CN106834161B - Bacillus subtilis Z12 and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to bacillus subtilis for protein expression and application thereof, which is characterized in that: the Bacillus subtilis Z12(Bacillus subtilis Z12) is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 12750. The strain of the invention is bacillus subtilis which can be transformed with high efficiency and has large protein secretion amount, and can be used as an original strain of a bacillus subtilis expression system. Its transformation efficiency can be up to 1.8X 106cfu/ug, about 2X 10 can be obtained by a single electric shock5The single bacterial colony has high transformation efficiency and can meet the requirements of harsh experiments such as library building and the like. The bacillus is confirmed to be biologically safe by biochemical identification and molecular identification. The secretion capacity of the strain is high and is more than 3 times that of the model strain 168.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an original strain-bacillus subtilis Z12 for protein expression and application thereof.
Background
The protein expression technology is one of the core technologies of modern biology, and the expressed protein can be used for biological research and can also provide commercial protein products, such as recombinant vaccines, recombinant insulin, cytokines and other products. Currently, common expression systems include Escherichia coli, yeast, insect cells, mammalian cells, and the like, but all of them have obvious advantages and disadvantages. Coli expression systems are the most well studied and have many options, the most commonly used being the pET expression system of Novagen, which uses bacteriophage T7RNA polymerase to specifically transcribe the target gene behind the T7 promoter. Under the optimal condition, the target protein can reach more than 50 percent of the total protein of the escherichia coli. Although the method has the advantages of high expression efficiency, low culture cost and the like, the disadvantages are also very obvious: the protein is easy to form inclusion bodies, and the renaturation difficulty and the cost are higher; escherichia coli can not carry out glycosylation modification on protein; the cell wall contains lipopolysaccharides (endotoxins) and is not easily removed completely. Another commonly used expression system is a yeast expression system, which has the advantages of high expression quantity, inducibility, easy purification of protein secreted to the extracellular space, certain post-translational modification capability and the like, but has the disadvantages that part of expression products are easy to degrade, the expression quantity is uncontrollable, and the protein more than 30KDa can not be secreted almost. The insect cell and animal cell expression system features complete modification system, natural activity of the expressed product, no endotoxin pollution, low expression amount, long period, high technological requirement and high production cost.
The bacillus subtilis is a gram-positive bacterium widely existing in water, air and soil, can produce spores when the environment is not suitable, can resist extreme environments such as high temperature, drought and the like, and germinates for vegetative growth when the environment is suitable. The bacillus subtilis has strong secretion capacity, and the protein secretion amount can reach 20-25 g/L when high-density fermentation is carried out. The products such as protease, amylase, inosine, riboside and the like produced by fermentation of the strain have already entered the daily life of people. Because of the biological safety, the probiotic produced by the additive is generally recognized as GRAS by FDA (food and drug administration) judges, and the fermentation product produced by the probiotic is widely applied to the medical breeding industry.
In the eighties of the last century, the bacillus subtilis is used for protein expression, and the expression products of the bacillus subtilis not only comprise various enzymes from bacteria, such as amylase, protease, endoglucanase, lipase and the like, but also comprise proteins from different sources, such as hEGF, IFN-alpha 2, Proinsulin, Streptavidin, cathelicidin-BF and the like. The expressed protein can be secreted into the fermentation liquor by utilizing the secretion route of the bacillus subtilis, thereby greatly reducing the difficulty of later separation and purification. The bacillus subtilis belongs to gram-positive bacteria, does not contain lipopolysaccharide, and is convenient for producing injection medicines. The bacillus subtilis has simple nutrition requirement, and has low difficulty of large-scale culture technology and low culture cost because the bacillus subtilis realizes large-scale industrial production. At present, genome sequencing work of multiple strains in bacillus subtilis and multiple strains in bacillus is completed, the genetic background is clear, the safety is high, and genome modification is facilitated. However, the Bacillus subtilis expression system is not a widely used expression system and has a number of disadvantages to overcome.
The wild type strain of the 168 Bacillus subtilis model strain has been lost, and all strains available at present are mutants thereof. Although it meets the requirements of scientific research, the protein secretion capacity is poor, and the protein is a characteristic of auxotrophic mutants and cannot meet the requirements of establishing commercial expression systems. The expression systems established at present are based on the model strain 168, although the strains of wild type origin and the Bacillus subtilis species already used are very numerous and difficult to transform, preventing further modification of these strains.
