CN117904161B - Cell surface display system constructed based on agrobacterium tumefaciens outer membrane protein Atuporin and application thereof - Google Patents
Cell surface display system constructed based on agrobacterium tumefaciens outer membrane protein Atuporin and application thereof Download PDFInfo
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- 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
- C12N15/743—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Agrobacterium; Rhizobium; Bradyrhizobium
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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Abstract
The invention discloses a cell surface display system constructed based on agrobacterium tumefaciens outer membrane protein Atuporin and application thereof. The invention firstly clones and obtains the outer membrane protein Atuporin from the agrobacterium tumefaciens, and the C end of the outer membrane protein is deleted by 11 amino acids, and the outer membrane protein serves as an anchoring protein to construct a cell surface display system, so that the blank that the plant pathogenic bacteria lack an effective cell surface display system is filled. The cell surface display system is a very efficient agrobacterium tumefaciens cell surface display system, can effectively display target proteins/polypeptides on the surface of agrobacterium tumefaciens cells, has the capability of identifying the toxicity of exogenous target proteins/polypeptides, builds an effective platform for pertinently screening antibacterial peptides with antibacterial activity on plant pathogenic bacteria, is hopeful to screen novel efficient antibacterial peptides by using the cell surface display system, opens up a new idea for researching peptide functions, developing green biopesticides or genetic breeding against diseases, and has wide application prospects.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a cell surface display system constructed based on agrobacterium tumefaciens outer membrane protein Atuporin and application thereof.
Background
The microbial cell surface display technology refers to a novel genetic engineering technology for displaying a target sequence on the surface of a microbial cell in the form of fusion protein by means of membrane localization polypeptide fragments (or protein domains), and specifically introduces a technology for fixing a target protein or polypeptide on the surface of the cell by means of an anchoring protein, wherein the displayed target protein or polypeptide usually maintains relatively independent spatial structure and biological activity. The microbial cell surface display system mainly comprises host bacteria, dockerin and target protein/polypeptide, wherein a section of linker (linker) sequence without obvious biological function is added between dockerin and target protein/polypeptide to ensure that the target protein/polypeptide normally functions. The microbial cell surface display system has very wide application prospect 1,2,3 in aspects of polypeptide screening and identification, enzyme whole cell catalyst, whole cell adsorbent, vaccine and antibody screening, production, protein interaction library screening, biosensor, ecological environment restoration and the like.
The 2018 nobel chemical prize was awarded to scientists who invented phage display technology. The main factors which are commonly used in the microbial cell surface display system at present are phage (Bacterionphages), gram-negative bacteria (such as escherichia coli ESCHERICHIA COIL), gram-positive bacteria (such as bacillus glutamicum Corynebacterium glutamicum, bacillus subtilis Bacillus subtilis and the like), saccharomycetes (such as saccharomyces cerevisiae Saccharomy cescerevisiae) and the like. The key anchoring proteins in microbial cell surface display systems generally have the following properties: (1) can stably express and fix on the surface of the cell; (2) Can be effectively fused with target protein/polypeptide without affecting the structure and biological functions of the target protein/polypeptide; (3) Can resist degradation of proteases in the culture medium or periplasmic space. The anchor proteins of the microbial cell surface display systems commonly used today mainly include: (1) an outer membrane localization protein having a transmembrane domain (e.g., ompA of E.coli), (2) an N-terminal domain of ice nucleoprotein (e.g., inaXN of Xanthomonas campestris Xanthomonas campestris pv. Campestris), 4, (3) alpha-floc of yeast, (4) a spore coat protein (e.g., cot B of Bacillus subtilis), and the like 5.
