WO2012122765A1 - 一种表面展示家蚕乙醇脱氢酶重组芽孢及其制备方法 - Google Patents

一种表面展示家蚕乙醇脱氢酶重组芽孢及其制备方法 Download PDF

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WO2012122765A1
WO2012122765A1 PCT/CN2011/076941 CN2011076941W WO2012122765A1 WO 2012122765 A1 WO2012122765 A1 WO 2012122765A1 CN 2011076941 W CN2011076941 W CN 2011076941W WO 2012122765 A1 WO2012122765 A1 WO 2012122765A1
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recombinant
spore
bacillus subtilis
gene
alcohol dehydrogenase
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陈克平
王楠
宁德刚
李国辉
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Jiangsu University
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)

Definitions

  • the invention belongs to the technical field of recombinant fusion proteins. Specifically, it relates to a method of displaying an alcohol dehydrogenase as a foreign active protein on the surface of Bacillus subtilis and a recombinant spore.
  • the active alcohol dehydrogenase gene can be activated by molecular biological methods.
  • the spore capsid protein gene is expressed after recombination, so that the alcohol dehydrogenase which is originally unstable to temperature and ra becomes an alcohol dehydrogenase fusion protein which can have relatively stable activity under different environmental conditions.
  • Alcohol dehydrogenase belongs to the oxidoreductase family and can catalyze the reversible reaction between primary alcohol and aldehyde by using nicotinamide adenine dinucleotide (NAD) as a coenzyme.
  • ADHs play an important role in many physiological processes, such as metabolic processes such as steroids, vitamins, lipid peroxidation products, fatty acids, and ethanol.
  • ADH and aldehyde dehydrogenase (ALDH) constitute an alcohol dehydrogenase system, which is involved in the metabolism of ethanol in the body and is an important metabolic enzyme in humans and animals.
  • ADH bioelectrode and ethanol biosensor which can be used for industrial analysis of ethanol concentration
  • ADH catalytic properties used in the chemical industry for the production of many raw materials and intermediate reactants
  • 3 ADH is an important physiological indicator for humans and animals for various studies
  • 4 With the development of biotechnology and enzyme industry, ADH is used as a new experimental material for various new research. Among them, the development of effective hangover products using ethanol dehydrogenase has received extensive attention. In the liver, the total activity of ADH and ALDH determines the rate of ethanol metabolism.
  • ADH activity has been used as an important indicator to observe whether hangover drugs have an induction effect on ADH, so as to know whether drugs can accelerate ethanol catabolism, thereby reducing ethanol Damage to the human body.
  • ADH and ALDH have become an important direction of hangover research.
  • Bacillus subtilis ⁇ is aerobic Gram-positive bacteria, which can form a stress-resistant dormant body, spores, under nutrient deficiencies or other stress conditions. Under suitable conditions, spores can Germinating into vegetative cells with reproductive capacity. The spores are easy to culture and easy to separate, eliminating the need for complicated separation and purification operations. Spore is the most stress-resistant structure in the life world.
  • Bacillus subtilis is not pathogenic to organisms and can be used as a probiotic to inhibit harmful bacteria (E. coli, Salmonella) by human or animal (Mazza, P. 1994. The use of Bacillus subtilis as an antidiarrhoeal Boll. Chim. Farm.
  • Bacillus subtilis live capsules available on the market, Beijing Zizhu Pharmaceutical Co., Ltd., Sinopharm S20030018 (hUp://www 77aaax >m binil /yao/20079182340271] 96 3569029.him) , indicating that Bacillus subtilis spores can be used orally to produce pharmacodynamics. Due to the above physical and chemical characteristics, coupled with the deciphering of its genome and the establishment of an operational genetic system, Bacillus subtilis spores have become a novel protein or enzyme drug carrier of great interest.
  • the three proteins CotB, CotC and CotG on the spore capsid have become a novel expression system for foreign proteins due to exposure to spore surface. It has been reported that this novel expression system has successfully expressed some foreign proteins, such as white spot synthesis of shrimp. Viral protein Vp28, etc., but due to biological specificity, gene expression is affected by many factors, etc., whether the expression system can successfully express other specific active proteins remains to be studied.
  • an alcohol dehydrogenase cloned from the model organism Bombyx mori is used as a foreign molecule
  • the Bacillus subtilis spore coat protein CotC is used as a carrier
  • the silkworm alcohol dehydrogenase (BmADH, GeneBank is registered by spore surface display technology). No.: NP—001037610. 1 ) Demonstrated on the surface of spores, which gives BmADH a large amount of expression and good resistance to stress, and provides a rapid and large-scale preparation of a stable active recombinant alcohol dehydrogenase product.
  • the product of the present invention maintains the physiological properties of Bacillus subtilis spores, and the catalytic stability to ethanol is significantly improved compared with the original silkworm alcohol dehydrogenase protein. So far, no similar reports and invention patents have been found. Summary of the invention
  • the present invention provides a recombinant spore of the surface display alcohol dehydrogenase ADH by taking the silkworm alcohol dehydrogenase BmADH as an example.
  • Preparation methods and products The invention is carried by the Bacillus subtilis spore coat protein CotC BmADH is displayed on the surface of spores of Bacillus subtilis by spore surface display technology to obtain a fusion protein with stable activity at different temperatures and ra conditions. It can be used directly in animals to become a new type of alcohol dehydrogenase drug. Nutrition.
  • the preparation method and the separation process of the surface display alcohol dehydrogenase protein of the invention are simple and rapid, and the produced fusion protein has more stable biological activity and has wider application value.
  • the Bacillus subtilis recombinant strain was obtained, and the recombinant strain was induced to produce spores, and a fusion protein having alcohol dehydrogenase activity was displayed on the surface of the spore.
  • the specific operation of the present invention is: the cDNA of the silkworm alcohol dehydrogenase BmADH is amplified from the silkworm cDNA library by reverse transcription PCR, and the obtained gene is ligated into the cloning vector and sequenced correctly, and then the silkworm ethanol is dehydrogenated.
  • the enzyme gene ⁇ 3 ⁇ 401 ⁇ 2 was ligated into the fusion expression plasmid pJS700 (preserved in the laboratory) to construct an integrated recombinant plasmid pJS700_BmADH.
  • the plasmid was used to transform Bacillus subtilis, and the host strain was induced to produce spores by depletion, and the fusion protein CotC-BmADH was displayed on the surface of the spore.
  • the present invention selects a Bacillus subtilis strain which has good transforming ability and can produce spores as a transformed strain of a recombinant plasmid, such as Bacillus subtil is 168 and a series of strains thereof (Bacillus Genetic Stock Center, Department of Biochemistry, The Ohio State University) , West 12th Avenue, Columbus, Ohio, 43210, USA); select the spore capsid protein gene (GenBank SEQ ID NO: NP-389653) as the molecular carrier of the surface display protein, and select the silkworm alcohol dehydrogenase gene 3 ⁇ 4so1 ⁇ 2 as the recombinant gene.
  • Bacillus subtilis is an environmentally friendly bacterium that differentiates into dormant spores in the absence of nutrients.
  • the spores are highly resistant, resistant to acids and alkalis, high in temperature, and highly viable; and because of their greater specificity, Separation and purification were carried out.
