CN104862297A - Genetic engineering-modified staphylococcus aureus staphylophage lyase as well as preparation method and application thereof - Google Patents
Genetic engineering-modified staphylococcus aureus staphylophage lyase as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a genetic engineering-modified staphylococcus aureus staphylophage lyase, the amino acid sequence of which is shown in SEQ ID NO:1. The invention also discloses a preparation method of the staphylococcus aureus staphylophage lyase, comprising the following concrete steps: (1) cloning the amino acid sequence shown in the SEQ ID NO:1 into a prokaryotic expression vector to obtain a recombinant plasmid; (2) transforming the recombinant plasmid obtained in the step (1) into host bacteria to obtain recombinant bacteria; (3) expressing the staphylococcus aureus staphylophage lyase by the recombinant plasmid; and (4) purifying the staphylococcus aureus staphylophage lyase obtained in the step (3). Multiple staphylococcus aureus can be specifically inactivated by independently using the genetic engineering-modified staphylococcus aureus staphylophage lyase or matching the genetic engineering-modified staphylococcus aureus staphylophage lyase with other compounds, thus providing a safe enzymic preparation source without toxic and side effects for staphylococcus aureus infection, particularly staphylococcus aureus caused dairy mastitis in the existing dairy farm control.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of staphylococcus aureus bacteriophage lyase and preparation method and application.
Background technology
Streptococcus aureus (Staphylococcus aureus) is a kind of important Zoonosis cause of disease bacterium, the various diseases such as wound infection, endocarditis, osteomyelitis, toxic shock syndrome can be caused, the mammitis of cow disease incidence caused diary farm streptococcus aureus up to 35-59%, greatly have impact on the milk yield of China milk cow, produce economic benefit even China's milk qualities competitive power in the world and the reputation of the quality of milk, diary farm.
At present, S. aureus L-forms infects the mammitis of cow of initiation mainly through 1) microbiotic extension water systemic injection, injection of breast or the mode such as perfusion, nipple infiltration, with 2) S. aureus L-forms vaccine immunity, 3) method such as herbal medicine use controls and treatment, although the bacteriological infection that microbiotic can inhibit streptococcus aureus to cause to a certain extent, antibiotic life-time service brings the harm of the pathogenic bacterium resistance that world wide inside irreversible turns.This harm not only causes huge financial loss, and serious health threat is caused to the mankind, mainly comprise two aspects: first, antibiotic use causes constantly producing multidrug resistant S. aureus L-forms for many years, as Methicillin-resistant Staphylococcus aureus (methicillin-resistant S.aureus, and VRSA (vancomycin-resistant S.aureus MRSA), and the appearance of these resistant organisms makes to need in a hurry research and development novel antibacterial medicine VRSA).And the appearance of new antibiotic can cause producing the S. aureus L-forms that more resistance is more difficult to prevent and treat, thus define vicious cycle.The second, because treatment S. aureus L-forms infects antibiotic remains in the milk caused, bring serious food safety hidden danger, the indirect hazard health of the mankind.Therefore, the study hotspot that novel antibacterials become extremely urgent social needs and various countries scientist is researched and developed.
Phage is as bacterium obligatory parasitism virus, be divided into lytic phage and lysogenic phage, wherein lytic phage can by absorption, inject genome, re-assembly virion, cracking bacterium release progeny phage virion completes a viral replacement cycle, reach fragmented for bacteria lysis object simultaneously.
Since 1915 find phage, phage has been used for the treatment of the treatment of the dysentery, wound infection etc. of soldier by the Soviet Union, Reichsweshr etc., and achieves good result for the treatment of.In the recent period, due to the appearance of bacterial resistance bacterium and superbacteria, phage the characteristic of cracking bacterium can be paid close attention to by various countries' scientists again and thinks a kind of potential novel antibacterial medicine and become study hotspot again because of it.2007, U.S. FDA have approved the preservation sterilization of listeria bacteria phage preparation for poultry and cheese, and within 2014, Government Of New Zealand have approved Escherichia coli O 157 phage for butchering the purification and sterilization of front milk cow.