Agrobacterium tumefaciens is one of the most serious plant pathogens that endanger crops and can induce the host to form crown gall 6. In nature, such soil-borne bacterial diseases seriously affect the yield and quality of crops. Meanwhile, because the agrobacterium tumefaciens has strong gene transfer capability, the agrobacterium tumefaciens is also developed by scientific researchers to be widely applied to genetic transformation of plants. At present, no cell surface display system for agrobacterium tumefaciens has been reported. In view of this, it is important to develop an efficient agrobacterium tumefaciens cell surface display system to rapidly screen and identify polypeptides that inhibit pathogenic bacteria. The technology is not only helpful for developing a green and environment-friendly plant disease control technology, but also provides valuable gene resources for disease-resistant breeding.
Reference is made to:
1、Chen,T.,et al.2019Construction of a bacterial surface display system based on outer membrane protein F.Microb Cell Fact 18(1):70.
2、Park,M.2020Surface Display Technology for Biosensor Applications:A Review.Sensors(Basel)20(10).
3、Anand,T.,et al.2021Phage Display Technique as a Tool for Diagnosis and Antibody Selection for Coronaviruses.Curr Microbiol 78(4):1124-1134.
4. Pair, li Youguo, li Yixing.2014 was built based on the surface display system at the N-terminus of Xanthomonas ice nucleoprotein Chin J Appl Environ Biol (3): 351-356.
5. Xie Zhidan, et al 2014, bacillus subtilis spore surface display research progress China Biotechnology (8): 105-111.
6、Mansfield,J.,et al.2012Top 10plant pathogenic bacteria in molecular plant pathology.Mol Plant Pathol 13(6):614-29.
Disclosure of Invention
The invention aims to provide a cell surface display system constructed based on agrobacterium tumefaciens outer membrane protein Atuporin and application thereof, so as to solve the defects in the prior art.
The invention adopts the following technical scheme:
The first aspect of the invention provides application of agrobacterium tumefaciens outer membrane protein Atuporin as an anchoring protein in constructing a cell surface display system, and the amino acid sequence of agrobacterium tumefaciens outer membrane protein Atuporin is shown as SEQ ID NO. 1.
Further, the Agrobacterium tumefaciens outer membrane protein Atuporin serves as an anchor protein, whose C-terminus is deleted by 11 amino acids.
Further, the gene sequence of the Agrobacterium tumefaciens outer membrane protein Atuporin is shown as SEQ ID NO. 2.
Furthermore, the cell surface display system is an agrobacterium tumefaciens cell surface display system which is formed by taking an agrobacterium tumefaciens outer membrane protein Atuporin as an anchoring protein and fixing target proteins/polypeptides on the surface of the agrobacterium tumefaciens cell.
Still further, the target protein/polypeptide comprises an antimicrobial peptide.
The invention provides an agrobacterium tumefaciens cell surface display system, which is formed by taking an agrobacterium tumefaciens outer membrane protein Atuporin as an anchoring protein and fixing target proteins/polypeptides on the surface of agrobacterium tumefaciens cells; the amino acid sequence of the Agrobacterium tumefaciens outer membrane protein Atuporin is shown as SEQ ID NO. 1.
Further, the Agrobacterium tumefaciens outer membrane protein Atuporin serves as an anchor protein, whose C-terminus is deleted by 11 amino acids.
Further, the gene sequence of the Agrobacterium tumefaciens outer membrane protein Atuporin is shown as SEQ ID NO. 2.
Further, the target protein/polypeptide comprises an antimicrobial peptide.
In a third aspect, the invention provides the use of the above-described Agrobacterium tumefaciens cell surface display system for identifying target protein/polypeptide toxicity.