  • the culture method of Bacillus subtilis is simple and convenient, the growth is rapid, the nutrition requirement is simple, and it is easy to carry out industrial scale-up culture.
  • the silkworm alcohol dehydrogenase is displayed on the surface of the spores of Bacillus subtilis, which has good resistance and stability while obtaining rapid and large-scale expression, and is easy and quick to separate and purify.
  • the present invention relates to a recombinant plasmid constructed by recombining a selected coding sequence of a spore-capsid protein gene cotC and a 3 ⁇ 4 a0 1 gene of the invention, wherein the recombinant plasmid contains a promoter of a spore-capsid protein gene, and a coding sequence containing no stop codon.
  • Bacillus subtilis recombined with the coding sequence of the 3 ⁇ 4so1 ⁇ 2 gene Fusion forms CotC-BmADH when differentiated to form spores.
  • the fusion gene co tC-Bmadh is integrated into a specific site on the chromosome of Bacillus subtilis and stably propagated genetically, thereby avoiding the disadvantage that the plasmid is easily lost in Bacillus subtilis cells.
  • Expression vectors bearing Bmadh can be transformed into host cells by heat bombardment or electroporation.
  • Cells that have been successfully transformed with the fusion gene constructed by the present invention are identified using methods well known in the art. Such as cell collection, lysis, starch plate identification, chromosome extraction and PCR identification, can also be identified by Western blotting using BmADH specific antibody after induction of expression.
  • the invention also relates to a recombinant strain obtained by transforming the constructed recombinant integrated plasmid into Bacillus subtilis screening, and the surface of the spore formed by the recombinant strain exhibits a recombinant silkworm alcohol dehydrogenase, which can be detected by SDS-PAGE and Western blotting.
  • the recombinant protein displayed on the surface of the spore was tested for its catalytic activity against ethanol.
  • the present invention relates to a primer sequence for cloning a gene ⁇ sofe coding sequence fragment:
  • B adJr-F 5 ' -GGGGTACCATGGCACCGGATTTCGTGAAGCGTT-3 ' , underlined Kpnl restriction site;
  • B adJ ⁇ R 5 ' -CGAGCTCCTATGTCTTGGAGAGTATTTGGA-3 ' , underlined for Sacl restriction sites.
  • the present invention relates to a primer sequence for fragment amplification of the Bacillus subtilis amylase gene aiwE (GenBank SEQ ID NO: NP-388186):
  • amyE-F ATTGCTCGGGCTGTATGACTGG
  • amyE-R GTTACACCATCACTGTTCGTTCCTT
  • the silkworm alcohol dehydrogenase BmADH is displayed on the surface of Bacillus subtilis, and the unique resistance to spores is utilized, so that the enzyme protein which is unstable to temperature and PH value becomes relatively stable ethanol.
  • the catalytically active fusion protein overcomes the disadvantages inherent in the instability of alcohol dehydrogenase.
  • the recombinant spore of Bacillus subtilis exhibiting silkworm alcohol dehydrogenase BmADH on the surface of the invention can be directly used for oral administration through the digestive tract barrier of the animal, and becomes a novel alcohol dehydrogenase nutrient or animal nutrition.
  • the product of the invention has large expression amount, preparation method and separation process, and is simpler and faster than other ethanol dehydrogenase samples, has better stability and wider application value, and expands its industrial application prospect to a certain extent, It provides a reference for improving the stability of other enzyme proteins.
  • Figure 1 is the nucleotide sequence and amino acid sequence of the silkworm alcohol dehydrogenase BmADH cloned in the GenBank cloned by the inventors, accession number NP-001037610. Lower case letters are nucleotide sequences, start codons and stop codons are indicated by boxes; uppercase letters are amino acid sequences, TATA-box is underlined in italics, and Poly-A signals are underlined in boxes. The sequence is a conservative amino acid.
  • Figure 2 is a construction method and a structural map of the fusion expression recombinant plasmid pJS700_BmADH expressing CotC-BmADH.
  • a W E 5, -end and a/ff 3 '-end represent the 5' and 3' ends of the amylase gene coding sequence, respectively
  • Anip r , ⁇ represent the ampicillin resistance gene and the erythromycin resistance gene, respectively.
  • cotC-Bmadh is a gene fragment in which a recombinant protein of CotC-BmADH is expressed in a spore of Bacillus subtil is 168 (trp).
  • oriC is an E. coli replicon fragment.
  • Figure 3 is a schematic representation of the integration of the ⁇ -co ⁇ - ⁇ 7ao1 ⁇ 2 gene fragment in the Bacillus subtil is 168 ( ⁇ ) chromosome.
  • Figure 4 is an amylase activity analysis of the recombinant strain Bacillus subtil is 168 ( trp ) /pJS700-BmADH.
  • A The wild type strain ac W ⁇ subtil is 168 ( ir/?- ) and the recombinant strain s J s 168 ( ⁇ ) /pJS700-BmADH were cultured on a starch plate;
  • B wild after being stained with iodine on a starch plate Type strains and recombinant strains.
  • Figure 5 is a PCR detection of the E-cotC-Bmadh fragment in recombinant strains.
  • Marker ⁇ -EcoT14 I digest Marker;
  • the chromosome of Bacillus subtil is 168 ( trp ) (Wi ld-type, W) and Bacillus subtil is 168 ( trp ) /pJS700-BmADH (Mutant, M) , using amyE-F/amyE-R, BmADH-F/BmADH-R, BmADH-F/ amyE-R and amyE-F/BmADH-R four pairs of primers (see Figure 3 for the position of the primer pair in the chromosome) PCR was performed to identify recombinant genes.
  • Figure 6 shows the SDS-PAGE and Western blotting of the CotC-BmADH fusion protein displayed on the surface of the transgenic recombinant spore.
  • M is Marker
  • lane 1 is the wild type strain Bacillus subtil is 168 (irp) control
  • lane 2 is the recombinant strain fusion protein.
  • Figure 7 is a comparison of the activity of ethanol dehydrogenase between the CotC-BmADH fusion protein displayed on the surface of the transgenic recombinant spore and the original BmADH protein at different temperatures and temperatures.
  • the solid line shows PJS700_BmADH which indicates the fusion protein obtained by the present invention, and the dotted line indicates that BmADH indicates the original BmADH protein.
  • the silkworm varieties used in the present invention are provided by the Jiangsu Academy of Life Sciences.
  • the midgut tissue of the silkworm was taken and placed in a mortar precooled with liquid nitrogen.
  • the liquid nitrogen was added for grinding, and the mRNA was isolated by an RNA extraction kit.
  • the cDNA sequence of the silkworm alcohol dehydrogenase gene was obtained by reverse transcription PCR.
  • the PCR amplification primers for the silkworm alcohol dehydrogenase ⁇ 01 ⁇ 2 gene fragment are:
  • Bmadlr-F 5 '-GGGGTACCATGGCACCGGATTTCGTGAAGCGTT-3, (SEQ ID NO. 1), underlined for Kpnl restriction sites;
  • Bmadlr-R 5, -CGAGCTCCTATGTCTTGGAGAGTATTTGGA-3, (SEQ ID NO. 2), underlined the Sacl restriction site.