Disclosure of Invention
In order to solve the technical problems, the invention provides bacillus subtilis Z12 which has the characteristics of high transformation efficiency and large protein secretion.
The invention discloses a bacillus subtilis Z12 for solving the technical problems, which is characterized in that: the Bacillus subtilis Z12 (Latin is Bacillus subtilis) is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.12750, the preservation date is 2016, 7 and 11 days, and the preservation address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The bacillus subtilis colony is round, white or light yellow, opaque, rough in surface, wrinkled and irregular in edge. The growth process of Z12 is aerobic, the optimum pH value for growth is 7.0-8.5, the optimum temperature is 30-45 ℃, and gram staining is positive.
The Bacillus subtilis is applied as an original strain of a Bacillus subtilis expression system.
The method for cultivating the bacillus subtilis comprises the following steps:
(1) collecting wild strains: collecting soil containing bacillus subtilis, wherein the ratio of the soil to the culture medium is 1: adding LB culture medium into 100, culturing at 37 deg.C and 200rpm for 24h to obtain bacterial suspension rich in Bacillus spore, and obtaining original strain.
The soil can be collected in original forest areas and places rich in degrading bacteria.
(2) Strain enrichment and screening: taking 1ml of bacterial liquid, heating at 85 ℃ for 30min, and killing all thalli; then press 103And 105Diluting and plating, LB culture medium, culturing at 37 deg.C overnight; the obtained single colony is streaked again, and purity identification is carried out.
(3) Strain transformation and screening:
the strain transformation method is referred to as hyperosmotic transformation method:
and (3) competent preparation: the single colony obtained in step (2) was inoculated into 2-3ml of GM (LB +0.5M sorbitol) medium and cultured overnight at 37 ℃ and 180 rpm. Inoculating to 50ml GM culture medium at 37 deg.C and 180rpm in the morning at a ratio of 1:100, culturing, and precooling the bacterial liquid on ice for 10min when the bacterial liquid grows to OD of 0.85-0.95; centrifugation at 5000g at 4 ℃ removed the supernatant and resuspension of the cells with an equal volume of precooled EM (0.5M sorbitol +0.5M mannitol + 10% glycerol in water) for a total of 4 replicates; adding about 1/40 volume of EM to resuspend the bacteria, ensuring the concentration of the bacteria liquid to be 1-1.3X 1010fu/ml.
And (3) electric conversion: 60ul of the bacterial liquid is taken, 1ul of plasmid to be transformed (pDG148 or pBAV1K-T5-GFP) is added, the concentration of the plasmid is more than 100ng/ul, and the mixture is blown and evenly mixed. Then add a pre-cooled 1mm shock cup. The electric conversion instrument (EppendorfEporator) has the parameter setting of 2.1kV, and the growth of the conversion colony is possible only when the actual electric shock time is 4.0-5.0 ms. After electric shock, 1ml of RM (LB +0.5M sorbitol +0.38M mannitol) was added immediately, blown up and mixed well, transferred into a 5ml sterile centrifuge tube, and cultured at 37 ℃ and 180rpm for 3 hours. The bacterial liquid was centrifuged, the supernatant was removed and diluted in a certain ratio, 200ul was spread on an LB plate containing 30mg/L kanamycin and cultured overnight at 37 ℃ while the bacterial liquid without electrotransformation was spread as a negative control. And observing the colony growth condition in the next morning, if the colony grows on the transformation plate and the negative control does not exist, indicating that the strain can be transformed, and numbering and recording the strain.
If the transformation efficiency needs to be detected, counting the total number of growing colonies, multiplying by the dilution times, and calculating the transformation efficiency according to the addition amount of the plasmid.
In the invention, a hypertonic electrotransformation method is adopted, the transformation plasmid is pDG148 or pBAV1K-T5-GFP, and the screened bacillus subtilis Z12 can be efficiently transformed and has large protein secretion amount, and can be used as an original strain of a bacillus subtilis expression system. Its transformation efficiency can be up to 1.8X 106cfu/ug, about 2X 10 can be obtained by a single electric shock5The single colony has high transformation efficiency and can meet the requirement of establishing a screening library.
