CN110590909A - Penetrating peptide and application thereof in knockout of staphylococcus aureus adhesion factor - Google Patents
Penetrating peptide and application thereof in knockout of staphylococcus aureus adhesion factor Download PDFInfo
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- 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
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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Abstract
The invention provides a penetrating peptide and application thereof in vitro knockout of a staphylococcus aureus adhesion factor, namely, a CRISPR-Cas9 system is firstly constructed according to a target sequence of a sgRNA recognition area of the staphylococcus aureus adhesion factor, the base sequence of the target sequence is shown as SEQ NO.1, in the constructed CRISPR-Cas9 system, a T7 promoter is inserted into the front of a sgRNA transcription initiation site to construct a prokaryotic expression vector of Cas9 protein, the T7 promoter is used for regulation and control, and then a cell penetrating peptide CPP5L with an amino acid sequence shown as SEQ ID NO.2 is used for bringing a staphylococcus aureus adhesion factor knockout vector pCas9-FnBP into a microbial cell so as to realize the in vitro knockout of the staphylococcus aureus adhesion factor.
Description
Technical Field
The invention relates to the field of microbial genetic engineering and biotechnology, in particular to a cell penetrating peptide CPP5L and a method for knocking out a staphylococcus aureus adhesion factor in vitro by using the cell penetrating peptide through a CRISPR-Cas9 system.
Background
CRISPR (clustered regulated short palindromic repeats) technology provides possibility for developing novel microbial gene therapy measures. The CRISPR-Cas9 system is a powerful gene site-directed editing technology newly discovered in 2013, and can conveniently and accurately edit, cut and modify DNA and nucleotide sequences. The working principle of the CRISPR-Cas9 system is that clustered regularly interspaced short palindromic repeats crrnas (CRISPR-derived RNAs) are combined with tracrrnas (trans-activating RNAs) through base pairing to form a tracrRNA/crRNA composition, and the composition guides the nuclease Cas9 protein to realize site-specific double-stranded DNA cleavage at a sequence target site paired with the crRNA. In practical applications, the tracrRNA/crRNA composition can be optimized as one sgRNA sequence, including the dual functions of tracrRNA/crRNA.
At present, the CRISPR-Cas9 system is most widely applied in the way that a DNA double-strand break is realized at the upstream of a target gene PAM (such as NGG motif) element by designing 1 sgRNA-Cas9 system, and the target DNA is deleted or inserted in the DNA damage repair process (homologous recombination or non-homologous end connection), so that a target gene is knocked out or a new gene is inserted. With the intensive research on the Cas9 protein, the off-target problem can be solved after 3 amino acids of the protein are changed, the non-target gene can not be cut at the metagenome level, and the support is provided for the application of the technology in treatment.
The CRISPR-Cas9 system has high gene editing efficiency and very simple system, and can be used for microbial gene modification technology. The technology can effectively knock out the genes related to drug resistance and virulence of the microorganism in vitro. Citorik and the like integrate a Cas9 encoding protein gene and an sgRNA gene in a phage genome or put in a transduction plasmid to realize site-directed knockout of a super drug resistance gene; it HAs recently been found that encapsulation of sgRNA-Cas9 protein compositions in liposomes or insertion of cell penetrating peptides CPP in Cas9 proteins can deliver sgRNA-Cas9 protein compositions into cells to achieve in vivo gene knockout (WadiaJS, Stan RV, Down SF. Transducible TAT-HA fusogenic peptide enghances of encampelof TAT-fusion proteins after lipoprotein secretion masropytosis. Nat. Med. 2004; 10: 310-.
Among the numerous drug carrier models, cell-penetrating peptides are one of a new class of drug carriers that have grown up rapidly since the 90 s of the last century. The cell-penetrating peptide can also carry larger functional molecules such as protein antibodies and the like into mammalian cells and play the function of the mammalian cells. There are also many ways of carrying protein polypeptides by cell-penetrating peptides, and it is common to construct fusion proteins of cell-penetrating peptides and target proteins by covalent attachment. At present, large-scale screening and design are not carried out on the polypeptide capable of carrying out macromolecule delivery in microorganisms, the application of the penetrating peptide is only used on mammals at present, and the penetrating peptide capable of carrying macromolecular substances such as nucleic acid or protein and the like into microbial cells is not reported.