  • the PCR reaction system is strictly carried out according to the instruction manual of 73 ⁇ 4 ⁇ 3 ⁇ 47& 73 ⁇ 4 ⁇ 7, 25 reaction system containing TaKaRa LA Taq 0. 2 ⁇ L, 10 X LA PCR Buffer II (Mg 2+ Plus) 2. 5 ⁇ L, dNTP Mixture (each 2. 5 mM) 2. 5 L, template DNA 1 ⁇ L, upstream and downstream primers (20 ⁇ M) each 0.5 ⁇ L, supplemented with sterile ultrapure water.
  • the PCR conditions were: denaturation at 94 °C for 5 min, 94 °C for lmin, 61 °C for 40 s, 72 °C for 2 min, 30 cycles.
  • the 825 bp PCR product was cloned into pMD18_T vector, transformed into E. coli DH5 ⁇ , and the cloned strain was screened.
  • the plasmid was extracted and identified by PCR and 7-71 and Sacl double digestion and agarose gel electrophoresis. After the positive clone, the recombinant plasmid was sequenced.
  • the cloned recombinant plasmid was named pMD18-T-BmADH, and the recombinant strain was named DH5 a /pMD18_T-BmADH.
  • the positive recombinant strain was stored in LB medium containing 15% glycerol and frozen at -80 °C.
  • the sequence of the alcohol dehydrogenase Bmadh gene in the recombinant plasmid was sequenced as shown in SEQ ID NO.
  • Example 2 Preparation of BmADH Bacillus subtilis recombinant spores using CotC as a vector
  • Plasmid pJS700 Li Qian. Study on the recombinant spores of Bacillus subtilis expressing WSSV envelope proteins Vpl9 and Vp28 on the surface of molecular carrier with CotX [D] .
  • Zhenjiang, Jiangsu: Jiangsu University, 2010 : 36-38 The size of the plasmid is about 5. 5kb.
  • amyE 5, -end and B amyE 3, -end represent the 5' and 3' ends of the amylase gene amyE (GenBank accession number: NP-388186), respectively, and are integrated in Bacillus by homologous recombination.
  • Sub til is 168 ( irp ) in the amylase gene of the chromosome.
  • the PCR amplification primer for the amylase gene a/zy ⁇ is:
  • a yE-F ATTGCTCGGGCTGTATGACTGG ( SEQ ID NO. 4 ) a yE-R: GTTACACCATCACTGTTCGTTCCTT ( SEQ ID NO. 5 )
  • Amp r and ⁇ represent the ampicillin resistance gene and the erythromycin resistance gene, respectively, and are used as screening markers in Escherichia coli and Bacillus subtil is 168 (ir/?-).
  • Co ⁇ is a Bacillus subtilis spore capsid protein CotC gene, and the gene fragment contains a promoter sequence of the co ⁇ gene (GenBank SEQ ID NO: NP-389653) and a CotC coding sequence which does not contain a stop codon.
  • oriC is an E. coli replicon fragment.
  • the Bmadh fragment and pJS700 were recovered by agarose gel recovery kit with Kpnl and the plasmids pMD18_T-BmADH and pJS700, respectively, and the two fragments were digested with T 4 DNA l igase, and the ligated product was transformed into Escherichia coli DH5.
  • ⁇ competent cells, transformants were plated in LB plates containing 50 g/mL ampicillin overnight at 37 ° C, and the next day selected positive monoclonal strains in LB liquid medium containing 50 ⁇ g / mL ampicillin, After incubating at 37 °C for 12 to 16 h, the plasmid was extracted and identified by PCR and 7 ⁇ 71 and feci digestion.
  • the positive clones were identified by agarose gel electrophoresis. After the identification, the recombinant integrated plasmid was named pJS700-BmADH, and the positive recombinant strain was named. Is DH5 a /pJS700_BmADH.
  • the recombinant integration plasmid pJS700_BmADH contains the recombinant gene cotC-Bmadh, and the construction process of the plasmid and its map are shown in FIG.
  • Transformation of the integrated recombinant plasmid pJS700_BmADH Subtil is 168 ( trp ) (Bacillus Genetic Stock Center, Department of Biochemistry, The Ohio State University, West 12th Avenue, Columbus, Ohio, 43210, USA), the homologous region of both recombines, and the recombinant gene is inserted into the amylase gene a sy destroyed its starch hydrolase activity, see Figure 3.
  • the recombinants were screened with LB plates containing 0.4 g/mL erythromycin (Sigma), single colonies were selected from the plates and inoculated into LB plates containing 1% starch, cultured overnight at 37 ° C, and iodine-potassium iodide was used the next day.
  • the solution was stained with starch plates to identify recombinant strains.
  • the preparation method of the 1% starch plate 100 mL LB solid medium is added with soluble starch 0. lg, and the plate is inverted after autoclaving.
  • iodine-potassium iodide solution 2 g of potassium iodide, lg of iodine, 300 mL of distilled water, first dissolve potassium iodide in a small amount of deionized water, add iodine after all dissolved, dilute to 300 mL by shaking, and store in a brown glass bottle protected from light. It can be diluted 10 times when used. Appropriate amount of iodine-potassium iodide solution was dropped on the starch plate. The transparent hydrolysis circle around the colony indicated that the recombinant gene was not inserted into the starch hydrolase gene s/sj ⁇ .
  • the inventors used the chromosome of Bacillus subtilis as a template, respectively, as afflj3 ⁇ 4-F/afflj3 ⁇ 4-R, BmADH-F/BmADH-R, BmADH-F/ Four pairs of primers of amyE-R and amyE-F/BmADH-R were subjected to PCR amplification.
  • the PCR reaction system is strictly carried out according to the instructions of 7 3 ⁇ 47 & LA 73 ⁇ 4.
  • the 25 ⁇ L reaction system contains 73 ⁇ 4A3 ⁇ 47& LA Taq 0.2 ⁇ L, 10XLA PCR Buffer II (Mg 2+ Plus) 2.5 L, dNTP Mixture (2.5 mM each) 2.5 ⁇ L, 1 ⁇ L of template DNA, 0.5 L of upstream and downstream primers (20 ⁇ ), and finally added with sterile ultrapure water.
  • the PCR reaction conditions were set separately according to the characteristics of each primer pair. Amplified fragments of about 4200 bp, 825 bp, 700 bp, and 2650 bp were detected in the recombinant strain, respectively, and only about llOObp of amylase gene was detected in the wild type strain, as shown in Fig. 5.
  • the recombinant strain contains the ⁇ -co td gene on the chromosome, and the ⁇ -co tC-Bmadti. fragment was successfully inserted into the starch hydrolase gene s/sj ⁇ .
  • the identified recombinant strain was named Bacillus subtil is 168 ( ⁇ — ) /pJS700_BmADH.
  • the method for extracting the chromosome of Bacillus subtilis is as follows: The single colony is connected to LB medium and cultured to 0D 6 in a shaker at 37 °C. . The value is around 1.0. Take 1.5 mL of the bacterial solution in an Eppendorf tube, centrifuge at 8000 rpm for 10 min, discard the supernatant, wash the pellet with 10 buffers (10 mM Tris-HCl, 1 mM EDTA), and centrifuge to remove the supernatant.