The protein concentration of the fermentation liquor of the invention is as follows: the Z12 strain obtained by screening can reach about 378mg/L of protein secretion when cultured in LB culture medium for 48h, while the model bacterium 168 can only reach about 121mg/L, and the protein secretion capacity of Z12 is far higher than that of the model bacterium 168.
The bacterial strain is characterized and identified, and the colony morphology of the screened bacillus subtilis Z12 is as follows: the colony is round, white or light yellow, opaque, rough, wrinkled and irregular in edge. The growth process of Z12 is aerobic, the optimum pH value for growth is 7.0-8.5, the optimum temperature is 30-45 ℃, and gram staining is positive. The bacillus subtilis is confirmed to be bacillus subtilis through physiological and biochemical identification and molecular identification. The bacillus is confirmed to be biologically safe by biochemical identification and molecular identification. The secretion capacity of the strain is high and is more than 3 times that of the model strain 168.
Drawings
FIG. 1 is a comparative PAGE electrophoresis chart of fermentation liquid of Z12 and 168 model strains in the invention
Detailed Description
The present invention will be described in further detail with reference to the following embodiments, wherein the original plasmid is commercially available, extracted and purified: the extraction and purification are carried out by adopting a kit (a universal plasmid miniprep kit DE-01001 of the Fujian organism). Example 1
Bacillus subtilis Z12, Latin Bacillus subtilis, and is preserved in China general microbiological culture Collection center (CGMCC), with the preservation number of CGMCC No.12750 and the preservation address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The screening and breeding steps of the strain are as follows:
(1) sampling
In the Zhao Gongshan forest area of the city of Jiangfan, Sichuan province, soil samples are randomly taken and numbered.
(2) Enrichment and screening of strains
About 1g of soil sample was inoculated into LB medium and cultured at 37 ℃ and 200rpm for 24 hours.
Taking 1ml of bacterial liquid, heating at 85 deg.C for 30min to kill all thalli, and then treating according to the formula of 103The plate was diluted, plated in LB medium and cultured overnight at 37 ℃. The obtained single colony is streaked again, and purity identification is carried out. Since the Bacillus was left, further transformation was possible as long as it was determined that different colonies were present.
Purity was determined by streaking, and all single colonies were morphologically identical.
(3) Strain transformation:
a single colony obtained in (2) was inoculated into about 3ml of GM (LB +0.5M sorbitol) medium and cultured overnight at 37 ℃ and 180 rpm. Inoculating to 50ml GM culture medium at 37 deg.C and 180rpm in the morning at a ratio of 1:100, culturing, and precooling the bacterial liquid on ice for 10min when the bacterial liquid grows to OD of 0.85-0.95; centrifugation at 5000g at 4 ℃ removed the supernatant and resuspension of the cells with an equal volume of precooled EM (0.5M sorbitol +0.5M mannitol + 10% glycerol in water) for a total of 4 replicates; adding about 1/40 volume of EM to resuspend the bacteria, ensuring the concentration of the bacteria liquid to be 1-1.3X 1010Between cfu/ml. Take 60uAdding 1ul of plasmid to be transformed (pDG148 or pBAV1K-T5-GFP) into the bacterial liquid, wherein the concentration of the plasmid is more than 100ng/ul, uniformly blowing, and then adding a precooled 1mm electric shock cup. The electric conversion instrument (Eppendorf Eporator) has the parameter setting of 2.1kV, and the growth of the conversion colony is possible only when the actual electric shock time is 4.0-5.0 ms. After electric shock. Immediately adding 1ml RM (LB +0.5M sorbitol +0.38M mannitol), pipetting, mixing, transferring into a 5ml sterile centrifuge tube, and culturing at 37 deg.C and 180rpm for 3 h. The bacterial liquid was centrifuged, the supernatant was removed and diluted in a certain ratio, 200ul was spread on an LB plate containing 30mg/L kanamycin and cultured overnight at 37 ℃ while the bacterial liquid without electrotransformation was spread as a negative control. And observing the growth condition of the colonies on the next morning, if the colonies grow on the transformation plate and the negative control is not available, indicating that the strain can be transformed and numbering the strain. If the transformation efficiency needs to be detected, counting the total number of growing colonies, multiplying by the dilution times, and calculating the transformation efficiency according to the addition amount of the plasmid.