The invention has the beneficial effects that:
According to the invention, an outer membrane protein Atuporin derived from agrobacterium tumefaciens is obtained by first cloning, analysis and experiments prove that the protein is positioned on the outer membrane of agrobacterium tumefaciens cells, 11 amino acids are deleted at the C end of the protein, and the protein is used as an anchoring protein to construct a cell surface display system, so that the gap that plant pathogenic bacteria lack an effective cell surface display system is filled. The cell surface display system is a very efficient agrobacterium tumefaciens cell surface display system, can effectively display target proteins/polypeptides on the surface of agrobacterium tumefaciens cells, can distinguish toxic proteins/polypeptides from non-toxic proteins/polypeptides after IPTG induction of protein/polypeptide expression, has the capability of identifying the toxicity of exogenous target proteins/polypeptides, builds an effective platform for targeted screening of antibacterial peptides with antibacterial activity on plant pathogenic bacteria, is hopeful to screen novel efficient antibacterial peptides by using the cell surface display system, opens up a new idea for researching peptide functions and developing green biopesticides or disease-resistant genetic breeding, and has wide application prospect.
Drawings
FIG. 1 is a graph of the predicted results of the extracellular membrane structure across proteins of Agrobacterium tumefaciens outer membrane protein Atuporin (left) and its C-terminal truncated version (C-terminal deletion of 11 amino acids) (right). The green box on the right panel shows that the C-terminus of Atuporin after expression truncation is exposed outside the extracellular membrane.
FIG. 2 is a schematic diagram of the carrier structure frame of an Agrobacterium tumefaciens cell surface display system constructed based on Agrobacterium tumefaciens outer membrane protein Atuporin. SIGNAL PEPTIDE signal peptide; pHM1: a carrier; bamH I/Nde I/Sal I/Xho I: restriction sites; lac repeater: lactose operon repressor protein; atuporin Δc11: the outer membrane protein Atuporin C end of the agrobacterium tumefaciens lacks 11 amino acids; the tether: a linker sequence; ccdB: removing toxin genes from the escherichia coli background to be used as screening markers; AMPs: inserting the target protein/polypeptide into the expression site; outer membrane: an outer membrane protein; PERIPLASM: periplasmic space.
FIG. 3 is a graph showing the results of laser confocal microscopy observations of a cell surface display system displaying the super-folded green fluorescent protein sfGFP with and without the Agrobacterium tumefaciens outer membrane protein Atuporin (left three) and the Agrobacterium tumefaciens outer membrane protein Atuporin (right three). Bright field is Bright field, GFP is green fluorescent protein excitation light conditions, and Merge is the overlap of the first two fields.
FIG. 4 is a graph showing the Western Blot results of the detection of HA antibody after exogenous proteinase K treatment of a wild strain containing the cell surface display system of Agrobacterium tumefaciens outer membrane protein Atuporin expressing the amino acid sequence (2 xHA) at positions 98-106 of the two fused human influenza virus hemagglutinin protein or the super-folded green fluorescent protein sfGFP or without the expression vector system. 2xHA corresponds to LBA4404 (pHM 1-ASD-2 xHA); GFP corresponds to LBA4404 (pHM 1-ASD-sfGFP); WT corresponds to LBA4404.
FIG. 5 shows that the cell surface display system containing the Agrobacterium tumefaciens outer membrane protein Atuporin displays the 98-106 amino acid sequence (2 xHA) (left three tubes) or the pig-derived antimicrobial peptide Protegrin-1 (right three tubes) of the two-segment fusion human influenza virus hemagglutinin protein on the cell surface, and after 0.1mM IPTG induction, the Agrobacterium tumefaciens is continuously cultured for 0/3/6h, and then the bacterial liquid becomes turbid.
FIG. 6 shows the change of OD600 of bacterial liquid after the two sections of fusion human influenza virus hemagglutinin protein 98-106 amino acid sequences (2 xHA) (left side) or swine antimicrobial peptide Protegrin-1 (right side) are induced on the cell surface by 0mM/0.1mM/1mM IPTG and the agrobacterium tumefaciens is continuously cultured for 0/3/6/9/12 hours by a cell surface display system containing the agrobacterium tumefaciens outer membrane protein Atuporin.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the application embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are conventional in the art. The experimental methods of the specific experimental conditions are not noted in the following examples, and generally follow the conventional experimental conditions. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
Interpretation of the terms
LB plate: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of NaCl and 20g/L of agar.