  • the recombinant strain ac Wis subtil is 168 ( ⁇ ) /pJS700_BmADH was induced to form spores by DSM medium.
  • the formulation of the DSM medium is: 0.8% nutrient broth (Difo), 0.1% KC1, 0.025% MgS0 4 ⁇ 7H 2 0, 1.
  • the recombinant strain was inoculated separately into DSM medium, and the host strain was inoculated with spores by a depletion method for 48 hours, and the culture was taken for treatment.
  • Method for treatment of spore protein The cells were collected by centrifugation at 12000 rpm for 10 min, resuspended in the same volume of GTE Buffer (50 mM glucose, 20 mM pH 7.5 Tris-HCl, 10 mM EDTA, 2 mg/mL lysozyme), and treated at 37 ° C for 30 minutes. To destroy the vegetative cells, the spores were precipitated at 12000 rpm ⁇ 10 min, washed 3 times with PBS of pH 7.4, and resuspended in the same volume of SDS-DTT buffer (0.1 mM pH 7.4).
  • GTE Buffer 50 mM glucose, 20 mM pH 7.5 Tris-HCl, 10 mM EDTA, 2 mg/mL lysozyme
  • the cells were treated with a water bath at 65 ° C for 10 min, and stained with Coomassie blue for SDS-PAGE to obtain a band of about 41 kD.
  • the rabbit anti-BmADH serum was used as the primary antibody for Western blotting to detect the spore protein of the recombinant strain and the wild type strain.
  • the spore protein of the wild type Bacillus subtil is 168 (trp) was used as a negative control. Consumption of ⁇ NAD + per minute is defined as 1 unit of alcohol dehydrogenase activity.
  • the inventors selected 0.1 M sodium acetate buffer, pH 4.0; 0.1 M phosphate buffer (NaH2P04 - Na2HP04), pH 7.0; 0.1 M NaOH / glycine
  • the buffer solution pH 8.0, 9.0, and 10.0, was added as a buffer solution to the reaction solution system, and the enzyme activity under different ra values at 25 ° C was measured.
  • the inventors measured the fusion protein at 16 ° C, 20 ° C, 25 ° C, 30 ° C with 0.1 mM NaOH/glycine as pH 9.0.

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Description

一种表面展示家蚕乙醇脱氢酶重组芽孢及其制备方法 技术领域
本发明属于重组融合蛋白技术领域。 具体涉及将乙醇脱氢酶作为外源活 性蛋白展示在枯草芽孢杆菌表面的方法和重组芽孢。而言是指： 利用枯草芽孢杆 菌在极端的温度、干燥以及暴露于有机溶剂和其他毒性化学物质等不利条件下能 长期存活的性质，通过分子生物学方法将有活性的乙醇脱氢酶基因与芽孢衣壳蛋 白基因重组后进行表达， 使原先对温度和 ra不稳定的乙醇脱氢酶成为可在不同 环境条件下具有相对稳定活性的乙醇脱氢酶融合蛋白。
背景技术
乙醇脱氢酶（alcohol dehydrogenase, ADH)属于氧化还原酶家族，能够以烟 酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide, NAD)为辅酶， 催化 伯醇和醛之间的可逆反应。 ADHs 在很多生理过程中都起着重要作用， 参与例如 类固醇、 类维生素 、 脂质过氧化产物、 脂肪酸、 乙醇等代谢过程。 在人和哺乳 动物体内， ADH 与乙醛脱氢酶 （aldehyde dehydrogenase, ALDH)构成了乙醇脱 氢酶系， 参与体内乙醇代谢， 是人和动物体内重要的代谢酶。 目前， ADH的应用研究主要体现在以下几个方面： ① ADH生物电极和乙醇生 物传感器， 可用于工业分析乙醇浓度； ② ADH的催化特性， 在化学工业中应用于 许多原材料及中间反应物的生产； ③ ADH作为人和动物重要的生理指标用于各项 研究； ④伴随生物技术和酶工业的发展， ADH被作为新的实验材料， 用于开展各 种新的研究。其中， 利用乙醇脱氢酶开发有效的解酒产品已受到广泛关注。在肝 脏中， ADH与 ALDH总活力的高低决定了乙醇代谢的速度。 因此， 在众多的解酒 药物研究方面， ADH活性已被作为一项重要的检测指标，用于观察解酒药物对 ADH 是否具有诱导作用， 以便了解药物能否加快乙醇分解代谢， 从而减少乙醇对人体 的损害。 同时开发高活性的 ADH 与 ALDH 也已经成为解酒药研究的重要方向之