Current, phage is as life virus also to have some scientists to think, making an addition in food for bacterial control and treatment has certain risk.Therefore, present many scientists turn to and start that the gene of expressing lyase in phage is carried out the higher lyase of recombinant expressed security and be used for the treatment of bacteriosis and the control of Bacterial Contamination.
Bacterial virus catenase (Bacteriophage lysin) is a class cell wall protein lytic enzyme of expressing by double-stranded DNA phage genome encoding and in the phage-infect bacterium later stage, can directly destroy the peptidoglycan of bacteria cell wall thus make bacteria lysis.It is generally acknowledged, lyase has two structural domains, the structural domain of the structural domain with cracking function being namely positioned at N end and the decision host specificity being positioned at C end.
Research in recent years shows, for gram-positive microorganism, the lyase of phage can identify bacteria cell wall acceptor and cracking bacterium.Bacterial virus catenase as a kind of antibacterials of new type of safe have efficient fast, fragmentation pattern is wide, security is high, be difficult to produce resistance bacterium, can the effective advantage such as cracking multi-drug resistant bacteria, more and more paid attention to.Both at home and abroad in increasing body, animal experiment shows that lyase has very high bactericidal properties, has wider fragmentation pattern and the raw resistance bacterium of difficult labour than phage, to the cracking performance of resistance pathogenic bacterium and the synergistic high efficiency of other antiseptic-germicides, and is not affected the effect of lyase by the antibody using lyase to cause producing in animal body in body.Therefore this research is expected to the research and development of staphylococcus aureus bacteriophage lyase to become the disease that a kind of control is caused by infection of staphylococcus aureus and the novel antibacterial preparation controlling S. aureus L-forms in animal derived food.
At present, about have S. aureus L-forms kill or the bacterial virus catenase report of splitting action few, the patent (ZL201210157894.6) that this laboratory member Zhang Hui declares is a kind of lyase gene coming from natural S. aureus L-forms phage, without transformation and restructuring, also different with aminoacid sequence from bacterial virus catenase LysS1 nucleotide sequence of the present invention, be two kinds of different lyase.
Summary of the invention
The technical issues that need to address of the present invention are, provide a kind of staphylococcus aureus bacteriophage lyase.
The technical problem that the present invention also will solve is, provides the preparation method of above-mentioned staphylococcus aureus bacteriophage lyase.
The technical problem that the present invention finally will solve is, provides the application of above-mentioned staphylococcus aureus bacteriophage lyase
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
Through a genetic engineering modified staphylococcus aureus bacteriophage lyase, the aminoacid sequence of this staphylococcus aureus bacteriophage lyase is as SEQ ID NO:1.
Through an encoding gene for genetic engineering modified staphylococcus aureus bacteriophage lyase, its nucleotide sequence is as shown in SEQ ID NO:2.
Comprise the plasmid of the encoding gene of above-mentioned staphylococcus aureus bacteriophage lyase also within protection scope of the present invention.
Comprise the recombinant bacterium of above-mentioned plasmid also within protection scope of the present invention.
A preparation method for staphylococcus aureus bacteriophage lyase, the method comprises the steps:
(1) nucleotide sequence shown in SEQ ID NO:2 is cloned in expression vector, obtains recombinant plasmid;
(2) by the recombinant plasmid transformed Host Strains that step (1) obtains, recombinant bacterium is obtained;
(3) recombinant bacterium is utilized to express staphylococcus aureus bacteriophage lyase;
(4) the staphylococcus aureus bacteriophage lyase that obtains of purification step (3).
In step (1), described expression vector is pET32b (+).
In step (2), described Host Strains is intestinal bacteria TransB (DE3).
In step (3), utilize IPTG to induce recombinant bacterium to express staphylococcus aureus bacteriophage lyase, the concentration of IPTG is 1.0 ~ 1.2mmol/L, and the temperature of abduction delivering is 20 ~ 26 DEG C.
Utilize the concrete grammar of recombinant bacterium pan staphylococcus aureus bacterial virus catenase as follows:
Nucleotide sequence shown in SEQ ID NO:2 is cloned on pET32b (+) plasmid, obtain recombinant plasmid called after: pET32b-LysS1, by pET32b-LysS1 plasmid transformation escherichia coli TransB (DE3), the recombinant bacterium called after TransB (pET32b-LysS1) obtained.