The adhesion of Staphylococcus aureus (Staphylococcus aureus) to epithelial cells is considered a key pathogenic mechanism of Staphylococcus aureus mastitis. Most importantly, staphylococcus aureus adheres to the surface of mammary epithelial cells by adhesins, subsequently colonizes and proliferates in large numbers. Current research indicates that adhesins FnBPs are important proteins in the pathogenesis of bovine mastitis Staphylococcus aureus (RIOLET C, RAINARD P, POUTRELL B. cells and cytokine in inflammation secretion of bovine Mammary gland and [ M ]. Biology of Mammary gland and. Springer 2002:247-58.) there is no method for knocking out the adhesion factors of Staphylococcus aureus at present. A method for knocking out adhesion factors in staphylococcus aureus and delivering the deleted genes into cells by cell penetrating peptides is also not reported.
Disclosure of Invention
In view of the above problems, the present invention provides a cell penetrating peptide CPP5L and a method for knocking out staphylococcus aureus and its adhesion factor (FnBP) in vitro by using the cell penetrating peptide through CRISPR-Cas9 system, which is achieved by:
first, the present invention provides a cell-penetrating peptide CPP5L having an amino acid sequence shown in SEQ ID No. 2.
Secondly, the invention provides the application of the cell penetrating peptide CPP5L in carrying protein or nucleic acid into microbial cells; further, the nucleic acid is preferably pCas9-FnBP plasmid, and the microorganism is preferably Staphylococcus aureus (Staphylococcus aureus);
thirdly, the invention provides a method for using CRISPR-Cas9 system to adhere staphylococcus aureus in vitro, which comprises the following steps:
A) synthesizing a DNA sequence according to the sequence with the nucleotide sequence shown as SEQ ID NO.5, and annealing to obtain a hybrid double strand for constructing the knockout vector pCas 9-FnBP.
An annealing system: 1 muL of synthetic DNA with the concentration of 100 muM and 1 muL of 10xPCR Buffer, and adding water to complement to 10 muL;
and (3) annealing: denaturation at 95 ℃ for 5min, followed by cooling to 25 ℃ at a rate of 2 ℃ per minute, completing the reaction.
B) The vector pCas9 is cut by BsaI enzyme;
and (3) enzyme digestion system: BsaI enzyme 1. mu.L (New England Biolabs Ltd.), 10x Buffer 5. mu.L, plasmid 2. mu.g supplemented to 50. mu.L with water;
enzyme digestion step, incubation is carried out for 12h at 37 ℃;
in this step, the vector pCas9 is a conventional vector in the art, as described in "Jiang W, Bikard D, Cox D, et al.RNA-guided encoding of bacterial genes using CRISPR-Cas systems [ J ]. Nature Biotechnology,2013,31(3): 233-.
C) Connecting the hybrid double strand obtained in the step A) with the vector pCas9 after enzyme digestion by using T4 ligase to obtain a vector pCas 9-FnBP;
the ligation system comprises 1 mu L of T4 enzyme (New England Biolabs Ltd.), 1 mu L of 10x Buffer, 0.5 mu g of plasmid, and 1 mu L of hybrid double strand supplemented with water to 10 mu L;
a connection step: incubating for 12h at 4 ℃;
D) in PBS, mixing the vector pCas9-FnBP and the cell penetrating peptide CPP5L with the staphylococcus aureus containing the adhesion factors, and incubating together at 4-37 ℃ to knock out the adhesion factors of the staphylococcus aureus.
The incubation is as follows: the vector pCas9-FnBP with the final concentration of 0.1-1 mug/ml, the cell penetrating peptide CPP5L with the final concentration of 5-45 mug/ml (the purity is 99 percent, the amino acid sequence is shown as SEQ ID NO. 2) and the final concentration of 10 are added8Mixing Staphylococcus aureus of each ml, and incubating at 4-37 deg.C for more than 6 hr.