然而， ADH对乙醇的催化活力对环境条件敏感， 如温度和 ra值不适则易失 活， 这一缺点极大地影响了 ADH的应用前景。随着生物工业生产对酶催化功能需 求的日益增加， 寻求一种新方法生产催化活性更稳定的酶蛋白是十分必要的。 枯草芽孢杆菌 ί Bacillus sub til is) 是好氧型的革兰氏阳性菌， 它在营养 缺乏或其他胁迫条件下， 能形成具有抗逆性的休眠体——芽孢， 在适宜条件下芽 孢又能萌发成具有繁殖能力的营养细胞。芽孢易培养易分离， 可以免去复杂的分 离纯化操作。芽孢是生命世界中抗逆性最强的一种结构，其稳定性好，能耐氧化， 耐高温， 并且在抗化学药物和抗辐射等方面也十分突出， 可在恶劣环境中存活几 年至几十年。 芽孢在酸性胃环境中能保持活性， 可以耐唾液和胆汁的攻击， 从而 顺利通过动物消化道。研究证明， 枯草芽孢杆菌对生物体无致病性， 能作为抑制 有害细菌（大肠杆菌、沙门氏杆菌）的益生素被人或动物口服（Mazza, P. 1994. The use of Bacillus subtilis as an antidiarrhoeal microorganism. Boll. Chim. Farm. 133:3-18.) 目前市面上已有口服枯草芽孢杆菌活菌胶囊产品，北京紫竹药业有限 公司， 国药准字 S20030018 (hUp://www 77aaax >m binil/yao/20079182340271 ] 96 3569029.him) , 表明枯草芽孢杆菌芽孢可以通过人口服使用而产生药效性。 由于 以上理化及生理特性， 再加上其基因组的破译和可操作的遗传系统的建立， 枯草 芽孢杆菌芽孢成为备受关注的新型蛋白或酶类药物载体。芽孢衣壳上的三种蛋白 CotB、 CotC和 CotG由于暴露在芽孢表面而成为外源蛋白的新型表达系统， 目前 已有报道利用这种新型表达系统成功表达了一些外源蛋白，如对虾白斑综合征病 毒蛋白 Vp28等， 但是由于生物的特异性、 基因表达受多种因素影响等原因， 利 用该表达系统是否能成功表达其他特定的活性蛋白仍有待研究。
本发明以从模式生物家蚕 Bombyx mori体内克隆得到的乙醇脱氢酶作为外源 分子， 以枯草芽孢杆菌芽孢衣壳蛋白 CotC为载体， 通过芽孢表面展示技术将家 蚕乙醇脱氢酶 （BmADH, GeneBank登录号： NP— 001037610. 1 ) 展示在芽孢表面， 使 BmADH获得大量表达的同时具有良好的抗逆性，提供了快速大量制备具有稳定 活性的重组乙醇脱氢酶产品的方法。本发明的产品在保持了枯草芽孢杆菌芽孢生 理特性的基础上， 对乙醇的催化稳定性较之原家蚕乙醇脱氢酶蛋白有明显提高， 迄今尚未见类似的报道和发明专利。 发明内容
为了解决乙醇脱氢酶因在极端条件下的不稳定性而在工业应用上存在的困 难， 本发明以家蚕乙醇脱氢酶 BmADH为例， 提供了一种表面展示乙醇脱氢酶 ADH 的重组芽孢的制备方法和产品。 本发明以枯草芽孢杆菌芽孢衣壳蛋白 CotC为载 体，通过芽孢表面展示技术将 BmADH展示在枯草芽孢杆菌的芽孢表面， 得到在不 同温度、 ra 条件下具有较稳定活性的融合蛋白， 可用于动物直接口服， 成为一 种新型的乙醇脱氢酶药物营养品。相对于其他乙醇脱氢酶蛋白产品， 本发明表面 展示乙醇脱氢酶蛋白的制备方法和分离过程简单快速，产生的融合蛋白生物活性 更稳定， 具有更广泛的应用价值。
本发明所采用的技术方案为：
一种表面展示家蚕乙醇脱氢酶 BmADH重组芽孢的制备方法，利用枯草芽孢杆 菌芽孢衣壳蛋白基因为分子载体， 与家蚕乙醇脱氢酶基因重组， 构建融合表达的 整合型重组质粒， 并转化枯草芽孢杆菌获得枯草芽孢杆菌重组菌株， 诱导重组菌 株产生芽孢， 在芽孢表面展示具有乙醇脱氢酶活性的融合蛋白。
本发明的具体操作是： 通过反转录 PCR的方法从家蚕 cDNA文库中扩增获得 家蚕乙醇脱氢酶 BmADH的 cDNA,将获得的基因连接到克隆载体中测序正确之后， 再将家蚕乙醇脱氢酶基因 ^¾0½连接到融合表达质粒 pJS700 (本实验室保存）， 构建出整合型重组质粒 pJS700_BmADH。 用该质粒转化枯草芽孢杆菌， 采用耗竭 法诱导宿主菌产生芽孢， 在芽孢表面展示有融合蛋白 CotC-BmADH。
本发明选择具有良好的转化能力并且可以产生芽孢的枯草芽孢杆菌菌株为 重组质粒的转化菌株， 例如 Bacillus subtil is 168 及其衍生的一系列菌株 (Bacillus Genetic Stock Center, Department of Biochemistry, The Ohio State University, West 12th Avenue, Columbus, Ohio, 43210， USA)；选择芽孢衣壳蛋白基 因 (GenBank序列号： NP— 389653) 作为表面展示蛋白的分子载体， 选择家 蚕乙醇脱氢酶基因 ¾so½为重组基因。枯草芽孢杆菌为环境友好型细菌， 其在营 养缺乏时会分化形成休眠体芽孢， 芽孢具有极强的抗逆性， 可耐酸碱、 耐高温， 存活能力较强； 并因其比重大， 容易进行分离纯化。枯草芽孢杆菌的培养方法简 单方便， 生长迅速， 营养要求简单， 容易进行工业放大培养。 在枯草芽孢杆菌的 芽孢表面展示家蚕乙醇脱氢酶，使其在获得快速大量表达的同时具有良好的抗逆 性和稳定性， 并且分离纯化简便快捷。
本发明涉及发明所选择的芽孢衣壳蛋白基因 cotC与 ¾a0½基因编码序列重 组后构建的重组质粒，在此重组质粒中含有芽孢衣壳蛋白基因的启动子、不含终 止密码子的编码序列与 ¾so½基因的编码序列重组后的基因，可在枯草芽孢杆菌 分化形成芽孢时融合表达 CotC-BmADH。 通过构建整合型重组质粒， 使得融合基 因 co tC-Bmadh整合于枯草芽孢杆菌染色体上的特定位点并稳定地进行遗传繁 殖， 避免了质粒在枯草芽孢杆菌细胞中容易丢失的缺点。
带有 Bmadh的表达载体可以通过热击法或电穿孔法转化宿主细胞。使用本领 域熟知的方法鉴定经过成功转化带有本发明构建的融合基因的细胞。如细胞的收 集、裂解、淀粉板鉴定、提取染色体和 PCR鉴定等，也可以在诱导表达后用 BmADH 特异抗体进行 Western blott ing鉴定。
本发明还涉及所构建的重组整合型质粒转化枯草芽孢杆菌筛选所得到的重 组菌株， 这些重组菌株诱导形成的芽孢表面展示有重组家蚕乙醇脱氢酶， 可通过 SDS-PAGE和 Western blott ing 检测其芽孢表面展示的重组蛋白， 并检测其对乙 醇的催化活力。
本发明涉及基因 ^sofe编码序列片段克隆的引物序列：
B adJr-F： 5 ' -GGGGTACCATGGCACCGGATTTCGTGAAGCGTT-3 ' ， 下划线表示 Kpnl限制性酶切位点；
B adJ^R: 5 ' -CGAGCTCCTATGTCTTGGAGAGTATTTGGA-3 ' ， 下划线表示 Sacl 限制性酶切位点。
本发明涉及枯草芽孢杆菌淀粉酶基因 aiwE (GenBank序列号: NP— 388186) 片 段扩增的引物序列：
amyE-F: ATTGCTCGGGCTGTATGACTGG
amyE-R: GTTACACCATCACTGTTCGTTCCTT
本发明的突出优点表现为：将家蚕乙醇脱氢酶 BmADH展示在枯草芽孢杆菌表 面， 利用芽孢所具有的独特抗逆性， 使得原本对温度、 PH值不稳定的酶蛋白成 为具有相对稳定的乙醇催化活力的融合蛋白，克服了乙醇脱氢酶普遍具有的不稳 定性缺点。本发明构建的表面展示家蚕乙醇脱氢酶 BmADH的枯草芽孢杆菌重组芽 孢可通过动物消化道屏障直接用于口服，成为一种新型的乙醇脱氢酶营养品或动 物营养品。本发明产品表达量大、制备方法和分离过程相对于其他乙醇脱氢酶样 品更为简单快速， 具有更好的稳定性和更广泛的应用价值， 在一定程度上拓展了 其工业应用前景， 也为提高其他酶蛋白的稳定性提供了参考。 附图说明