PBS-MgCl 2 buffer: 4g NaCl,0.1g KCl,0.71g Na 2HPO4、0.12g KH2PO4、1.19g MgCl2 the above reagents were dissolved in 400ml of water, the pH was adjusted to 7.4 and the volume was set to 500ml.
The amino acid sequence of the outer membrane protein Atuporin of Agrobacterium tumefaciens is shown as SEQ ID NO. 1, and the gene sequence is shown as SEQ ID NO. 2 (including the final stop codon tga).
The agrobacterium tumefaciens surface display vector taking agrobacterium tumefaciens outer membrane protein Atuporin as anchoring protein is vector pHM1-ASD, which is obtained by connecting the gene sequence of agrobacterium tumefaciens outer membrane protein Atuporin (the last 11 amino acids shown in SEQ ID NO:1, as shown in figure 1) with the modified pHM1 vector (pHM 1 vector is conventionally used in the field and is provided with a spectinomycin resistance gene and can be used for protein expression in agrobacterium tumefaciens) through a homologous recombination method, wherein the gene sequence is shown in SEQ ID NO:9 and comprises Lac Repressor sequence, atuporin (the last 11 amino acids shown in SEQ ID NO:1, as shown in SEQ ID NO: 11) sequence, NO obvious functional linker 2xtether sequence, ccdB sequence and a spectinomycin resistance marker gene sequence. The construction of the Agrobacterium tumefaciens cell surface display system is to clone and connect the gene sequence of target protein/polypeptide to the downstream of the gene sequence of Agrobacterium tumefaciens outer membrane protein Atuporin in the vector pHM1-ASD by homologous recombination (as shown in FIG. 2), and form a fusion gene open reading frame by a linker sequence. The vector pHM1-CK involved differs from vector pHM1-ASD in that it does not include Atuporin (C-terminal deletion of 11 amino acids, i.e., the last 11 amino acids shown in SEQ ID NO: 1) sequence.
Example 1: construction of Agrobacterium tumefaciens outer membrane protein Atuporin cell surface display sfGFP vector pHM1-ASD-sfGFP
(1) Acquisition of target protein/polypeptide sfGFP Gene
The amplification primers were designed based on the gene sequence (shown as SEQ ID NO:4, including the final stop codon tga) of the target protein/polypeptide sfGFP (super-folded green fluorescent protein, amino acid sequence shown as SEQ ID NO: 3) and the sequence features on the pHM1-ASD vector:
sfGFP-F1(SEQ ID NO:10):
5’-CTCCAGCTGCGGGTATCGCATATGCCATGCGTAAAGGCGAGGAG-3’
sfGFP-R1(SEQ ID NO:11):
5’-CAAGCTTGCATGCCTGCAGGTCGACTCATTTGTACAGTTCATC-3’
the plasmid vector containing the complete open reading frame of the sfGFP gene is used as a template, the sfGFP-F1 and the sfGFP-R1 are used as primers, and the target protein/polypeptide sfGFP gene is amplified by a PCR method under the following amplification conditions: pre-denaturation at 98℃for 3min; the following 30 cycles were further performed: denaturation at 98℃for 10s, annealing at Tm-5℃for 30s and extension at 72℃for 15s; finally, the extension is carried out for 10min at 72 ℃.
(2) Digestion recovery of vector pHM1-ASD
The selectable marker fragment ccdB on the vector pHM1-ASD was removed by appropriate cleavage sites (Nde I and SalI) based on the sequence of the vector pHM1-ASD, and the vector pHM1-ASD fragment was recovered by agarose gel.