Although the cell-penetrating peptides known so far theoretically have a cell-penetrating function, no function of carrying nucleic acids or proteins into microbial cells has been found so far. The application realizes the in-vitro knockout of the staphylococcus aureus adhesion factor by using the CRISPR-Cas9 system for the first time.
Arginine in the sequence of the penetrating peptide CPP5L provided by the invention can realize the function of cell penetration, the delivery protein enters the interior of a microorganism, the self-composed structure can interact with nucleic acid, and the peptide is a good delivery element. Compared with intracellular knockout, the realization of the in vitro knockout of the cell can apply the technology to the prevention and control of clinical super-drug-resistant bacteria, and has wide application prospect.
Drawings
FIG. 1 is an electrophoresis chart of monoclonal detection results of Staphylococcus aureus after successful adhesion factor knockout.
FIG. 2 is a schematic view of in vitro knockout of Staphylococcus aureus JYG2 adhesion factor by penetrating peptide CPP5L-SgRNA in a fluorescence confocal microscope.
Detailed Description
The following examples are given for the purpose of illustration only and are not intended to limit the scope of the invention.
In the examples, materials, reagents and techniques used, unless otherwise specified, were those conventionally known to those skilled in the art, and were commercially available.
pCas9 was purchased from addrene;
Alexa FluorR488Protein Labeling Kit (A10235, ThermoFisher corporation);
staphylococcus aureus JYG2(Zhang L, Sun L, Wei R, et al, intracellular Staphylococcus aureus Control by Virulent Bacteriophages with MAC-T Bovine Mammary epipolar Cells [ J ]. Antirhizobiological Agents & Chemotherapy,2017,61(2): AAC.01990-16.) was isolated and stored by the academy of agricultural sciences of Jiangsu province.
The cell-penetrating peptide CPP5L was synthesized by Gill Biochemical technology Ltd.
The molecular biological experiments, which are not specifically described in the following examples, were performed by referring to the methods described in molecular cloning, a laboratory Manual (third edition), scientific Press, J. SammBruker, or according to the kit and product instructions.
Examples relate to the sequence:
SEQ ID NO.1 (adhesion factor gene specific targeting sequence of Staphylococcus aureus):
agcagaaacacaagttgaag；
SEQ ID No.2 (cell penetrating peptide CPP5L amino acid sequence):
RQRQQRQQFKKLFKKL；
SEQ ID NO.3 (adhesion factor gene upstream primer of Staphylococcus aureus):
aagcaccacaaactgcacaac；
SEQ ID NO.4 (adhesion factor gene downstream primer of Staphylococcus aureus):
tctgtacctttaatttctgaag；
SEQ ID No.5 (DNA sequence corresponding to sgRNA):
gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc。
the examples relate to the culture medium:
TSB culture medium formula: adding tryptone 1.5% (g/100ml), soybean peptone 0.5% (g/100ml), and sodium chloride 0.5% (g/100ml), and adding distilled water to balance; adjusting pH to 7.2 + -0.2, and sterilizing with steam at 121 deg.C for 15 min.
Example 1 construction of CRISPR-Cas9 system for adhesion factors against staphylococcus aureus
1. Comparing an adhesion factor sequence of staphylococcus aureus, searching a relatively conservative area, designing sgRNA of a specific target sequence of the area as shown in SEQ ID No.1, and obtaining the sgRNA, wherein the corresponding DNA sequence information is shown in SEQ ID No. 5.
2. Construction of pCas 9-FnBP:
(1) designing and synthesizing sgRNA DNA sequence oligo DNA for identifying staphylococcus aureus adhesion factor gene according to SEQ ID NO. 5;
(2) carrying out gradient cooling annealing on the synthesized oligo DNA sequence, and specifically comprising the following steps:
after mixing the synthesized oligo DNA (100. mu.M, 1. mu.L) with 10xPCR Buffer (1. mu.L), 10. mu.L of water was added to make up the system; then denaturation is carried out for 5min at 95 ℃, and then the temperature is reduced to 25 ℃ at the rate of 2 ℃ per minute to complete the reaction; after annealing, a synthetic oligo DNA hybrid duplex is formed.