Recombinant bacterium TransB (pET32b-LysS1) is inoculated in the LB nutrient solution containing penbritin (50 μ g/mL), 37 DEG C of shaking culture 12h; Be 1% be forwarded in 100mL LB substratum by volume, 37 DEG C of shaking culture are to OD
600when value is about 0.5, add IPTG to final concentration 1.0mmol/L, 26 DEG C of induction 20h.Collect thalline, ultrasonic disruption cell, collect supernatant supernatant His affinity chromatography ni-sepharose purification.The lyase product of purifying is carried out detoxification process (≤0.01EU/ μ g intracellular toxin) through detoxification element test kit.
The staphylococcus aureus bacteriophage lyase that the preparation method of above-mentioned staphylococcus aureus bacteriophage lyase prepares is within protection scope of the present invention.
Above-mentioned staphylococcus aureus bacteriophage lyase suppresses being applied within protection scope of the present invention in the medicine of staphylococcus aureus growth in preparation.
Above-mentioned staphylococcus aureus bacteriophage lyase being applied within protection scope of the present invention in preparation control mammitis of cow medicine.
Above-mentioned staphylococcus aureus bacteriophage lyase is preparing the application in dairy cow farm epidemic prevention drug.
Above-mentioned staphylococcus aureus bacteriophage lyase is suppressing the application in Staphylococcus aureus in food growth, and staphylococcus aureus bacteriophage lyase prepared by the present invention can suppress staphylococcus aureus in milk to grow.
Beneficial effect:
(1) streptococcus aureus lyase is cloned in pET32b (+) plasmid by the present invention, and by recombinant plasmid transformed in intestinal bacteria, enters abduction delivering and prepare streptococcus aureus lyase.
(2) the streptococcus aureus lyase that prepared by the present invention has splitting action to multiple streptococcus aureus.
Accompanying drawing explanation
The structure of Fig. 1 lyase LysS1 gene and qualification; Wherein, swimming lane 1 ~ 5 is the qualification of recombinant expression plasmid pET32b-LysS1Nde I/Xho I double digestion, and swimming lane 6 is the qualification of empty control plasmid pET32b (+) Nde I/XhoI double digestion, and Ml is 2Kb DNA ladder, Mr is 15Kb DNA ladder.
Fig. 2 recombinates the identification and analysis of lyase LysS1 expression product; Swimming lane 1 is the restructuring lyase LysS1 of purifying, swimming lane 2 is abduction delivering TransB (pET32b) 1.0mmol/L IPTG, swimming lane 3 is abduction delivering TransB (pET32b-LysS1) 1.0mmol/L IPTG, and M is protein molecular mass M arker.
Fig. 3 recombinates the lytic activity analysis of lyase LysS1 to streptococcus aureus; A: streptococcus aureus X12 drips restructuring lyase LysS1 and empty carrier pET32b (+) contrast outward, and B: streptococcus aureus S6 bacterium drips restructuring lyase LysS1 and empty carrier pET32b (+) contrast outward.
Fig. 4 recombinates the sterilizing ability analysis of lyase LysS1 to streptococcus aureus; A: streptococcus aureus S6 bacterium adds restructuring lyase LysS1 outward; B: contrast, streptococcus aureus S6 does not add lyase.
Fig. 5 recombinates the sterilizing ability analysis of lyase LysS1 to streptococcus aureus S6; PBS: damping fluid, Elutionbuffer: damping fluid, S: thick enzyme LysS1, P: restructuring lyase LysS1, Control: do not add enzyme contrast.