(3) Construction of vector pHM1-ASD-sfGFP
The PCR product of sfGFP gene obtained in step (1) was ligated with the vector pHM1-ASD fragment recovered in step (2) by Gibson homologous recombination (using Seamless Assembly seamless cloning kit from Saint Biotechnology (Shanghai) Inc.), and the ligation was transformed into E.coli T1 strain (Shanghai Weidi Biotechnology Co., ltd.) by heat shock. Transformants were screened with LB plates containing 100mg/L spectinomycin, positive clones in the transformants were picked up using the identification primers (PCR primers for the corresponding gene sfGFP), plasmids were extracted, identified and sequenced (Zhejiang Kagaku Biotechnology Co., ltd.), sequencing results showed that the obtained pHM1-ASD-sfGFP vector was constructed successfully, the sfGFP gene sequence was correct, and the sfGFP sequence was located downstream of the Agrobacterium tumefaciens outer membrane protein Atuporin, and fused into a complete open reading frame by the linker sequence.
(4) Construction and identification of recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-sfGFP)
The plasmid pHM1-ASD-sfGFP constructed successfully in step (3) was transformed into Agrobacterium tumefaciens LBA4404 by heat shock, and positive transformants were selected on LB plates containing 100mg/L spectinomycin: colony PCR amplification was performed using sfGFP-F1 and sfGFP-R1 as primers, and the results demonstrated that plasmid pHM1-ASD-sfGFP was successfully transformed into LBA4404 strain, resulting in recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-sfGFP).
(5) Construction and identification of recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-CK-sfGFP) negative control Strain
The vector pHM1-ASD was replaced with vector pHM1-CK, see steps (1) - (4) for the rest.
(6) Laser confocal microscopic analysis of recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-sfGFP) and LBA4404 (pHM 1-CK-sfGFP)
Recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-sfGFP) and LBA4404 (pHM 1-CK-sfGFP) strains were inoculated into 5ml of LB liquid medium containing 100mg/L spectinomycin, respectively, and cultured at 28℃and 220rpm for 12 hours; then diluting the bacterial liquid with 5ml of fresh LB liquid medium containing 100mg/L spectinomycin, dividing the bacterial liquid into two parts, adding IPTG (isopropyl-beta-D-thiogalactoside) into one part with the final concentration of 0.1mM, and adding no reagent into the other part; then inducing and culturing for 12h at 28 ℃ and 220 rpm; finally, 10ul of each sample was taken out and coated on a microscope slide, covered with a cover slip, finger pressed and dried for 1h for observation by a laser confocal microscope. As shown in FIG. 3, the results showed that sfGFP protein in the negative control strain LBA4404 (pHM 1-CK-sfGFP) was mainly localized in the cytoplasm after induced expression, whereas sfGFP protein in strain LBA4404 (pHM 1-ASD-sfGFP) was significantly localized on the cell membrane after induced expression, indicating that successful expression of sfGFP protein on the cell surface of the recombinant strain LBA4404 (pHM 1-ASD-sfGFP) containing the cell surface display system of Agrobacterium tumefaciens outer membrane protein Atuporin.
Example 2: construction of Agrobacterium tumefaciens outer membrane protein Atuporin cell surface display 2xHA vector pHM1-ASD-2xHA
(1) Acquisition of target protein/polypeptide 2xHA Gene
According to the 98-106 amino acid sequence (2 xHA, the amino acid sequence is shown as SEQ ID NO:5, the gene sequence is shown as SEQ ID NO: 6) of two-segment fusion human influenza virus hemagglutinin protein, the two-segment fusion human influenza virus hemagglutinin protein is directly synthesized by commercial company (Beijing department of biotechnology Co., ltd.) and according to the sequence characteristics on pHM1-ASD carrier, proper homology arms are added at two ends.
(2) Digestion recovery of vector pHM1-ASD
The selectable marker fragment ccdB on the vector pHM1-ASD was removed by appropriate cleavage sites (Nde I and SalI) based on the sequence of the vector pHM1-ASD, and the vector pHM1-ASD fragment was recovered by agarose gel.
