阅读说明：本技术 用于提取金黄色葡萄球菌总蛋白的方法及试剂盒 (Method and kit for extracting total protein of staphylococcus aureus ) 是由 聂子梅 黄非 宋乐元 �田润 于 2020-06-03 设计创作，主要内容包括：本发明涉及金黄色葡萄球菌检测,具体涉及一种用于提取金黄色葡萄球菌总蛋白的方法及试剂盒。所述方法包括：培养金黄色葡萄球菌并收集菌体,用反应缓冲液重悬菌体,然后加入由酿酒酵母菌表达的溶葡球菌酶,于37℃温育30-60min,得到酶解产物；用PBS溶液稀释所述酶解产物,即得金黄色葡萄球菌总蛋白提取物。该方法具有简便快捷、蛋白提取效率高的优点,解决了金黄色葡萄球菌全蛋白难提取的问题。(The invention relates to staphylococcus aureus detection, in particular to a method and a kit for extracting total protein of staphylococcus aureus. The method comprises the following steps: culturing staphylococcus aureus, collecting thalli, resuspending the thalli by using a reaction buffer solution, then adding lysostaphin expressed by saccharomyces cerevisiae, and incubating for 30-60min at 37 ℃ to obtain an enzymolysis product; and diluting the enzymolysis product by using a PBS solution to obtain the total protein extract of the staphylococcus aureus. The method has the advantages of simplicity, convenience, rapidness and high protein extraction efficiency, and solves the problem of difficult extraction of whole protein of staphylococcus aureus.)

1. A method for extracting total protein of Staphylococcus aureus, comprising: culturing staphylococcus aureus and collecting thalli, and is characterized in that: resuspending the thallus with reaction buffer solution, adding lysostaphin expressed by Saccharomyces cerevisiae, and incubating at 37 deg.C for 30-60min to obtain enzymolysis product; and diluting the enzymolysis product by using a PBS solution to obtain the total protein extract of the staphylococcus aureus.

2. The method of claim 1, wherein: the preparation method of the lysostaphin comprises the following steps: adding a soluble sequence at the N end of the amino acid sequence of the lysostaphin and adding an enzyme protein stability sequence at the C end to obtain the amino acid sequence of the recombinant lysostaphin; synthesizing the encoding gene of the recombinant lysostaphin, constructing an expression vector, and then converting saccharomyces cerevisiae to obtain recombinant saccharomyces cerevisiae; and culturing the recombinant saccharomyces cerevisiae, inducing protein expression and performing protein purification.

3. The method of claim 2, wherein: the amino acid sequence of the lysostaphin is shown as SEQ ID NO.1, the soluble sequence is shown as SEQ ID NO. 3, and the enzyme protein stability sequence is shown as SEQ ID NO. 4; preferably, the nucleotide sequence of the coding gene of the recombinant lysostaphin is shown as SEQ ID NO. 2.

4. The method of claim 1, wherein the step of removing the metal oxide is performed in a batch processThe method comprises the following steps: the reaction buffer comprises the following components: 15-30mM pH7.0-8.0Tris-Cl, 0.5% -1.2% g/ml NaCl, 5-15mM EDTA, 2-8mM MnCl₂，0.05％-0.12％g/ml SDS。

5. The method of claim 4, wherein: the formula of the reaction buffer solution is as follows: 20mM pH7.4Tris-Cl, 0.9% g/ml NaCl, 10mM EDTA, 5mM MnCl₂，0.1％g/ml SDS。

6. The method of claim 1, wherein: the PBS solution comprises the following components: 137mM NaCl, 2.7mM KCl, 8mM NaH₂PO₄，2mM K₂HPO₄，pH7.4。

7. The method according to any one of claims 1 to 6, wherein: 400 mul of reaction buffer, 10 mul of lysostaphin solution and 1mL of PBS solution are used for each milliliter of thalli collected from the staphylococcus aureus culture solution; the concentration of the lysostaphin solution is 1mg/mL, and the purity is more than 90%.