Fig. 6 recombinates lyase LysS1 sterilization effect analysis in food; PBS: damping fluid, LysS1: restructuring lyase, Control: do not add enzyme contrast.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
The present invention tests and uses streptococcus aureus YZ-12, YZ-15, YZ-16, YZ-19, YZ-34, YZ-42, YZ-49, YZ-56, XG-9, XG-25, SF-16, SF-22, SF-37, SF-46, SF-54, SF-56, A14, A16, X5-1, X12, M-9, S4 and S6 is this room isolation identification and preserves, Salmonella typhimurium (HNER178, Salmonella Typhimurium is also that this laboratory is separated preservation, streptococcus aureus (ATCC25923), Salmonella enteritidis (ATCC13076, Salmonella Enteritidis) and intestinal bacteria (ATCC25922) all purchased from ATCC.The conventional bacterial strain that intestinal bacteria TransB (DE3) is this area, the conventional plasmid that pET32b (+) is this area.Embodiment 1: the clone of lyase LysS1 gene (lysS1), the structure of expression vector.
A, first design lyase LysS1 gene, by its called after lyase LysS1 gene, its nucleotides sequence is classified as Seq IDNO.1;
B, lyase LysS1 gene are transferred to the raw work synthetic gene in Shanghai and carry out subclone to obtain recombinant expression plasmid and called after pET32b-LysS1; Next day transformation of E. coli Trans1-T1 competent cell, coat the flat board containing penbritin (50 μ g/ml), 37 DEG C of overnight incubation, picking positive colony is inoculated in the LB liquid nutrient medium containing penbritin (50 μ g/ml), after 37 DEG C of overnight incubation, extract recombinant plasmid (specifically illustrate by test kit and carry out) with Dongsheng plasmid extraction kit and carry out Nde I and the qualification of Xho I double digestion, 3h is hatched in 37 DEG C of water-baths, 1% agarose electrophoresis, uses pET32b (+) empty plasmid in contrast simultaneously;
C, double digestion identified correct plasmid delivers the order-checking of order-checking company, by plasmid called after pET32b-LysS1 correct for order-checking qualification; Next day, transformation of E. coli TransB (DE3) competent cell, coated the LB flat board containing penbritin (50 μ g/ml), 37 DEG C of overnight incubation by the bacterium liquid of conversion.Picking positive colony is inoculated in the LB liquid nutrient medium containing penbritin (50 μ g/ml), 37 DEG C of overnight incubation, recombinant bacterium called after TransB (pET32b-LysS1).
As shown in Figure 1, after recombinant plasmid pET32b-LysS1 Nde I and Xho I carries out double digestion, occur pET32b (+) belt carrier and lysS1 genonema at 5.4kb and 1kb place respectively, size conforms to expection result, shows to build correctly.
Embodiment 2: the abduction delivering of lyase LysS1 albumen and purifying
Be inoculated into by recombinant bacterium TransB (pET32b-LysS1) in the LB nutrient solution containing penbritin (50 μ g/mL), 37 DEG C of shaken overnight are cultivated; Next day, be forwarded in 100mL LB substratum in 1:100 ratio, 37 DEG C of shaking culture are to OD
600when value is about 0.5, add IPTG to final concentration 1.0mmol/L, 26 DEG C of induction 20h.Collect thalline, ultrasonic disruption cell, 4 DEG C, the centrifugal 10min of 10,000rpm/min, collect supernatant, and by supernatant through 0.22 μm of membrane filtration, the protein expression situation in SDS-PAGE analytical pyrolysis supernatant.By cracking supernatant His affinity chromatography nickel post (GE Healthcare, the Sweden) purifying filtered, specifically illustrate that step is poly-by test kit and carry out.Obtaining protein designations is lyase LysS1, and the lyase LysS1 product of purifying is carried out detoxification process (≤0.01EU/ μ g intracellular toxin) through detoxification element test kit (Novagen company).
SDS-PAGE analytical results as shown in Figure 2, recombinant bacterium TransB (pET32b-LysS1) is after IPTG induction, thicker induced protein bands is had in the position of about 50kD in its supernatant, conform to expection size, thus show, recombinant bacterium TransB (pET32b-LysS1) builds correct, and the lyase protein product LysS1 expressed is soluble proteins.
