were digested and identified with MluI and BsmB1 restriction enzymes, and the size of the product DNA fragments were compared by agarose gel electrophoresis.
  • the electrophoresis diagram of the obtained plasmid digested products is shown in Figure 8, where Among them, L1, L3 represent plasmid pXMJ19; L2, L4 represent plasmid pXMJ19C1786T; L1 ⁇ L2 represent the product after digestion with MluI; L3 ⁇ L4 represent the product after digestion with BsmB1.
  • L1 ⁇ L2 represent the product after digestion with MluI; L3 ⁇ L4 represent the product after digestion with BsmB1.

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Abstract

提供了一种具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体及其构建方法，该表达质粒的核苷酸序列如SEQ ID NO:9所示。该表达质粒是通过在pXMJ19质粒的棒状杆菌复制子调节部位，将第1786位点的核苷酸C进行人工突变而获得的。该表达质粒比pXMJ19质粒拷贝能力高约12.5倍。

Description

一种具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体及其构建方法 技术领域
本发明属于生物技术领域，具体涉及一种具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体及其构建方法。
背景技术
好的基因工程疫苗最大的特点在于它不带任何感染性病毒，因此不会引起由病毒感染带来的毒副作用，从而更加安全可靠。此外基因工程疫苗相对于传统疫苗生产过程来说具有更加简便、质量更易控制、更便于储存与运输等优势。作为一类新兴生物技术,基因工程疫苗的应用正在不断拓展,逐渐成为经济社会中疫病防治的发展新趋势。基因工程疫苗主要有两类：一类是核酸类包括DNA和RNA疫苗，另一类是含特异性抗原蛋白的亚单位疫苗。二者均通过抗原蛋白诱导宿主细胞产生应对该抗原蛋白的免疫应答，以达到预防和治疗疾病的目的。
基因工程疫苗的制备虽可通过生物合成，如直接合成多肽(亚单位疫苗)或合成能在宿主细胞内表达的DNA序列(DNA疫苗)，但无论生物合成多肽还是合成核酸其造价都很高，远不能用于规模化生产，目前基因工程疫苗的制备还完全依赖于细菌或细胞这些相对便宜且易于控制的生物工厂来生产。如蛋白亚单位疫苗的生产途径主要包括制备表达质粒以及蛋白表达与纯化，抗原蛋白的制备过程包括将质粒转化进原核细胞，主要是大肠杆菌或真核细胞(针对需要三维空间或糖基化修饰的抗原蛋白)进行转录和翻译从而产生抗原蛋白作为蛋白亚单位疫苗的原材料。DNA疫苗制作过程包括把抗原基因克隆到能在真核内表达的载体拟获得表达该抗原基因的质粒，再将该质粒转化进大肠杆菌，让质粒先在细菌这个廉价工厂中大量扩增，再通过一系列步骤将质粒DNA从细菌工厂中提取出来，DNA疫苗经肌肉注射或微弹轰击等方法导入宿主体内，通过宿主细胞将疫苗携带的抗原基因表达成抗原蛋白。比之蛋白疫苗其优点是抗原蛋白在宿主体内合成，能被正确地折叠和修饰，抗原性强，由于疫苗的主要成分是DNA因此更易储存和运输。但由于进入宿主细胞的外源DNA不能在宿主细胞内复制因此能进入宿主细胞的DNA的量是除抗原自身的效价外影响疫苗效力的另一关键因素。
目前无论直接用作DNA疫苗的质粒还是用于生产抗原蛋白的质粒都是带大肠杆菌复制子的质粒，这类质粒只能在大肠杆菌中复制，由于大肠杆菌是格兰式阴性菌，含有大量内毒素，通过该菌种制备质粒最严重的问题就是产生内毒素污染，因为内毒素是重要的致热源，能产生严重毒副作用，国家食品药品监管局对其有严格的要求。此外，目前用于表达抗原蛋白的宿主细胞主要也是大肠杆菌，由大肠杆菌进行大规模抗原蛋白表达常用的纯化方法也很难避免内毒素污染，为此寻找新的更好的抗原表达质粒仍然是基因工程疫苗生产的重大攻关难题之一。