(3) The vector pCas9 was digested with BsaI to generate cohesive ends.
And (3) enzyme digestion system: BsaI enzyme 1. mu.L (New England Biolabs Ltd.), 10x Buffer 5. mu.L, plasmid 2. mu.g supplemented to 50. mu.L with water;
enzyme digestion step, incubation is carried out for 12h at 37 ℃;
and (3) connecting the oligo DNA hybrid double strand obtained in the step (2) with the digested vector pCas9 by using T4 ligase, and obtaining the vector pCas9-FnBP of the CRISPR-Cas9 system after connection.
The ligation system comprises 1 mu L of T4 enzyme (New England Biolabs Ltd.), 1 mu L of 10x Buffer, 0.5 mu g of plasmid, and 1 mu L of hybrid double strand supplemented with water to 10 mu L;
a connection step: incubating for 12h at 4 ℃;
(4) synthesizing a cell penetrating peptide CPP5L, wherein the amino acid sequence of the cell penetrating peptide is shown as SEQ ID NO. 2; the synthesized polypeptide is purified by Gell Biochemical company, and the purity of the purified cell penetrating peptide CPP5L reaches 99%.
(6) Mixing the vector pCas9-FnBP (final concentration: 1. mu.g/ml) obtained in step (4) with the purified cell-penetrating peptide CPP5L (final concentration: 45. mu.g/ml) obtained in step (5), and mixing at 4-37 ℃ with Staphylococcus aureus (final concentration: 10)8One/ml) of the strain, and incubating for more than 6 hours to obtain the staphylococcus aureus JYG2 monoclonal thallus with the adhesion factor gene knocked out by using the adhesion factor gene.
(7) Screening single clone of transformed staphylococcus aureus JYG2, designing primer sequences shown in SEQ ID NO.3 (upstream primer) and SEQ ID NO.4 (downstream primer) aiming at adhesion factor genes, taking the obtained staphylococcus aureus JYG2 single clone thallus as a template (simultaneously taking staphylococcus aureus JYG2 wild-type thallus as a template for a control test), and carrying out PCR amplification:
PCR reaction (50. mu.L): 1. mu.L of upstream primer (10 pmol/. mu.L), 4. mu.L of downstream primer (10 pmol/. mu.L) 1. mu. L, dNTP (2.5. mu.M), 1. mu.L of Taq DNA Polymerase, and template (about 10. mu.L of template was added to the reaction system)6One), 10 × Reaction Buffer 5 μ L, ddH2O is complemented to 50 mu L;
PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; 35 cycles of 95 ℃ 20S, 55 ℃ 30S, 72 ℃ 30S; finally, extension is carried out for 10min at 72 ℃.
And (3) performing electrophoresis on the amplification product by using 0.8% agarose gel at a voltage of 120V, wherein the electrophoresis result is shown in FIG. 1, lane M in the FIG. 1 is a DNA marker, lane 5 is a staphylococcus aureus JYG2 wild type thallus control, and lanes 2-4 are staphylococcus aureus JYG2 monoclonal thallus obtained in the step (6). As can be seen in FIG. 1, the adhesion gene was cleared in Staphylococcus aureus.
The electrophoresis PCR detection screens the mutant clone without the adhesion gene, and proves that the penetrating peptide can carry nucleic acid into a microbial cell, and the CRISPR-Cas9 system constructed in the embodiment can knock out the adhesion factor gene of staphylococcus aureus.
Example 2 penetration peptide CPP5L-SgRNA knockout in vitro staphylococcus aureus JYG2 adhesion factor assay procedure is as follows:
1. with Alexa FluorR488Protein labelling Kit (the procedure is strictly as described), labelling staphylococcus aureus JYG 2; the labeled compound can react with N-terminal NH of the protein2 -And residues are connected, so that the marked protein emits green fluorescence under the excitation of laser, and bacteria emit green fluorescence.