Embodiment 3: the enzyme spectrum analysis of lyase LysS1
LB (containing 1.2% agarose) flat board is divided into several regions, draws different host strain overnight culture: streptococcus aureus YZ-12, YZ-15, YZ-16, YZ-19, YZ-34, YZ-42, YZ-49, YZ-56, XG-9, XG-25, SF-16, SF-22, SF-37, SF-46, SF-54, SF-56, A14, A16, X5-1, X12, M-9, S4, S6, more than be this room isolation identification and preserve and streptococcus aureus (ATCC25923), 0.1mL drips in the dull and stereotyped centre of TSB, and bacterium liquid is spreadable equably, cultivates 1h for 37 DEG C; Simultaneously by Salmonella typhimurium (HNER178, Salmonella Typhimurium (this laboratory be separated preserve) and Salmonella enteritidis (ATCC13076, Salmonella Enteritidis) and intestinal bacteria (ATCC25922) 0.1mL drip in TSB is dull and stereotyped and coating is even; Get lyase product, empty carrier plasmid (pET32b) abduction delivering product and each 10 μ L of enzyme place buffer control, drip respectively in the flat board scribbling different host strain, just putting after naturally drying, after being placed in 37 DEG C of incubators cultivation 10h respectively, observe lyase product to the splitting action of different host strain.
Result is as Fig. 3 (part bacterial strain) and table 1, and lyase product only has specific lytic activity to streptococcus aureus, and reactionless to other kind bacterial strains.
The antimicrobial spectrum of table 1 staphylococcus aureus bacteriophage lyase LysS1
In table 1, "+" represents faint inhibition zone, and " ++ " represents remarkable inhibition zone, and "-" represents without inhibition zone.
Embodiment 4: lyase LysS1 cracking streptococcus aureus effect test.
If add lyase and do not add lyase contrast, lyase is added in the Staphylococcus aureus S6 (S.aureus) cultivated in liquid TSB, bacterium liquid 1ml is got respectively when 7min, centrifugal thalline, 2.5% glutaraldehyde is fixed, ethanol gradient concentration is dewatered, and makes sample, with scanning electron microscopic observation lyase product to the splitting action of S6 (S.aureus).
As shown in Figure 4, lyase product causes aureus cell wall to be out of shape coming off result, and occur ghost phenomenon, bacterial cell form changes, and it is normal not add ne ar in lyase contrast, without this phenomenon.
The Staphylococcus aureus S6 (S.aureus) cultivated in liquid TSB is cultured to OD
600when value is about 0.1, add 100 μ L lyase respectively, thick enzyme, Elution buffer (purification buffer) and damping fluid PBS (damping fluid), room temperature leaves standstill, and observes the change of bacterium liquid.
As shown in Figure 5, lyase product causes streptococcus aureus liquid to be clarified to result, and thick enzyme causes the broken cracking of streptococcus aureus, and bacterium liquid well-grown in damping fluid and blank.
Embodiment 5: the sterilization functions of lyase in milk
Pasteurized milk is packed as three groups, often organizes 5ml pasteurized milk, in A group, only add 10
4cfu/mL Host Strains (streptococcus aureus S6, S.aureus) in contrast, inoculates Host Strains (streptococcus aureus S6) 10 in B group
4cfu/mL, adds PBS in contrast, C group inoculation Host Strains (streptococcus aureus S6) 10
4cfu/mL, adds the lyase LysS1 (the lyase LysS1 of about 1U) of 100 μ g, the sample prepared is placed in 25 DEG C respectively, detects Host Strains quantity respectively at 30min and 60min.
As shown in Figure 6, after 25 DEG C of effect 30min, Host Strains quantity declines result to some extent; To 60min, Host Strains quantity decline about 3.5 log10, are significant difference with control group, thus show that lyase LysS1 has good germicidal action in food.
Claims (10)
1. through a genetic engineering modified staphylococcus aureus bacteriophage lyase, it is characterized in that, the aminoacid sequence of this staphylococcus aureus bacteriophage lyase is as SEQ ID NO:1.
2. through an encoding gene for genetic engineering modified staphylococcus aureus bacteriophage lyase, it is characterized in that, the nucleotide sequence of the encoding gene of this staphylococcus aureus bacteriophage lyase is as shown in SEQ ID NO:2.