图 1是发明人克隆的家蚕乙醇脱氢酶 BmADH在 GenBank中检索到的核苷酸序 列和氨基酸序列， 登录号 NP— 001037610. 1。 小写字母为核苷酸序列， 起始密码 子和终止密码子用方框标出； 大写字母为氨基酸序列， TATA-box 用斜体带下划 线标出， Poly-A信号用下划线标出， 方框中的为保守氨基酸序列。
图 2是融合表达 CotC-BmADH整合型重组质粒 pJS700_BmADH的构建方法及其 结构图谱。 aWE 5， -end和 a/ff 3 ' -end分别表示淀粉酶基因编码序列的 5 ' 端和 3 ' 端， Anipr， ^分别表示氨苄青霉素抗性基因和红霉素抗性基因。 cotC-Bmadh为在 Bacillus subtil is 168 ( trp )的芽孢中融合表达 CotC-BmADH 重组蛋白的基因片段。 oriC为大肠杆菌复制子片段。
图 3是 ^ -co^-^7ao½基因片段在 Bacillus subtil is 168 ( ίι )染色体 中的整合示意图。
图 4是重组菌株 Bacillus subtil is 168 ( trp ) /pJS700-BmADH的淀粉酶活 性分析。 A: 在淀粉平板上培养野生型菌株 ac W^ subtil is 168 ( ir/?— )和重 组菌株 s J s 168 ( ίι ) /pJS700-BmADH; B: 在淀粉平板上被碘液 染色后的野生型菌株和重组菌株。
图 5是 PCR检测重组菌株中的 E -cotC-Bmadh片段。 Marker： λ -EcoT14 I digest Marker； 分另 lj以 Bacillus subtil is 168 ( trp ) (Wi ld-type, W)禾口 Bacillus subtil is 168 ( trp ) /pJS700-BmADH (Mutant, M)的染色体为模板， 用 amyE- F/amyE- R、 BmADH- F/ BmADH -R、 BmADH -F/ amyE-R和 amyE- F/ BmADH -R 四对引物 （引物对的在染色体中的位置见附图 3 ) 进行 PCR鉴定重组基因。
图 6 是转基因重组芽孢表面展示的 CotC-BmADH 融合蛋白的 SDS-PAGE 和 Western blotting鉴定。 M为 Marker, 泳道 1为野生型菌株 Bacillus subtil is 168 ( irp )对照， 泳道 2为重组菌株融合蛋白。
图 7是转基因重组芽孢表面展示的 CotC-BmADH融合蛋白与原 BmADH蛋白在 不同 1¾值和温度条件下的乙醇脱氢酶活力比较。实线所示 PJS700_BmADH表示本 发明所得融合蛋白， 虚线所示 BmADH表示原 BmADH蛋白。
具体实施方式
实验材料： 各种限制性内切酶、 T4 DNA l igase、 LA Tag, Tag , pMD18_T载体均购至宝 生物工程 （大连） 有限公司， PCR引物由上海捷瑞生物工程有限公司合成， 测序 由百奥迈科 (Biomics)生物技术有限公司完成，用于本发明的所有 DNA片段的回收 均采用 Omega Bio- Tek Gel Extract ion Kit分离纯化， 大肠杆菌菌株 DH5 α (中 国普通微生物保藏管理中心 CGMCC, 北京市朝阳区北辰西路 1号院， 中科院微生物 研究所， 100101 ) 为本实验室保存， 其它试剂均为国产分析纯。
在本发明中所使用的术语， 非特别说明的情况下， 一般为本领域普通技术人 员所理解的含义。 以下实施例中未作具体说明的实验操作方法均参照《分子克隆 实验指南》（Sambrook等编著， 科学出版社， 1992年版）、 试剂盒说明书、 及产品 说明书进行。 以下的实施例只是为了举例说明本发明， 并非以任何方式限制本发 明的范围。在以下实施例中， 未详细描述的一些过程和方法都为本领域技术人员 熟悉和了解的常规方法。 所用试剂的来源、 商品名， 均在首次出现时标明， 其后 所用相同试剂如无特殊说明，均与首次标明的相同。本发明实施例中涉及的由发 明人保存的生物材料， 均可向公众提供 20年。
实施例 1: 家蚕乙醇脱氢酶 基因的克隆、 测序及鉴定
本发明所用家蚕品种由江苏大学生命科学院伺养提供。取家蚕中肠组织， 放 入用液氮预冷的研钵中， 加入液氮进行研磨， 用 RNA提取试剂盒分离 mRNA， 通 过反转录 PCR技术获得家蚕乙醇脱氢酶基因的 cDNA序列。
家蚕乙醇脱氢酶 ^0½基因片段的 PCR扩增引物为：
Bmadlr-F: 5 ' - GGGGTACCATGGCACCGGATTTCGTGAAGCGTT- 3， ( SEQ ID NO. 1 ) , 下划线表示 Kpnl限制性酶切位点；
Bmadlr-R: 5， - CGAGCTCCTATGTCTTGGAGAGTATTTGGA- 3， ( SEQ ID NO. 2 ) , 下 划线表示 Sacl限制性酶切位点。
PCR反应体系按 7¾Α¾7& 7¾ί7使用说明书严格进行， 25 反应体系中含 TaKaRa LA Taq 0. 2 μ L, 10 X LA PCR Buffer II (Mg2+ Plus) 2. 5 μ L, dNTP Mixture (各 2. 5 mM) 2. 5 L, 模板 DNA 1 μ L， 上下游引物（20 μ M)各 0. 5 μ L,加灭菌超纯 水补齐。 PCR条件为： 94 °C变性 5min， 94 °C lmin , 61 °C 40 s， 72 °C 2min , 30 个循环。 将大小为 825bp的 PCR产物克隆于 pMD18_T载体中， 转化大肠杆菌 DH5 α， 筛选克隆菌株， 提取质粒， 通过 PCR及 7^ 71和 Sacl双酶切及琼脂糖凝胶电泳鉴定 阳性克隆后， 对重组质粒进行测序。 克隆的重组质粒命名为 pMD18-T-BmADH， 重 组子菌株命名为 DH5 a /pMD18_T-BmADH。 阳性重组菌株保存在含有 15%甘油的 LB 培养基中， 冻存于 -80°C。
经测序重组质粒中乙醇脱氢酶 Bmadh基因序列为 SEQ ID NO. 3所示