Example 2
Determination and analysis of protein content in fermentation liquor
(1) Bacterial liquid culture
A single colony obtained in step (3) of example 1, which was highly transformed, was inoculated into 3ml of LB medium and cultured overnight at 37 ℃ and 180 rpm. The next day, the cells were inoculated into 50ml of LB medium at a ratio of 1:100, and cultured at 37 ℃ and 180rpm for 48 hours. Taking all the bacteria liquid, centrifuging, and taking the supernatant for subsequent tests.
(2) Determination of protein content in bacterial liquid
The bacterial liquid obtained in the step (1) is taken, protein is concentrated by adopting a general protein precipitation reagent (No. c510012), and the polypeptide added in the culture medium is removed.
Protein concentration was determined using a modified BCA protein concentration assay kit (biologies, No. c 503051).
(3) PAGE electrophoresis
100ul of the bacterial solution obtained in the above (1) was added with 2 Xloading buffer and treated at 95 ℃ for 5 min. Electrophoresis was performed on a 10% SDSPAGE gel, with a loading of 20 ul.
Wherein the fermentation supernatant of the model bacteria 168 and the Bacillus subtilis Z12 is shown in an electrophoresis chart 1.
When the bacillus subtilis Z12 is cultured in an LB culture medium for 48 hours, the protein secretion amount can reach about 378mg/L, the model bacterium 168 can only reach about 121mg/L, and the protein secretion capacity of the bacillus subtilis Z12 is far higher than that of the model bacterium 168.
Example 3
Identification of strains
(1) The original colonies and the transformed strains in example 1 were streaked onto LB plates, cultured at 37 ℃ for 24 hours, and the colony morphology was observed.
(2) The colonies obtained in (1) were subjected to gram staining and observed.
(3) Taking the single colony in the step (1), inoculating about 3ml of LB culture medium, and culturing overnight. Bacterial genome DNA extraction kit (Chengdu Fuji organisms, DE-05311) was used to extract bacterial genome. According to the literature (Phylogenetic biology of the genetic transformed by synthetic analysis of small-subbunit-ribosomal RNA sequences, 1991), 16S DNA fragments were amplified, sequenced and aligned on genbank, and the similarity of the 16S DNA sequence of Z12 to several species of Bacillus was found to be greater than 99.5%.
Wherein the primer sequence for amplifying the 16S DNA fragment, F: AGAGTTTGATCCTGGCTCAG, R: GGTTACCTTGTTACGACTT, annealing temperature 60 deg.C, and extension for 2 min.
Primers were synthesized according to the literature (genomic analysis of Bacillus subtilis and related tax based partial gyrA gene sequences, 2000), the gyrA fragments were amplified, sequenced and aligned on GenBank, and the sequence was found to have a similarity of more than 99.5% to the gyrA sequence of multiple strains of Bacillus subtilis species.
Wherein the primer sequence for amplifying the gyrA fragment, F: CAGTCAGGAAATGCGTACGTCCTT, R: CAAGGTAATGCTCCAGGCATTGCT, annealing temperature 60 ℃, and extending for 2 min.
The above results indicate that the Z12 strain is Bacillus subtilis belonging to the genus Bacillus.
Example 4
The single colony obtained in (1) of example 3 was collected and subjected to the method described in Bergey's Manual of bacteria identification, ninth edition, and the results of physiological and biochemical tests of Z12 and model bacterium 168 are shown in the following table:
| feature(s) | |
168 | Feature(s) | |
168 |
| Starch hydrolysis | + | + | VP reaction | + | + |
| Gelatin hydrolysis test | + | + | Catalase enzyme | + | + |
| Fermentation test of glucose | + | + | Xylose fermentation experiment | + | + |
| Lactose fermentation experiment | - | - | Citric acid salt | + | + |
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (2)
1. A bacillus subtilis Z12 characterized in that: the Bacillus subtilis Z12 and the Latin Bacillus subtilis Z12 are preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is 12750; the bacillus subtilis colony is round, white or light yellow, opaque, rough in surface, wrinkled and irregular in edge.
2. The use of Bacillus subtilis Z12 according to claim 1, wherein: the Bacillus subtilis is applied as an original strain of a Bacillus subtilis expression system.
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| PCT/CN2017/091056 WO2018099063A1 (en) | 2016-11-30 | 2017-06-30 | Method for efficiently secreting and expressing foreign protein using bacillus |
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