为了避免内毒素污染，最直接的方法就是避免使用大肠杆菌来生产质粒或表达蛋白，然而目前已商业化的质粒基本上全是只能在大肠杆菌内复制的质粒。棒状杆菌基于它不产生内毒素越来越受到科学工作者的青睐，谷氨酸棒状杆菌不仅已广泛用于微生物发酵工程生产谷氨酸(味精)，近年来人们也开始探讨，研发它在蛋白生产方面的应用，并受到越来越广泛的关注。然而有关棒状杆菌生物代谢和细菌复制的研究远不及大肠杆菌那样深入和全面，因而推广到市面上的能在棒状杆菌内复制的商业化载体非常少见，并且它们的拷贝能力属于中低范围，低的拷贝能力直接导致产量低下从而推高成本，很难用于大规模生产。
申请人天津大学于2019年7月15日提出了发明专利申请201910636614.1，公开了一种合成香叶醇的谷氨酸棒状杆菌及构建方法及应用，其构建方法为：将酿酒酵母来源的突变香叶基焦磷酸合成酶基因ERG20F96W-N127W和截短C端53个氨基酸残基的缬草来源的香叶醇合成酶基因tVoGES通过融合PCR的方法连接，同源区加入核糖体结合位点序列，插入谷氨酸棒状杆菌表达质粒pEC-XK99E的SacI和XbaI酶切位点得到质粒1；将质粒1转化进入谷氨酸棒状杆菌，得到合成香叶醇的谷氨酸棒状杆菌1；该发明为香叶醇的合成提供更多的前体，发酵时间短42-48小时，经气质检测，未检测出相应的副产物。
申请人天津科技大学于2018年4月20日提出了发明专利申请201810356892.7，公开了一种棒状杆菌诱导型启动子及含有该启动子的表达载体与应用，所述表达载体转化至谷氨酸棒状杆菌获得的5-氨基乙酰丙酸生产菌株并利用该菌株发酵法生产5-氨基乙酰丙酸。用该发明获得的启动子构建的表达载体，通过肌醇诱导实现基因的可控表达，克服了现用诱导剂异丙基-β-D-硫代半乳糖苷(Isopropylβ-D-Thiogalactoside，IPTG)对菌体细胞生长的抑制作用以及因其价格高昂造成的生产成本高等不足，同时具有稳定性等特性。利用该启动子整合于宿主基因组或将表达载体转化至宿主谷氨酸棒状杆菌构建获得5-氨基乙酰丙酸 生产菌株，其5-氨基乙酰丙酸产量达到24.2g/L。
以上两个发明专利申请都是通过对质粒pXMJ19进行酶切并插入目的基因得到重组质粒，并将重组质粒导入至谷氨酸棒状杆菌，分别用于生产香叶醇和5-氨基乙酰丙酸，其都是上述背景技术中提到的微生物发酵工程领域的应用，并未涉及到本发明提到的用于直接合成多肽(亚单位疫苗)或合成能在宿主细胞内表达的DNA序列(DNA疫苗)领域的应用。pXMJ19质粒是具中低拷贝能力(约20)的质粒。
本发明要解决的技术问题是：如何构建一种在棒状杆菌里具高拷贝能力的DNA复制载体。
发明内容
本发明目的在于提供一种具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体及其构建方法，通过在pXMJ19载体的棒状杆菌复制子调节部位，将第1786位点的核苷酸C进行人工突变，成功获得比pXMJ19质粒拷贝能力高12.5倍的棒状杆菌/大肠杆菌双用载体pXMJ19C1786T质粒，与pXMJ19质粒相比，pXMJ19C1786T质粒的拷贝数从20提高到了251。
本发明的技术方案为：
一种具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体，所述表达载体的核苷酸序列如SEQ ID NO:9所示。
表达载体pXMJ19C1786T的序列(SEQ ID NO:9)：
Figure PCTCN2020096714-appb-000001
Figure PCTCN2020096714-appb-000002
Figure PCTCN2020096714-appb-000003
同时，本发明还公开了一种如上所述的表达载体的构建方法，包括以下步骤：
步骤1：以质粒pXMJ19为模板，分别设计引物通过PCR扩增将质粒pXMJ19第1786位点的核苷酸C突变为T的长度为2283bp的DNA片段1和长度为4348bp的DNA片段2；所述引物为C1786T-F、C1786T-R、V-C1786T-F、V-C1786T-R且其核苷酸序列依次如SEQ ID NO:1～SEQ ID NO:4所示；所述DNA片段1的核苷酸序列如SEQ ID NO:10所示，所述DNA片段2的核苷酸序列如SEQ ID NO:11所示；