3. one kind comprises the plasmid of the encoding gene of staphylococcus aureus bacteriophage lyase according to claim 2.
4. one kind comprises the recombinant bacterium of plasmid according to claim 2.
5. the preparation method of staphylococcus aureus bacteriophage lyase according to claim 1, it is characterized in that, the method comprises the steps:
(1) nucleotide sequence shown in SEQ ID NO:2 is cloned in expression vector, obtains recombinant plasmid;
(2) by the recombinant plasmid transformed Host Strains that step (1) obtains, recombinant bacterium is obtained;
(3) recombinant bacterium is utilized to express staphylococcus aureus bacteriophage lyase;
(4) the staphylococcus aureus bacteriophage lyase that obtains of purification step (3).
6. the preparation method of staphylococcus aureus bacteriophage lyase according to claim 4, is characterized in that, in step (1), described expression vector is pET32b (+).
7. the preparation method of staphylococcus aureus bacteriophage lyase according to claim 4, is characterized in that, in step (2), described Host Strains is intestinal bacteria TransB (DE3).
8. the preparation method of staphylococcus aureus bacteriophage lyase according to claim 4, it is characterized in that, in step (3), IPTG is utilized to induce recombinant bacterium to express staphylococcus aureus bacteriophage lyase, the concentration of IPTG is 1.0 ~ 1.2mmol/L, and the temperature of abduction delivering is 20 ~ 26 DEG C.
9. the staphylococcus aureus bacteriophage lyase that the preparation method of the staphylococcus aureus bacteriophage lyase described in any one of claim 4 ~ 7 prepares.
10. staphylococcus aureus bacteriophage lyase according to claim 9 suppresses the application in the medicine of staphylococcus aureus growth in preparation.
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| CN105238773A (en) * | 2015-11-27 | 2016-01-13 | 江苏省农业科学院 | Wide spectrum bacteriophage chimeric lytic enzyme capable of resisting staphylococcus, preparation method and appliance thereof |
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| CN107670023A (en) * | 2017-10-10 | 2018-02-09 | 中国科学院武汉病毒研究所 | A kind of new application of V12CBD albumen and its encoding gene |
| CN109666667A (en) * | 2019-01-22 | 2019-04-23 | 上海交通大学 | The chimaeric enzyme antibiotic of killing staphylococcus aureus and its preparation and application |
| CN110684760A (en) * | 2019-09-27 | 2020-01-14 | 吉林大学 | Gene engineering lyase for killing staphylococcus and preparation method and application thereof |
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| CN105238773A (en) * | 2015-11-27 | 2016-01-13 | 江苏省农业科学院 | Wide spectrum bacteriophage chimeric lytic enzyme capable of resisting staphylococcus, preparation method and appliance thereof |
| CN106754849A (en) * | 2017-03-13 | 2017-05-31 | 江苏省农业科学院 | A kind of multi-functional lyases and its preparation method and application |
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| CN107670023A (en) * | 2017-10-10 | 2018-02-09 | 中国科学院武汉病毒研究所 | A kind of new application of V12CBD albumen and its encoding gene |
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| CN110684760A (en) * | 2019-09-27 | 2020-01-14 | 吉林大学 | Gene engineering lyase for killing staphylococcus and preparation method and application thereof |
| CN110951715A (en) * | 2019-12-28 | 2020-04-03 | 王中华 | Staphylococcus-resistant broad-spectrum phage coding lyase and preparation method and application thereof |
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| CN114736894B (en) * | 2022-05-26 | 2023-10-31 | 华中农业大学 | Chimeric enzyme ClyQ for degrading staphylococcus biofilm and preparation method and application thereof |
| CN115820616A (en) * | 2022-07-22 | 2023-03-21 | 昆明理工大学 | Bacteriophage lyase with fluorescent label and application thereof |
| CN115927270A (en) * | 2022-08-29 | 2023-04-07 | 山东大学 | Staphylococcus aureus phage lyase, and variant and application thereof |
| CN115927270B (en) * | 2022-08-29 | 2024-05-31 | 山东大学 | Staphylococcus aureus phage lyase, variant thereof and application thereof |
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