8. A staphylococcus aureus total protein extraction kit is characterized in that: comprises reaction buffer solution and lysostaphin expressed by saccharomyces cerevisiae; the reaction buffer comprises the following components: 15-30mM of Tris-Cl with pH of 7.0-8.0, 0.5% -1.2% g/ml of NaCl, 5-15mM of EDTA and 2-8mM of MnCl₂，0.05％-0.12％g/ml SDS。

9. The kit of claim 8, wherein: the preparation method of the lysostaphin comprises the following steps: adding a soluble sequence at the N end of the amino acid sequence of the lysostaphin and adding an enzyme protein stability sequence at the C end to obtain the amino acid sequence of the recombinant lysostaphin; synthesizing the encoding gene of the recombinant lysostaphin, constructing an expression vector, and then converting saccharomyces cerevisiae to obtain recombinant saccharomyces cerevisiae; and culturing the recombinant saccharomyces cerevisiae, inducing protein expression and performing protein purification.

10. The kit of claim 9, wherein: the amino acid sequence of the lysostaphin is shown as SEQ ID NO.1, the soluble sequence is shown as SEQ ID NO. 3, and the stability sequence of the enzyme protein is shown as SEQ ID NO. 4; preferably, the nucleotide sequence of the coding gene of the recombinant lysostaphin is shown as SEQ ID NO. 2.

11. The kit of claim 8, wherein: the PBS solution is also included, and the formula is as follows: 137mM NaCl, 2.7mM KCl, 8mM NaH₂PO₄，2mM K₂HPO₄，pH7.4。

12. The kit according to any one of claims 8 to 11, wherein: the formula of the reaction buffer solution is as follows: 20mM Tris-Cl, pH7.4, 0.9% g/ml NaCl, 10mM EDTA, 5mM MnCl₂0.1% g/ml SDS; purity of said lysostaphin>90％。

Technical Field

The invention relates to staphylococcus aureus detection, in particular to a method and a kit for extracting total protein of staphylococcus aureus.

Background

Staphylococcus aureus (s. aureus), also known as "Staphylococcus aureus", belongs to the genus Staphylococcus, is a representative of gram-positive bacteria, and is a common food-borne pathogenic microorganism; the staphylococcus aureus has certain tolerance to high temperature, can be completely killed in a high-temperature environment of more than 80 ℃ for 30min, can survive in a high-salt environment, and can tolerate a NaCl solution with 15% of concentration to the maximum; the staphylococcus aureus has very high cell wall tolerance, and the conventional physical and mechanical methods, detergents and other methods are difficult to break the cell wall to release intracellular nucleic acid and protein, which influences the application of modern molecular biological detection methods in the low-concentration rapid detection of the staphylococcus aureus.

Lysostaphin, also known as Lysostaphin, cleaves staphylococcus aureus. At present, escherichia coli or pichia pastoris is usually adopted to express lysostaphin, but the prepared lysostaphin has low activity and cannot be used for breaking the walls of staphylococcus aureus independently, and the staphylococcus aureus can be cracked only by combining lysozyme or by means of a physical thallus wall breaking method, so that the operation steps are very complicated and the consumed time is long.

At present, the detection of staphylococcus aureus is mainly a culture medium biochemical method, and a PCR method using DNA as a detection target and an immunological method using a thallus surface protein as a detection target cannot be widely applied due to the limitations of methodologies, and the main obstacles are that: the cell wall of the staphylococcus aureus has high tolerance capability to protease, detergent and high salt penetration, time consumption and low efficiency in the wall breaking stage, and complete and considerable DNA is difficult to extract; in the total protein extraction process, it is extremely difficult to obtain the total protein by breaking cells by methods such as ultrasonic waves, freeze-thaw and the like, and cell surface protein is often detected in a compromised manner to avoid wall breaking, but staphylococcus aureus outer membrane protein proteinA can be specifically combined with antibody protein, so that the effect of immunodetection is weakened, and false negative is caused.

Disclosure of Invention

In order to solve the problem of difficult extraction of staphylococcus aureus holoprotein, the invention provides the method and the kit for extracting staphylococcus aureus total protein, and the method and the kit have the advantages of simplicity, convenience, rapidness and high protein extraction efficiency.

In one aspect, the invention provides a method for extracting total protein of staphylococcus aureus, comprising the following steps: culturing staphylococcus aureus and collecting thalli, and is characterized in that: resuspending the thallus with reaction buffer solution, adding lysostaphin expressed by Saccharomyces cerevisiae, and incubating at 37 deg.C for 30-60min to obtain enzymolysis product; and diluting the enzymolysis product by using a PBS solution to obtain the total protein extract of the staphylococcus aureus.