(3) Construction of vector pHM1-ASD-2xHA
The two-segment fusion human influenza virus hemagglutinin protein 98-106 amino acid sequence (2 xHA) obtained in the step (1) was ligated with the vector pHM1-ASD fragment recovered in the step (2) by Gibson homologous recombination (using Seamless Assembly seamless cloning kit from the company of Santa Biotechnology, inc., shanghai), and the ligation system was transformed into E.coli T1 strain (Shanghai Biotechnology, inc.) by a heat shock method. Transformants were screened with LB plates containing 100mg/L spectinomycin, positive clones in the transformants were picked using the identification primers (AMP-NdeI-F and AMP-SalI-R), plasmids were extracted, identified and sequenced (Zhejiang Kagaku Biotechnology Co., ltd.), and the sequencing results indicated that the obtained pHM1-ASD-2xHA vector was constructed successfully, 2xHA gene sequence was correct, and 2xHA sequence was located downstream of Agrobacterium tumefaciens outer membrane protein Atuporin, fused into a complete open reading frame by linker sequences.
AMP-NdeI-F (as shown in SEQ ID NO: 12): 5'-CGCTGAGGCTCCAGCTGCGGGTATC-3' A
AMP-SalI-R (as shown in SEQ ID NO: 13): 5'-GCCAAGCTTGCATGCCTGCAGGTCG-3' A
(4) Construction and identification of recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-2 xHA)
The plasmid pHM1-ASD-2xHA constructed successfully in step (3) was transformed into Agrobacterium tumefaciens LBA4404 by heat shock, and positive transformants were selected on LB plates containing 100mg/L spectinomycin: colony PCR amplification was performed using AMP-NdeI-F and AMP-SalI-R as primers, and the results demonstrated that plasmid pHM1-ASD-2xHA was successfully transformed into LBA4404 strain, resulting in recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-2 xHA).
(5) Western Blot identification after proteinase K treatment of recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-2 xHA)
Recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-2 xHA) and LBA4404 (pHM 1-ASD-sfGFP) strains were inoculated into 5ml of LB liquid medium containing 100mg/L spectinomycin, respectively, while Agrobacterium tumefaciens LBA4404 (i.e., wild LBA4404 strain) containing no exogenous plasmid was cultured with LB liquid medium containing no antibiotic, and cultured at 28℃and 220rpm for 12 hours; then diluting the bacterial liquid with 5ml of fresh LB liquid medium containing 100mg/L spectinomycin, dividing the bacterial liquid into two parts, adding IPTG to the final concentration of 0.1mM in one part, and adding no reagent in the other part; then inducing and culturing for 12h at 28 ℃ and 220 rpm; then centrifugally collecting thalli at 4 ℃ for 1500g for 10min, centrifugally washing thalli twice by using PBS buffer solution (pH=7.4), finally adding protease K (Proteinase K) buffer solution to gently resuspend the collected thalli, adding protease K to a final concentration of 400ug/ml, and carrying out water bath treatment at 37 ℃ for 0, 30, 40, 50 and 60min, wherein a negative control strain LBA4404 (pHM 1-ASD-sfGFP) is only selected for 0 and 60min, and a negative control strain LBA4404 without exogenous plasmid is also selected for 0 and 60min; then, 1ul of PMSF (phenylmethylsulfonyl fluoride) was immediately added, incubated at room temperature for 10min, the proteinase K reaction was terminated, the cells were washed by resuspension with PBS-MgCl 2 buffer for 2 times, the disrupted cells were removed, the cells were resuspended with 40ul of PBS-MgCl 2, 10ul 5x SDS Loading Buffer protein loading buffer was added, the cell-denatured proteins were disrupted by boiling for 10min, and finally 5ul was removed for Western Blot analysis (HA antibody detection). As a result, as shown in FIG. 4, when the protease K treatment was not performed, each of LBA4404 and LBA4404 (pHM 1-ASD-sfGFP) and LBA4404 (pHM 1-ASD-2 xHA) had a single protein band at about 25 kDa; whereas, after 2xHA was displayed by the cell surface display system containing Agrobacterium tumefaciens outer membrane protein Atuporin, a unique protein band was present between 35kDa and 50kDa, and negative controls LBA4404 and LBA4404 (pHM 1-ASD-sfGFP) were absent between 35kDa and 50kDa, indicating that this band was unique after induction, atuporin-2xHA fusion expression; after 30, 40, 50 and 60min of proteinase K external application treatment, the protein quantity between 35kDa and 50kDa is found to be reduced, and at the same time, the protein band of about 25kDa is not changed obviously after 60min treatment. The 25kDa protein band is an intracytoplasmic protein that is unaffected by treatment with exogenous proteinase K and that can reflect successful display of 2xHA expression on the cell surface by Agrobacterium tumefaciens outer membrane protein Atuporin.