DNA片段1(2283bp，SEQ ID NO:10)的序列：
Figure PCTCN2020096714-appb-000004
DNA片段2(4348bp，SEQ ID NO:11)序列：
Figure PCTCN2020096714-appb-000005
Figure PCTCN2020096714-appb-000006
Figure PCTCN2020096714-appb-000007
步骤2：将步骤1得到的两个DNA片段进行同源重组，得到表达表达载体pXMJ19C1786T。
在上述的表达载体的构建方法中，所述步骤1中：
DNA片段1、DNA片段2的PCR扩增方法为：
DNA聚合酶1uL，dNTP Mixture 1uL，其中dATP、dCTP、dGTP和dTTP的浓度均为2.5mM，引物C1786T-F为10pmoL，引物C1786T-R为10pmoL，模板pXMJ19为30ng，缓冲液25uL，加无核酸酶灭菌水至50uL；PCR反应条件为：95℃预变性3min，95℃变性15s，56℃退火15s，72℃延伸，变性、退火、延伸进行共30个循环，72℃保温5min；
DNA聚合酶1uL，dNTP Mixture 1uL，其中dATP、dCTP、dGTP和dTTP的浓度均为2.5mM，引物V-C1786T-F为10pmoL，引物V-C1786T-R为10pmoL，模板pXMJ19为30ng，缓冲液25uL，加无核酸酶灭菌水至50uL；PCR反应条件为：95℃预变性3min，95℃变性15s，61℃退火15s，72℃延伸，变性、退火、延伸进行共30个循环，72℃保温5min。
在上述的表达载体的构建方法中，所述步骤2中，同源重组的体系为：DNA片段1加入量为DNA片段1的碱基数×0.02ng，DNA片段2加入量为DNA片段2的碱基数×0.02ng，重组酶2uL，缓冲液4uL，加无核酸酶灭菌水至20uL。
本发明的有益效果如下：
1、本发明通过在pXMJ19载体的棒状杆菌复制子调节部位，将第1786位点的核苷酸C进行人工突变，成功获得比pXMJ19质粒拷贝能力高12.5倍的棒状杆菌/大肠杆菌双用载体pXMJ19C1786T。与pXMJ19质粒相比，pXMJ19C1786T质粒的拷贝数从20提升至251，并且经过突变的pXMJ19C1786T具备较好的稳定性。
2、本发明制备的表达质粒分别可应用于亚单位抗原的表达，使用者可根据表达的特定 抗原对棒状杆菌的毒性来选择具体该使用哪一种质粒；一般来说特定抗原蛋白对宿主细菌无毒的可选用高复制力的载体拟提高抗原蛋白的表达产量，而对于那些对宿主细菌具较强毒性的则选用复制能力较低的质粒拟减轻对宿主细菌的伤害，由于这些蛋白是在棒状杆菌内产生的因此在纯化过程中能完全地避免内毒素的污染，合理地减小成本并提高产品的质量。
3、本发明制备的表达质粒中具倍高复制能力的棒状杆菌复制子可替换常用的真核细胞表达载体中的大肠杆菌复制子，可以获得能在真核细胞内表达的具高复制能力的质粒，这些质粒可用于生产DNA疫苗，由于这些质粒是在棒状杆菌内复制的因而能完全避免内毒素的污染。
附图说明
附图1为pXMJ19的质粒构造图；
附图2为实施例1中PCR扩增后产物的电泳图，其中左L1和右L1分别来自PCR扩增后产生的两个片段；
附图3为实施例1中制备的表达质粒的双酶切产物的电泳图；
附图4为实施例1中制备的表达质粒pXMJ19C1786T的构造图；
附图5为实施例1中制备的表达质粒的测序验证结果图；
附图6为拷贝能力测试中PCR扩增后产物的电泳图，其中L1,L2来自pXMJ19，L3,L4来自pXMJ19C1786T；
附图7为拷贝能力测试中将qPCR结果用软件Grapd prism 5作的柱状图；
附图8为稳定性测试中质粒酶切产物的电泳图，其中L1，L3代表质粒pXMJ19；L2，L4代表质粒pXMJ19C1786T；L1～L2为MluI酶切后的产物；L3～L4为BsmB1酶切后的产物。
具体实施方式
下面结合具体实施方式，对本发明的技术方案作进一步的详细说明，但不构成对本发明的任何限制。
实施例1
一种具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体的构建方法，包括以下步骤：