In a preferred embodiment of the method of the present invention, the method for preparing the lysostaphin comprises the steps of: adding a soluble sequence at the N end of the amino acid sequence of the lysostaphin and adding an enzyme protein stability sequence at the C end to obtain the amino acid sequence of the recombinant lysostaphin; synthesizing the encoding gene of the recombinant lysostaphin, constructing an expression vector, and then converting saccharomyces cerevisiae to obtain recombinant saccharomyces cerevisiae; and culturing the recombinant saccharomyces cerevisiae, inducing protein expression and performing protein purification.

In a preferred embodiment of the method of the present invention, the amino acid sequence of the lysostaphin is shown as SEQ ID NO.1, the soluble sequence is shown as SEQ ID NO. 3, and the protein stability sequence of the enzyme is shown as SEQ ID NO. 4; preferably, the nucleotide sequence of the coding gene of the recombinant lysostaphin is shown as SEQ ID NO. 2.

In a preferred embodiment of the method of the invention, the reaction buffer comprises the following components: 15-30mM pH7.0-8.0Tris-Cl, 0.5% -1.2% g/ml NaCl, 5-15mM EDTA, 2-8mM MnCl₂，0.05％-0.12％g/mlSDS。

In a preferred embodiment of the method of the present invention, the reaction buffer is formulated as: 20mM pH7.4Tris-Cl, 0.9% g/ml NaCl, 10mM EDTA, 5mM MnCl₂，0.1％g/ml SDS。

In some embodiments of the methods of the invention, the PBS solution is formulated as: 137mM NaCl, 2.7mM KCl, 8mM NaH₂PO₄，2mM K₂HPO₄，pH7.4。

In some embodiments of the methods of the invention, 400. mu.L of the reaction buffer, 10. mu.L of the lysostaphin solution, and 1mL of the PBS solution are used per mL of the collected cells of the Staphylococcus aureus culture; the concentration of the lysostaphin solution is 1mg/mL, and the purity is more than 90%.

On the other hand, the invention provides a staphylococcus aureus total protein extraction kit, which is characterized in that: comprises reaction buffer solution and lysostaphin expressed by saccharomyces cerevisiae; the reaction buffer comprises the following components: 15-30mM pH7.0-8.0Tris-Cl, 0.5% -1.2% g/ml NaCl, 5-15mM EDTA, 2-8mM MnCl₂，0.05％-0.12％g/ml SDS。

In a preferred embodiment of the kit of the present invention, the method for preparing the lysostaphin comprises the following steps: adding a soluble sequence at the N end of the amino acid sequence of the lysostaphin and adding an enzyme protein stability sequence at the C end to obtain the amino acid sequence of the recombinant lysostaphin; synthesizing the encoding gene of the recombinant lysostaphin, constructing an expression vector, and then converting saccharomyces cerevisiae to obtain recombinant saccharomyces cerevisiae; and culturing the recombinant saccharomyces cerevisiae, inducing protein expression and performing protein purification.

In a preferred embodiment of the kit of the present invention, the amino acid sequence of the lysostaphin is shown as SEQ ID NO.1, the soluble sequence is shown as SEQ ID NO. 3, and the protein stability sequence of the enzyme is shown as SEQ ID NO. 4; preferably, the nucleotide sequence of the coding gene of the recombinant lysostaphin is shown as SEQ ID NO. 2.

In some embodiments of the kit of the present invention, the kit further comprises a PBS solution having a formulation of: 137mM NaCl, 2.7mM KCl, 8mM NaH₂PO₄，2mM K₂HPO₄，pH7.4。

In a preferred embodiment of the kit of the present invention, the reaction buffer is formulated as follows: 20mM pH7.4Tris-Cl, 0.9% g/ml NaCl, 10mM EDTA, 5mM MnCl₂0.1% g/ml SDS; purity of said lysostaphin>90％。

The inventor finds that the activity (552U/mg) of the obtained recombinant lysostaphin is obviously higher than that of the traditional lysostaphin (376U/mg) expressed by pichia pastoris and the lysostaphin (124U/mg) expressed by escherichia coli, and the protein stability is far higher than that of the lysostaphin expressed by pichia pastoris and escherichia coli and the commercially available lysostaphin by adding a soluble sequence at the N end and adding an enzyme protein stability sequence at the C end of the lysostaphin amino acid sequence and adopting a saccharomyces cerevisiae expression system to carry out protein expression.