Example 3: identification of antibacterial peptide toxicity Using cell surface display System containing Agrobacterium tumefaciens outer Membrane protein Atuporin
(1) Acquisition of target protein/polypeptide Protegrin-1 Gene
According to the sequence of pig antimicrobial peptide Protegrin-1 (amino acid sequence is shown as SEQ ID NO:7, gene sequence is shown as SEQ ID NO: 8), the polypeptide is directly synthesized by commercial company (Beijing qing family Biotech Co., ltd.) and according to the sequence characteristics on pHM1-ASD carrier, two ends of the polypeptide are added with proper homology arms.
(2) Digestion recovery of vector pHM1-ASD
The selectable marker fragment ccdB on the vector pHM1-ASD was removed by appropriate cleavage sites (Nde I and SalI) based on the sequence of the vector pHM1-ASD, and the vector pHM1-ASD fragment was recovered by agarose gel.
(3) Construction of vector pHM1-ASD-Protegrin-1
The pig-derived antimicrobial peptide Protegrin-1 sequence obtained in the step (1) was ligated with the vector pHM1-ASD fragment recovered in the step (2) by Gibson homologous recombination (using Seamless Assembly seamless cloning kit from Saint Biotechnology (Shanghai)) and the ligation was transformed into E.coli T1 strain (Shanghai Biotechnology Co., ltd.) by a heat shock method. Transformants were screened with LB plates containing 100mg/L spectinomycin, positive clones in the transformants were picked using identification primers (AMP-NdeI-F and AMP-SalI-R), plasmids were extracted, identified and sequenced (Zhejiang Kagaku Biotechnology Co., ltd.), and the sequencing results indicated that the obtained pHM1-ASD-Protegrin-1 vector was constructed successfully, that the Protegrin-1 gene sequence was correct, that the Protegrin-1 sequence was located downstream of the Agrobacterium tumefaciens outer membrane protein Atuporin, and that it was fused into a complete open reading frame by a linker sequence.
(4) Construction and identification of recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-Protegrin-1)
The plasmid pHM1-ASD-Protegrin-1 constructed successfully in step (3) was transformed into Agrobacterium tumefaciens LBA4404 by heat shock method, and positive transformants were selected on LB plates containing 100mg/L spectinomycin: colony PCR amplification was performed using AMP-NdeI-F and AMP-SalI-R as primers, and the results demonstrated that plasmid pHM1-ASD-Protegrin-1 was successfully transformed into LBA4404 strain, resulting in recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-Protegrin-1).