(1)以质粒pXMJ19(江南大学白仲虎实验室惠赠)为模板，其质粒构造图如附图1所示，设计引物通过PCR扩增将质粒pXMJ19第1786位点的核苷酸C突变为T的长度为2283bp的DNA片段1和长度为4348bp的DNA片段2；
所述引物的核苷酸序列如下所示：
构建DNA片段1引物：
C1786T-F：gtttctacaaactcttttgtttatttttctaaatac(SEQ ID NO:1)
C1786T-R：agttagtagctcgcacggg(SEQ ID NO:2)
构建DNA片段2引物：
V-C1786T-F：tgcgagctactaactcatatgcacg(SEQ ID NO:3)
V-C1786T-R：gaattcgagctcggtacccgg(SEQ ID NO:4)
所述PCR反应体系如下表1所示：
表1 PCR反应体系
Figure PCTCN2020096714-appb-000008
Figure PCTCN2020096714-appb-000009
所述PCR反应条件为：
Figure PCTCN2020096714-appb-000010
将PCR扩增后的产物做琼脂糖凝胶电泳，回收正确分子量的琼脂糖凝胶条带，测定浓度后备用；其PCR扩增后产物的电泳图如附图2所示，其中左L1和右L1分别来自PCR扩增后产生的两个片段，由附图2可知，左L1片段2283bp；右L1片段4348bp。
(2)胶回收后利用诺维赞同源重组试剂盒进行同源重组，得到表达质粒pXMJ19C1786T。
所述同源重组体系如下表2所示：
表2同源重组体系
Figure PCTCN2020096714-appb-000011
Figure PCTCN2020096714-appb-000012
将二片段重组后通过单菌落提取质粒DNA，重组正确的质粒使用EcoR V(Takara 1042A)和Nde Ⅰ(Takara 1161A)双酶切应产生3944bp和2657bp的两个片段，其双酶切产物的电泳图如附图3所示，由附图3暗示该质粒可能为正确质粒pXMJ19C1786T，其构造图如附图4所示。
将实施例1制备的质粒送生工测序验证，结果如图5所示，证明实施例1制备的质粒在1786位点的核苷酸C突变成T。
性能测试
一、质粒pXMJ19和pXMJ19C1786T在棒状杆菌的拷贝能力测试
1、分别电转pXMJ19和pXMJ19C1786T至谷氨酸棒状杆菌ATCC13032野生型菌株，具体方法如下：
(1)取100ng质粒(质粒pXMJ19或pXMJ19C1786T)加入自己制备的感受态细胞100u冰浴10min，转入0.1cm电转杯；
(2)1800Kv电击5ms；
(3)立即加入1mL 46℃预热的LBHIS培养液(详见LBHIS标准配方)，46℃水浴6min(不要摇晃)，转入30℃摇床200rpm摇菌2h；
(4)涂布于含有5ug/mL氯霉素抗性培养皿培养12h；
感受态细胞恢复培养基LBHIS配方如下：(g/L)
酵母粉2.5，胰蛋白胨5.0，氯化钠5.0，脑心浸液18.5，山梨醇90.0,pH 6.8-7.0去离 子水定容至1L,121℃高压灭菌。
试剂 品牌 货号
酵母粉 OXOID LP0021
胰蛋白胨 OXOID LP0042
氯化钠 大茂 7647-14-5
脑心浸液BHI 环凯微生物 024053
山梨醇 sigma BCBW8882
制作感受态细胞的方法
(1.)从新鲜平板挑取2-3个单个菌落在10ml BHI培养基中30℃过夜培养，同时添加终浓度为2％的葡萄糖。
(2.)在500mL摇瓶中加入100mL EPO培养液(建议EPO提前37度预热)，接种上述菌种2ml，30℃，200rpm，培养3-5h.使OD600约0.35.
(3.)将摇瓶里的感受态细胞转移到50mL离心管中，冰浴30min.
(4.)4000rpm 4℃离心10min,弃上清。
(5.)用30mL冰上预冷的TG缓冲液(1M Tris-HCL,pH 7.5+10％甘油)充分洗涤1次，4000rpm 4℃离心10min,弃上清。
(6.)加预冷的10％甘油重复洗涤2次。
(7.)最后用1ml10％甘油重悬，分装每管100ul每管，液氮快速冷冻后置-80℃保存。
BHI培养基：(g/L)(环凯微生物货号024053)
蛋白胨10.0，脱水小牛脑浸粉12.5，脱水牛心浸粉5.0，氯化钠5.0，葡萄糖2.0，磷酸氢二钠2.5,PH＝7.4±0.2.