Aiming at the recombinant lysostaphin prepared by the invention, the invention also develops a reaction buffer solution matched with the recombinant lysostaphin for extracting the staphylococcus aureus protein. The recombinant lysostaphin and the reaction buffer solution are used for extracting the total protein of staphylococcus aureus, and the method has high efficiency and convenience which are difficult to achieve by the traditional protein extraction method. As shown in Table 3, the total protein of Staphylococcus aureus extracted by the method of the present invention released the intracellular nuclease protein at a level much higher than that of the ultrasonication method and the lysozyme method. Therefore, the protein extraction method has very remarkable specific advantages on the extraction of total protein of staphylococcus aureus, and particularly shows extremely high efficiency in the cell lysis process. The method has the advantages of simple and rapid operation, high protein release rate and low viscosity of the lysate, is suitable for various conventional immunological detection methods, and can improve the detection efficiency and the detection accuracy of staphylococcus aureus.

Drawings

FIG. 1 is a SDS-PAGE pattern of recombinant lysostaphin expressed using Saccharomyces cerevisiae;

wherein M is a protein Marker and is 116, 66.2, 45, 35, 25, 18.4 and 14.4kDa from top to bottom in sequence; 1: induction sample (total protein of thallus extracted after induction of saccharomyces cerevisiae expression protein, without protein purification), 2: breakthrough sample (breakthrough sample refers to the fraction of the column that saturates during and after the saturation of the column during purification), 3: 10mM imidazole eluent, 4: 20mM imidazole eluent, 5: 50mM imidazole eluent, 6: 100mM imidazole eluent, 7: 200mM imidazole eluent; the black arrow in the figure indicates the protein band of the recombinant lysostaphin, which has a molecular weight of 28 kDa.

FIG. 2 is a SDS-PAGE pattern of recombinant lysostaphin expressed using Pichia pastoris;

wherein M is a protein Marker and is 116, 66.2, 45, 35, 25, 18.4 and 14.4kDa from top to bottom in sequence; 1: induction medium not concentrated, 2: induction medium concentration 5-fold, 3: concentrating the induction culture medium by 20 times; the black arrow in the figure indicates the protein band of the recombinant lysostaphin, which has a molecular weight of 28 kDa.

FIG. 3 is a SDS-PAGE pattern of recombinant lysostaphin expressed using E.coli;

wherein M is a protein Marker and is 116, 66.2, 45, 35, 25, 18.4 and 14.4kDa from top to bottom in sequence; 1: no induction of the expressing strain (total protein of E.coli without induction of protein expression) 2: induction of intact cell (total cell protein extracted after induction of escherichia coli expression protein, without protein purification), 3: breakthrough sample (breakthrough sample refers to the fraction of the column that saturates during and after the saturation of the column during purification), 4: 10mM imidazole eluent, 5: 20mM imidazole eluent, 6: 50mM imidazole eluent, 7: 100mM imidazole eluent, 8: 200mM imidazole eluent, 9: 300mM imidazole eluent; the black arrow in the figure indicates the band of recombinant lysostaphin protein, which has a molecular weight of 28 kDa.

FIG. 4 shows an electrophoretogram of total DNA of Staphylococcus aureus extracted by the DNA extraction method of the present invention.

FIG. 5 shows an electrophoretogram of total DNA of Staphylococcus aureus extracted by a conventional DNA extraction method.

Detailed Description

The present invention is described in detail below with reference to examples, it being understood that the following examples are only illustrative and illustrative of the present invention and do not limit the scope of the present invention in any way.

Escherichia coli (DH 5 alpha) is purchased from China general microbiological culture Collection center (CGMCC), and has a strain number of 1.12873 and a platform resource number of 1511C 0002100009047.

Escherichia coli (BL 21) is purchased from China general microbiological culture Collection center (CGMCC), and has a strain number of 1.12875 and a platform resource number of 1511C 0002100009049.

Saccharomyces cerevisiae (INVSC 1) was purchased from Saimer Feishell Scientific Co., Ltd (Thermo Fisher Scientific) under the catalog number C81000.

Pichia pastoris (GS 115) was purchased from ThermoFisher Scientific, Inc. under product catalog number C18100.

Staphylococcus aureus (Staphylococcus aureus, Cowan I) was purchased from China general microbiological culture Collection center (CGMCC), and the strain number is 1.1476, and the platform resource number is 1511C 0002100001789.

The above-described biological materials are also stored in the laboratory and claimed by the applicant to be publicly available for validation testing within twenty years from the filing date.

The pYES2-NT vector is a known yeast expression vector available from Siemens Feishel Scientific, Inc. (ThermoFisher Scientific) under the catalog number V825220. The bacterial resistance of pYES2-NT vector (6.0kb) was Ampicillin resistance (Ampicillin), the cloning procedure was restriction endonuclease/multiple cloning site, promoter GAL1, protein tag 6 XHis tag, V5 epitope tag and Xpress epitope tag.