(5) Antibacterial Activity detection of recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-Protegrin-1)
Recombinant Agrobacterium tumefaciens LBA4404 (pHM 1-ASD-2 xHA) and LBA4404 (pHM 1-ASD-Protegrin-1) strains were activated on LB plates containing 100mg/L spectinomycin, respectively, for 48h; then, the monoclonal strains are respectively transferred into 5ml of LB liquid medium containing 100mg/L spectinomycin, and cultured for 12 hours at 28 ℃ and 220 rpm; then diluted to od600=0.05 with fresh LB liquid medium containing 100mg/L spectinomycin, treated one without IPTG, treated two with IPTG to a final concentration of 0.1mM, treated three with IPTG to a final concentration of 1mM, each treatment set three replicates separately; then culturing for 12h at 28 ℃ with a 220rpm incubator, and measuring the concentration of the bacterial liquid by using a spectrophotometer every 3h, wherein the photographing record is carried out for 0h, 3h and 6h, as shown in figure 5, the bacterial liquid can proliferate and grow after the LBA4404 (pHM 1-ASD-2 xHA) is induced by IPTG, and the bacterial liquid growth of the bacterial strain LBA4404 (pHM 1-ASD-Protein-1) expressing the pig antimicrobial peptide Protein-1 is obviously inhibited after the bacterial liquid is induced by IPTG. Finally, the statistical data is used for preparing a growth curve, as shown in fig. 6, the same result can be observed, the bacterial liquid can proliferate and grow after the LBA4404 (pHM 1-ASD-2 xHA) is induced by IPTG, and the bacterial liquid growth of the strain LBA4404 (pHM 1-ASD-Protein-1) for expressing the pig antimicrobial peptide Protein-1 is obviously inhibited after the induction by IPTG. The result shows that the pig-derived antibacterial peptide protegrin-1 acts on bacterial cell membranes to play a toxic function, and the pig-derived antibacterial peptide protegrin-1 is expressed on the cell surface to inhibit the growth of agrobacterium tumefaciens, while the nontoxic 2xHA is expressed on the cell surface to not inhibit the growth of agrobacterium tumefaciens.
The cell surface display system constructed based on the agrobacterium tumefaciens outer membrane protein Atuporin can effectively display target proteins/polypeptides on the cell surface of the agrobacterium tumefaciens, and can distinguish toxic proteins/polypeptides from non-toxic proteins/polypeptides after the expression of IPTG induced proteins/polypeptides, so that the system has the capability of identifying the toxicity of exogenous target proteins/polypeptides, and an effective platform is built for targeted screening of antibacterial peptides with antibacterial activity on plant pathogenic bacteria.
Claims (7)
1. The application of the agrobacterium tumefaciens outer membrane protein Atuporin as an anchor protein in constructing a cell surface display system is characterized in that the amino acid sequence of the agrobacterium tumefaciens outer membrane protein Atuporin is a sequence shown as SEQ ID NO.1, and 11 amino acids are deleted at the C end; the cell surface display system is an agrobacterium tumefaciens cell surface display system which is formed by taking an agrobacterium tumefaciens outer membrane protein Atuporin as an anchoring protein, fusing target proteins/polypeptides at the C end of the agrobacterium tumefaciens outer membrane protein Atuporin so as to be fixed on the surface of the agrobacterium tumefaciens cell.
2. The use according to claim 1, wherein the gene sequence of the outer membrane protein Atuporin of agrobacterium tumefaciens is shown in SEQ ID No. 2.
3. The use of claim 1, wherein the target protein/polypeptide comprises an antimicrobial peptide.
4. The agrobacterium tumefaciens cell surface display system is characterized by being formed by taking an agrobacterium tumefaciens outer membrane protein Atuporin as an anchoring protein, fusing target protein/polypeptide at the C end of the agrobacterium tumefaciens outer membrane protein Atuporin so as to be fixed on the surface of the agrobacterium tumefaciens cell; the amino acid sequence of the Agrobacterium tumefaciens outer membrane protein Atuporin is a sequence shown in SEQ ID NO. 1, and 11 amino acids are deleted at the C-terminal.
5. The system of claim 4, wherein the gene sequence of the Agrobacterium tumefaciens outer membrane protein Atuporin is shown in SEQ ID NO. 2.
6. The agrobacterium tumefaciens cell surface display system of claim 4, wherein the target protein/polypeptide includes an antimicrobial peptide.
7. Use of the agrobacterium tumefaciens cell surface display system of any of claims 4-6 for identifying target protein/polypeptide toxicity.
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