EPO培养基配方：(g/L)
酵母粉5.0，胰蛋白胨10.0，氯化钠10.0，甘氨酸25.0，异烟肼5.0，Tween80 1mL，
pH 6.8-7.0，去离子水定容至1L,121℃高压灭菌。
试剂 品牌 货号
酵母粉 OXOID LP0021
胰蛋白胨 OXOID LP0042
氯化钠 大茂 7647-14-5
甘氨酸 sigma G8898
异烟肼 sigma MKBQ8553V
Tween 80 scientific research special 9005-65-6
2、提取总DNA(包括细菌DNA和质粒DNA)，具体方法如下：
(1)在LB培养基中过夜培养3mL谷氨酸棒状杆菌；
(2)收集不大于3mL的菌液，室温、4000×g离心10min，沉淀细菌；
(3)弃培养基，加入100uL的TE Buffer重悬细菌，然后加入10uL Lysozyme(溶菌酶)，37℃水浴30min；
(4)加25mg的玻璃珠，在室温条件下高速涡旋5min,静置让玻璃珠沉淀下来，转移上清液至新的1.5mL的离心管中；
(5)加入100uL BTL Buffer(Omega BTL buffer 101QG)和20uL Proteinase K Solution涡旋混匀，将样品放置在55℃水浴摇床中使细胞完全裂解，一般1小时左右；
(6)加入5uL RNase A(全式金GE101-01)并反复颠倒混匀，在室温条件下孵育5min；
(7)室温条件下，10000×g离心2min，沉淀不溶解的物质，转移上清液到新的离心管中；
(8)加入220uL BDL Buffer涡旋混匀，65℃孵育10min；
(9)加入220uL 96％～100％的无水乙醇，高速涡旋20秒，如有沉淀出现，用枪头反复吹打以破碎沉淀；
(10)将
Figure PCTCN2020096714-appb-000013
DNA Mini结合柱套在一个2mL收集管上，将步骤(9)得到的所有液体转至于柱子內，室温10000×g离心1min，弃收集管和滤液；
(11)将
Figure PCTCN2020096714-appb-000014
DNA Mini结合柱套在新的2mL收集管上，加入500uL的HBC Buffer，室温10000×g离心1min，弃滤液；
(12)将
Figure PCTCN2020096714-appb-000015
DNA Mini结合柱套在同一个2mL收集管上，加入700uL DNA Wash Buffer，室温10000×g离心1min，弃滤液；
(13)重复步骤(12)；
(14)将
Figure PCTCN2020096714-appb-000016
DNA Mini结合柱套在同一个2ml收集管上，高速离心2min(≥10000×g)以干燥
Figure PCTCN2020096714-appb-000017
DNA Mini结合柱的基质，该步骤是用来除去可能会影响下游实验的乙醇；
(15)将
Figure PCTCN2020096714-appb-000018
DNA Mini结合柱套在1.5ml离心管上，加入50～100uL预热至65℃的Elution Buffer，室温下放置3～5min；
(16)室温10000×g离心1min，洗脱基因组DNA；
(17)重复步骤(15)和(16)，加入预热至65℃的Elution Buffer，进行二次洗脱以提高产率；
3、通过荧光定量PCR测定质粒的相对复制能力
(1)选择以下引物进行荧光定量PCR测定质粒pXMJ19和pXMJ19C1786T的相对复制能力，所述引物的核苷酸序列如下所示：
检测棒状杆菌内源基因组DNA的引物：
A(DnaA_1F)：ggtcgatgacatccagttcc(SEQ ID NO:5)
A(DnaA_1R)：gcttatctgcctggtgcaat(SEQ ID NO:6)
检测棒状杆菌内源质粒DNA的引物：
C(p_3_F)：tgcgtggataaggctaagga(SEQ ID NO:7)
C(p_3_R)：tccccatgagtaggcagaac(SEQ ID NO:8)
(2)反应液的配制：
Figure PCTCN2020096714-appb-000019
Figure PCTCN2020096714-appb-000020
每个样品做3个平行孔，总体系配好之后，用枪吸打混匀，然后每管分装20uL至八联管中，加Dye发光液时避免强光直射；将10ng样品总DNA用灭菌纯水梯度稀释至10 -3和10 -4，相当于10pg/uL和1pg/uL即可加入至刚配好的反应体系中；将各排八联管在掌上离心机离心数秒；