The pPIC9K vector is a known Pichia vector available from ThermoFisher Scientific, Inc. under catalog number V17520. The bacterial resistance of the pPIC9K vector (9.3kb) was Ampicillin resistance (Ampicillin) and Gentamicin resistance (Gentamicin), and the cloning method was restriction endonuclease/multiple cloning site, promoter AOX 1.

pET-28a (+) is a commonly used prokaryotic expression vector of fusion protein type, available from Solambio, Inc., having a cargo number: p3110. pET-28a (+) size 5369bp, contains kanamycin resistance gene, expression by host cell provided T7 RNA polymerase induction.

BamH I endonuclease (Code No.1010S), Xho I endonuclease (Code No.1094S), EcoR I endonuclease (Code No.1040S), Not I endonuclease (Code No.1166S), Sac I endonuclease (Code No.1078S), TaKaRaMiniBEST Agarose Gel DNA Extraction Kit Ver.4.0(Code No.9762), T4 DNA Ligase (Code No.2011A), all available from Baozi physician's technology (Beijing) LimitedCompany (Takara). Plasmid miniprep kit was purchased from tiangen biochemistry technology (beijing) limited, catalog No.: and DP 106. Dialysis bag MD25 (8000-: YA 1071. HisPrep FF16/10 prepacked column was purchased from GEHealthcare Life Sciences, product number 17-5256-01. Casamino acids (Casamino acids, Sigma-Aldrich 70172), galactose (galactose, Sigma-Aldrich V900922), glucose (glucose, Sigma-Aldrich V900392), YNB (amino-free yeast nitrogen source, Sigma-Aldrich V900895). Yeastextract (yeast extract, OXOID LP0021), Peptone (Peptone, Solibao P8450), Biotin (Biotin, Sigma-Aldrich V900418), Tryptone (Tryptone, OXOID LP0042B), EDTA (ethylenediaminetetraacetic acid, chemical formula: C)₁₀H₁₆N₂O₈CAS registry number: 60-00-4, Sigma-Aldrich V900106), SDS (sodium dodecyl sulfate, formula: c₁₂H₂₅SO₄Na, CAS accession No.: 151-21-3, Sigma-Aldrich V900859); sarcosyl (sodium N-lauroyl sarcosinate, CAS number 137-16-6, linear formula CH)₃(CH₂)₁₀CON(CH₃)CH₂COONa) purchased from Sigma-Aldrich, cat #: l9150. 4M GuSCN (guanidinium isothiocyanate, CAS number 593-84-0, linear formula NH)₂C(＝NH)NH₂HSCN) purchased from Sigma-Aldrich, cat #: 11685929001. lysozyme (CAS number 12650-88-3, EC number 3.2.1.17) was derived from chicken protein and was purchased from Sigma-Aldrich, cat # L6876. Proteinase K (CAS number 39450-01-6, EC number 3.4.21.64) was derived from Candida albicans, purchased from Sigma-Aldrich, cat # P6556.

LB Medium

Each 100ml of LB medium contained: 1g tryptone, 0.5g yeast extract, 1g sodium chloride, pH 7.4.

The preparation method comprises the following steps: at 950ml ddH₂Dissolving 10g tryptone, 5g yeast extract, 10g sodium chloride in O, adjusting pH to 7.4 with NaOH, and adding ddH₂And O is metered to 1L. If a solid medium is prepared, 15g of agar per liter are added. Sterilizing with high pressure steam at 121 deg.C for 20 min.

YEPD medium

The formula is as follows: yeast cream: peptone: glucose was 1:2:2 (mass ratio).

The preparation method comprises the following steps: dissolving 10g of yeast extract, 20g of peptone in 900ml of ddH₂In O, 20g of agar is added if the plate is prepared; sterilizing with high pressure steam at 121 deg.C for 20 min; after cooling, 100ml of a separately sterilized (autoclaved at 115 ℃ C. for 15min) glucose solution (containing 20g of glucose) was added.

Unless otherwise specified, the reagents used in the following examples are conventional in the art, and are either commercially available or formulated according to methods conventional in the art, and may be of laboratory pure grade. Unless otherwise specified, the experimental methods and experimental conditions used in the following examples are all conventional in the art, and reference may be made to relevant experimental manuals, well-known literature, or manufacturer's instructions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.