gctgcaggaa ttcgaattcg gcaacagtac agtgcactgc gtcgctatct acggacacca gtaacatata aatatgatgt aattgttggt ttccaaatgg caccggattt cgtgaagcgt tttgtggatg tcgacgataa agtgttcttg gtgaccggtg gtgcggccgg cgtcggcgct ggcttggtca aggcgttgct gttcgaaaat gcacggcacg tcgcgtttct agatgtcgct gaccgagaag gggccgccct ggaggcccaa cttataatca agttcggtgc cttgagagtg aagttcataa agtgcgacgt aggagacgaa cgtcaactcg cttctgcata caaacaagtg ttggacaaat acaaaaggct tgacggcgtc ataaacagcg ctgcagtact tagcgtcgat gataactctt tcaacagaat gattgatatc aattttacgg gcacagtgaa tagtactctg aaagcgctcg atattatggg ggcagataaa ggcggttctg gcggtgttat agtgaatata tcgtccctac ttgctcttaa tctcaccagc catttacctg tttacgccgc tactaaagca gcggtattac agttcagcat tagaatgggc acgcaggaac aatttacccg gactaaagta cgggtattgt ccgtatgcct cgggcccact gacactgcta tcctttacaa gaacaactta accaagtttg atacagagta cgctccgtgt ttgtcttcgc gtgcccccgt gagacaaaga gtcgagtcag ccgttaaggg tattttggag gtgatcaaca aagcgagcac cggagatacg tggatcgtgg aaagcgacaa accggcatac gattttactc aaaatatatc cgaagccttc caaatactct ccaagacata__gatttacgag ttattataaa tcggctatac ggcgaaatcg attctgaggc ttcctgcgca aagctaatat tttccttgat gctatatttt ctcataatgt aagatattcg aataggtaaa tattgtgaat cgtattgtta ttggattttg tattacacgt 下划线处分别为起始密码子和终止密码子。 测序结果与 GenBank中
(http : //b la.st. ncbi. nlm. nih. gov/B iast. cgi)的核苷酸序列一致， 见图 1。
实施例 2 : 以 CotC为载体展示 BmADH的枯草芽孢杆菌重组芽孢的制备
1. 重组整合型质粒 pJS700_BmADH的构建
质粒 pJS700(李倩.以 CotX为分子载体表面展示 WSSV囊膜蛋白 Vpl9和 Vp28 的枯草芽孢杆菌重组芽孢的研究 [D] . 江苏镇江： 江苏大学， 2010 : 36-38 ) 由江 苏大学环境学院宁德刚副教授惠赠， 该质粒大小约为 5. 5kb。 在 PJS700质粒中， amyE 5， -end禾 B amyE 3， -end分别表示淀粉酶基因 amyE (GenBank登录号： NP— 388186)编码序列的 5 ' 端和 3 ' 端，通过同源重组整合在 Bacillus sub til is 168 ( irp )染色体的淀粉酶基因中。
淀粉酶基因 a/zy^的 PCR扩增引物为：
a yE-F: ATTGCTCGGGCTGTATGACTGG ( SEQ ID NO. 4 ) a yE-R: GTTACACCATCACTGTTCGTTCCTT ( SEQ ID NO. 5 ) Ampr, ϋ分别表示氨苄青霉素抗性基因和红霉素抗性基因， 在大肠杆菌和 Bacillus subtil is 168 ( ir/?— )中作为筛选标记。 co^为枯草芽孢杆菌芽孢衣壳 蛋白 CotC基因， 该基因片段含有 co^基因（GenBank序列号： NP— 389653)的启 动子序列、 不含有 终止密码子的 CotC编码序列。 oriC为大肠杆菌复制子 片段。
分别用 Kpnl和 酶切质粒 pMD18_T-BmADH和 pJS700， 用琼脂糖凝胶回 收试剂盒回收 Bmadh片段和 pJS700， 用 T4 DNA l igase连接两双酶切后纯化好的 片段， 连接产物转化大肠杆菌 DH5 α 感受态细胞， 转化物涂布于含有 50 g/mL 氨苄青霉素的 LB平板中 37°C培养过夜， 次日挑选阳性单克隆菌株于含有 50 μ g/mL氨苄青霉素的 LB液体培养基中， 37°C培养 12至 16h， 提取质粒， 通过 PCR 及 7^ 71和 feci双酶切， 琼脂糖凝胶电泳鉴定阳性克隆， 鉴定正确后将重组整合 型质粒命名为 pJS700-BmADH， 阳性重组菌株命名为 DH5 a /pJS700_BmADH。 该重 组整合型质粒 pJS700_BmADH中含有重组基因 cotC- Bmadh, 该质粒的构建过程及 其图谱见图 2。
2. 融合表达重组芽孢的枯草芽孢杆菌菌株的筛选与鉴定
将整合型重组质粒 pJS700_BmADH转化
Figure imgf000010_0001
subtil is 168 ( trp ) (Bacillus Genetic Stock Center, Department of Biochemistry, The Ohio State University, West 12th Avenue, Columbus, Ohio, 43210, USA)，两者的同源区域发生 重组， 且重组基因插入淀粉酶基因 a sy 破坏了其淀粉水解酶活性， 见图 3。用 含有 0. 4 g/mL红霉素（Sigma)的 LB平板筛选重组子， 从平板上挑选单菌落接种 于含 1%淀粉的 LB平板中， 37°C过夜培养， 次日用碘-碘化钾溶液对淀粉平板染色 来鉴定重组菌株。 1%淀粉平板的制备方法： 100mL LB固体培养基中加可溶性淀粉 0. lg， 高压蒸汽灭菌后倒制平板。 碘-碘化钾溶液的配制： 碘化钾 2g， 碘 lg， 蒸 馏水 300mL， 先将碘化钾溶于少量去离子水中， 待全部溶解后再加入碘， 振荡溶 解后稀释至 300mL， 避光保存在棕色玻璃瓶中， 用时可稀释 10倍。 取适量碘- 碘化钾溶液滴于淀粉平板上，菌落周围产生透明水解圈表明重组基因没有插入淀 粉水解酶基因 s/sj^中， 菌落及其周围颜色变蓝表明重组基因成功的插入到了 Bacillus subtil is 168 ( ίι )染色体上的淀粉酶基因 a/zy^中， 因而导致淀粉水 解酶失去活性， 见图 4。 为了进一步鉴定重组菌株中正确插入了 E - co tC-Bmad 因， 发明人以枯草芽孢 杆菌的染色体为模板，分别以 afflj¾-F/afflj¾-R、BmADH-F/BmADH-R、BmADH-F/amyE-R 和 amyE-F/BmADH-R四对引物进行 PCR扩增。 PCR反应体系按 7 ¾7& LA 7¾使用说 明书严格进行， 25 μ L反应体系中含 7¾A¾7& LA Taq 0.2 μ L， 10XLA PCR Buffer II (Mg2+ Plus) 2.5 L, dNTP Mixture (各 2· 5 mM) 2.5μ L, 模板 DNA 1 μ L， 上 下游引物（20μΜ)各 0.5 L，最后加灭菌超纯水补齐。 PCR反应条件根据各个引物 对的特征分别设置。 在重组菌株中分别检测到大小约为 4200bp、 825bp、 700bp 和 2650bp的扩增片段， 而野生型菌株中只检测到约 llOObp的淀粉酶基因， 见图 5。 这表明重组菌株染色体上含有^ ^ -co td應且基因， 并且^ -co tC-Bmadti . 组片段成功的插入到了淀粉水解酶基因 s/sj^中。 将鉴定后的重组菌株命名为 Bacillus subtil is 168 ( ίι — ) /pJS700_BmADH。