(3)上机qPCR反应体条件如下：
Figure PCTCN2020096714-appb-000021
将扩增后的PCR产物做琼脂糖凝胶电泳，结果如附图6所示，产物为140bp；L1，L2来自pXMJ19，L3，L4来自pXMJ19C1786T。
将qPCR结果通过用2-△△Ct定量，将质粒DNA的Ct值除以基因组DNA的Ct值，计算出两组之间的差异倍数，再用pXMJ19的拷贝数乘以差异倍数即得到相对的质粒拷贝数。其结果为pXMJ19C1786T的拷贝数为251.6。用软件Grapd prism 5作图如附图7所示。
4、通过质粒提取质粒DNA验证上述质粒的复制能力
用革兰氏阳性菌DNA提取试剂盒(M2350)分别从10mL上述菌液提取质粒DNA并测定其含量，其结果与上述qPCR结果吻合，进一步证明上述在1786位点的突变能提高pXMJ19的复制能力。
二、质粒在棒状杆菌的稳定性测试
用MluI和BsmB1限制性内切酶对质粒pXMJ19和pXMJ19C1786T作酶切鉴定，通过琼脂糖凝胶电泳比较其产物DNA片段的大小，得到的质粒酶切产物的电泳图如附图8所示，其中其中L1，L3代表质粒pXMJ19；L2，L4代表质粒pXMJ19C1786T；L1～L2示MluI酶切后的产物；L3～L4示BsmB1酶切后的产物，其结果显示这些突变株与其母体pXMJ19的酶切结果没有差别，间接证明在1786位点的点突变没有对质粒构成明显不稳定的因素。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。

Claims (4)

  1. 一种具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体，其特征在于，所述表达载体的核苷酸序列如SEQ ID NO:9所示。
  2. 一种如权利要求1所述的具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体的构建方法，其特征在于，包括以下步骤：
    步骤1：以质粒pXMJ19为模板，分别设计引物通过PCR扩增将质粒pXMJ19第1786位点的核苷酸C突变为T的长度为2283bp的DNA片段1和长度为4348bp的DNA片段2；所述引物为C1786T-F、C1786T-R、V-C1786T-F、V-C1786T-R且其核苷酸序列依次如SEQ ID NO:1～SEQ ID NO:4所示；所述DNA片段1的核苷酸序列如SEQ ID NO:10所示，所述DNA片段2的核苷酸序列如SEQ ID NO:11所示；
    步骤2：将步骤1得到的两个DNA片段进行同源重组，得到表达载体pXMJ19C1786T。
  3. 根据权利要求2所述的具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体的构建方法，其特征在于，所述步骤1中：
    DNA片段1、DNA片段2的PCR扩增方法为：
    DNA聚合酶1uL，dNTP Mixture 1uL，其中dATP、dCTP、dGTP和dTTP的浓度均为2.5mM，引物C1786T-F为10pmoL，引物C1786T-R为10pmoL，模板pXMJ19为30ng，缓冲液25uL，加无核酸酶灭菌水至50uL；PCR反应条件为：95℃预变性3min，95℃变性15s，56℃退火15s，72℃延伸，变性、退火、延伸进行共30个循环，72℃保温5min；
    DNA聚合酶1uL，dNTP Mixture 1uL，其中dATP、dCTP、dGTP和dTTP的浓度均为2.5mM，引物V-C1786T-F为10pmoL，引物V-C1786T-R为10pmoL，模板pXMJ19为30ng，缓冲液25uL，加无核酸酶灭菌水至50uL；PCR反应条件为：95℃预变性3min，95℃变性15s，61℃退火15s，72℃延伸，变性、退火、延伸进行共30个循环，72℃保温5min。
  4. 根据权利要求2所述的具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体的构建方法，其特征在于，所述步骤2中，同源重组的体系为：DNA片段1加入量为DNA片段1的碱基数×0.02ng，DNA片段2加入量为DNA片段2的碱基数×0.02ng，重组酶2uL，缓冲液4uL，加无核酸酶灭菌水至20uL。
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