2. Staphylococcus aureus JYG2 was added to the TSB medium, and the penetrating peptide CPP5L (same as example 1) was added to the medium to a final concentration of 0.1mg/ml, and after co-culturing at room temperature for 1 hour, the recombinant protein was washed off with PBS.
3. Taking the staphylococcus aureus JYG2 co-cultured in the step (2) to infect the MAC-T cells for 1 hour as an experimental group (CPP5L), and simultaneously setting the staphylococcus aureus JYG2 which is not co-cultured in the step (2) to infect the MAC-T cells for 1 hour as a control Group (GFP); the MAC-T cells were then washed three times and observed under a 60-fold fluorescence confocal microscope, the results of which are shown in FIG. 2.
Fig. 2A and 2B are pictures of the control group under a green fluorescence microscope and a normal microscope (visible light), respectively, and fig. 2C and 2D are pictures of the experimental group under a green fluorescence microscope and a normal microscope (visible light), respectively. FIG. 2A shows green fluorescence in the experimental group, indicating that Staphylococcus aureus JYG2 can adhere to MAC-T cells; fig. 2C shows no green fluorescence, which indicates that the penetrating peptide CPP5L-SgRNA composition can enter staphylococcus aureus JS17 to knock out adhesion factors, resulting in the loss of adhesion ability of staphylococcus aureus JYG2, and this example also verifies that the penetrating peptide can carry nucleic acid into the microbial cell membrane.
Sequence listing
<110> agricultural science and academy of Jiangsu province
<120> penetrating peptide and application thereof in knockout of staphylococcus aureus adhesion factor
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Arg Gln Arg Gln Gln Arg Gln Gln Phe Lys Lys Leu Phe Lys Lys Leu
1 5 10 15
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aagcaccaca aactgcacaa c 21
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gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60
ggcaccgagt cggtgc 76
Claims (8)
1. A penetrating peptide, wherein the amino acid sequence of the penetrating peptide is shown as SEQ ID NO. 2.
2. Use of the penetrating peptide of claim 1 for carrying a protein or nucleic acid into a microbial cell.
3. The use of claim 2, wherein the nucleic acid is a pCas9-FnBP plasmid.
4. The use of claim 2, wherein the microorganism is staphylococcus aureus.
5. The use of claim 2, wherein said use is for knocking out an adhesion factor of Staphylococcus aureus using said penetration peptide.
6. The application of claim 5, wherein the application specific steps are as follows:
A) synthesizing a DNA sequence according to SEQ ID NO.5 and annealing to obtain a hybrid double chain;
B) the vector pCas9 is cut by BsaI enzyme;
C) connecting the hybrid double strand with the enzyme-digested vector pCas9 by using T4 ligase to obtain a vector pCas 9-FnBP;
D) mixing the carrier pCas9-FnBP, the cell penetrating peptide CPP5L and the microorganism of the staphylococcus aureus adhesion factor gene, and incubating together at 4-37 ℃ to knock out the staphylococcus aureus adhesion factor;
the amino acid sequence of the cell penetrating peptide CPP5L is shown as SEQ ID NO. 2.
7. The use according to claim 6, wherein the annealing treatment of step A) is:
an annealing system: 1 μ L of synthetic DNA with a concentration of 100 μ M, 1 μ L of 10xPCR Buffer, and water to make up 10 μ L;
and (3) annealing process: denaturation at 95 ℃ for 5min, followed by cooling to 25 ℃ at a rate of 2 ℃ per minute, completing the reaction.
8. Use according to claim 6, wherein the incubation of step D) is:
the vector pCas9-FnBP was added to a final concentration of 0.1-1. mu.g/ml, the cell-penetrating peptide CPP5L was added to a final concentration of 5-45. mu.g/ml, and a final concentration of 108The microorganisms are mixed and incubated at 4-37 deg.C for at least 6 hr.
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