枯草芽孢杆菌染色体的提取方法为： 挑单菌落接于 LB培养基中， 在 37°C摇床 中培养至 0D6。。值为 1.0左右。 取 1.5mL菌液于 Eppendorf管中， 8000rpm离心 10min， 弃上清， 力口10(^1^ 118.0的了5缓冲液 （10mM Tris-HCl， 1 mM EDTA) 洗沉淀， 离 心去上清， 再加 100 μ L ΤΕ悬浮沉淀； 向悬浮液中加 30 μ L 100mg/ml溶菌酶溶液， 不要摇晃， 在 37°C培养箱中放 lh， 加 50 L 10%SDS和 20 L 20mg/mL蛋白酶 K， 37 °〇继续培养 lh; 补 TE至 300 L， 分别加 150 苯酚和氯仿抽提， 12000rpm离心 lOmin, 取上清； 再加 300 μ ΐ氯仿， 12000rpm离心 5min， 取上清， 加 1/4体积 5M 的 NaCl, 2倍体积的无水乙醇， 室温下沉淀 DNA2h， 12000rpm离心 10min， 收集沉 淀， 用 500 70%的乙醇洗沉淀， 待挥发干后， 加 lO LTE缓冲液溶解沉淀， 取 1 μ L用琼脂糖凝胶电泳检测 DNA浓度， 剩余的可放于 -20°C中保存备用。
3. 表面展示家蚕乙醇脱氢酶重组芽孢的诱导及鉴定
用 DSM培养基诱导重组菌株 ac Wis subtil is 168 ( ίι ) /pJS700_BmADH形 成芽孢。 DSM培养基的配方为: 0.8% nutrient broth (营养肉汤 Difo)， 0.1%KC1， 0.025% MgS04 · 7H20, 1. OmM Ca (N03) 2 · 4H20, 10μΜ MnCl2， 1. ΟμΜ FeS04。 将和重 组菌株分别接种于 DSM培养基中， 采用耗竭法培养 48小时诱导宿主菌产生芽孢， 取培养物进行处理。 芽孢蛋白的处理方法： 12000rpmX10min离心收集菌体， 重 悬于相同体积的 GTE Buffer (50mM葡萄糖， 20mM pH7.5 Tris-HCl, lOmM EDTA, 2mg/mL溶菌酶） 中， 37°C处理 30分钟用以破坏营养细胞， 12000rpmX lOmin沉淀 芽孢， 用 PH7.4的 PBS洗 3次后重悬于相同体积的 SDS-DTT缓冲液 （0. ImM pH 7.4 PBS, 50mM DTT, 1.5%SDS) 中， 65°C水浴处理 lOmin, 进行 SDS-PAGE后用考马斯 亮蓝染色得到大小约为 41kD的条带。为了鉴定该条带是否为目标融合蛋白， 以兔 抗 BmADH血清为一抗做 Western blotting检测重组菌株和野生型菌株的芽孢蛋 白。 杂交结果显示： 野生型菌株 48h的培养物中没有检测到蛋白条带， 而重组菌 株泳道出现大小约为 41kD的条带， 表示在芽孢形成的过程中， BmADH随着芽孢衣 壳蛋白 CotC—同得到表达，因而能作为融合蛋白被 BmADH特异抗体检测到，见图 6。 这表明重组芽孢中家蚕乙醇脱氢酶通过衣壳蛋白载体 CotC被展示在了重组芽孢 的表面。并且从 PAGE胶上可以看出， 孢子上的其他蛋白相对目标融合蛋白表达量 十分小， 即杂蛋白量少， 体现了本发明纯化步骤简单的优点。
4. 融合蛋白的乙醇脱氢酶活力测定
由于乙醇脱氢酶在催化乙醇的过程中消耗辅酶 NAD+产生 NADH, 其酶活力可通 过用紫外分光光度计在 340nm处检测 NAD7NADH的含量测得。 反应液成分及体积比 为： 2 M 乙醇： 0.5M NAD+: 缓冲液 =1: 3： 2。 将本反应体系在一定温度下孵育 10分钟后， 按 30: 1的体积比加入重组孢子蛋白。 在第 1、 4、 6、 8、 10分钟分别 取混合液离心后记录上清液在 340nm处吸光值， 读数差值记为 AA34。。 以野生型菌 Bacillus subtil is 168 ( trp)的孢子蛋白作为负对照。 每分钟消耗 Ιμπιοΐ NAD+ 被定义为 1个单位 （unit) 的乙醇脱氢酶活力。 计算公式： （AA34。XV) I (6.22 XbXWXAt) , V为反应液总体积， b为比色皿厚度， W为样本蛋白含量， 用 BCA 蛋白定量试剂盒 （Pierce) 测得， At为吸光值改变的前后时间差 （分钟）。
为了测得融合蛋白酶活力在不同 ra值下的稳定性，发明人选取了 0.1M醋酸钠 缓冲液， pH4.0; 0.1M磷酸盐缓冲液 （NaH2P04 - Na2HP04)， pH 7.0； 0.1M NaOH/ 甘氨酸缓冲液， pH8.0、 9.0、 10.0， 分别作为缓冲液加入反应液体系， 测定 25 °C时不同 ra值条件下的酶活力。 为了测得融合蛋白酶活力在不同温度下的稳定 性， 发明人以 PH9.0的 0.1M NaOH/甘氨酸为缓冲液， 分别测定融合蛋白在 16°C、 20°C、 25°C、 30°C、 37°C下的酶活力。 将结果与原来的 BmADH蛋白酶活力做比较 (Nan Wang, et al.2011. Genetics and Molecular Biology, accepted), 结果显 本发 明所的产品的酶活力稳定性大大增加， 见图 7。

Claims

权利要求书
1. 一种表面展示家蚕乙醇脱氢酶重组芽孢的制备方法， 其特征在于： 利用枯草 芽孢杆菌芽孢衣壳蛋白基因为分子载体， 与家蚕乙醇脱氢酶基因重组， 构建 融合表达的整合型重组质粒， 并转化枯草芽孢杆菌得到枯草芽孢杆菌重组菌 株， 诱导重组菌株产生的芽孢表面展示家蚕乙醇脱氢酶的重组芽孢。
2. 按照权利要求 1所述的表面展示家蚕乙醇脱氢酶重组芽孢的制备方法， 其特 征在于： 所述的枯草芽孢杆菌芽孢衣壳蛋白基因为 C。
3. 按照权利要求 2所述的表面展示家蚕乙醇脱氢酶的重组芽孢的制备方法， 其 特征在于： 所述的融合表达的整合型重组质粒是将枯草芽孢杆菌芽孢衣壳蛋 白基因 与家蚕乙醇脱氢酶基因重组构建的质粒。
4. 按照权利要求 3所述的表面展示家蚕乙醇脱氢酶的重组芽孢的制备方法， 其 特征在于： 所述的枯草芽孢杆菌重组菌株是整合型重组质粒转化枯草芽孢杆 菌所得到的菌株，该菌株染色体上含有适用于枯草芽孢杆菌的选择标记基因、 以及芽孢衣壳蛋白基因与家蚕乙醇脱氢酶基因的编码序列重组后的基因， 这 些基因插入淀粉酶基因中导致重组菌株的淀粉酶基因失活。
5. 按照权利要求 4所述的表面展示家蚕乙醇脱氢酶的重组芽孢， 其特征在于， 重组芽孢是枯草芽孢杆菌重组菌株诱导形成的枯草芽孢杆菌的芽孢， 芽孢表 面展示有家蚕乙醇脱氢酶 BmADH。
6. 一种表面展示家蚕乙醇脱氢酶重组芽孢，其特征在于： 是通过以下方法制备: 利用枯草芽孢杆菌芽孢衣壳蛋白基因为分子载体， 与家蚕乙醇脱氢酶的基因 重组， 构建融合表达的整合型重组质粒， 并转化枯草芽孢杆菌得到枯草芽孢 杆菌重组菌株， 诱导重组菌株产生的芽孢表面展示家蚕乙醇脱氢酶的重组芽 孢。
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