CN114716516B

CN114716516B - Phage display polypeptide VS specifically bound by chicken DEC-205 and application thereof

Info

Publication number: CN114716516B
Application number: CN202210501605.3A
Authority: CN
Inventors: 马孙婷; 王晓丽; 欧阳伟; 吕立新; 徐彬; 钱晶; 王永山; 毕振威; 诸玉梅; 夏兴霞; 王晶宇
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2022-05-10
Filing date: 2022-05-10
Publication date: 2023-06-13
Anticipated expiration: 2042-05-10
Also published as: CN114716516A

Abstract

A phage display polypeptide VS specifically combined with chicken DEC-205 and application thereof relate to the field of biological medicine, and the amino acid sequence of the phage display polypeptide VS is ValSerProAlaTrpAspAlaArgThrArgSerALa. The phage display polypeptide VS specifically combined by the chicken DEC-205 has small relative molecular mass, and is easy to realize synthesis, expression, modification and the like. The polypeptide VS can specifically bind to chicken DEC-205 recombinant protein; the binding capacity of the polypeptide VS and 293t cells expressing the chicken DEC-205 recombinant protein is stronger; the polypeptide VS can be well combined with chicken bone marrow-derived DCs. The phage display polypeptide VS specifically combined with the chicken DEC-205 screened by the invention can be combined with various drug delivery vectors, can be used as a vaccine adjuvant to prepare chicken bone marrow-derived DCs targeting vaccine, and can improve the immunization effect of chicken bone marrow-derived DCs targeting vaccine.

Description

Phage display polypeptide VS specifically bound by chicken DEC-205 and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to a phage display polypeptide VS specifically combined by chicken DEC-205 and application thereof.

Background

Dendritic Cells (DCs) are professional antigen presenting cells that can effectively deliver drugs to corresponding sites by targeted bio-immunotherapy of dendritic cells, and can reduce the dosage and the number of times of administration, improve the therapeutic effect of the drugs, and reduce adverse reactions. Studies have shown that a targeting peptide of human peripheral blood DCs is obtained by screening by using phage display linear 12 peptide library technology, and after fusion expression of the targeting peptide and antigen, a better immune effect is obtained (Curiel T J, morris C, brumlik M, et al Peptides identified through phage display direct immunogenic antigen to dendritic cells [ J ]. Journal of Immunology,2004,172 (12): 7425-7431.).

Targeting antigens to endocytic receptors on the surface of DCs is a new strategy to enhance the adaptive immune response. Studies have shown that the use of prokaryotic expression antigens (newcastle disease virus hemagglutinin-neuraminidase) in fusion proteins with the single chain variable region of anti-chicken DEC-205 antibodies resulted in higher titers of neutralizing antibodies in the immunized group fused with the single chain variable region of anti-chicken DEC-205 antibodies after immunization (Shretha A, sadeyen J R, lukosaiyte D, et al targeting Haemagglutinin Antigen of Avian Influenza Virus to Chicken Immune Cell Receptors Dec and CD11c Induces Differential Immune-Potentiating Responses [ J ]. Vaccines,2021,9 (7): 784.).

Up to now, there has been no report on a dominant polypeptide capable of specifically binding to chicken bone marrow-derived DCs receptor DEC-205.

Disclosure of Invention

The invention aims to provide phage display polypeptide VS specifically combined with chicken DEC-205 and application thereof, so as to fill the blank of dominant polypeptide related to chicken bone marrow-derived DCs receptor DEC-205 specifically combined in the field of poultry targeting vaccine.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the amino acid sequence of phage display polypeptide VS specifically bound by chicken DEC-205 is ValSerProAlaTrpAspAlaArgThrArgSerA.

In a preferred embodiment, the nucleotide sequence encoding the amino acid sequence of the phage display polypeptide VS is GTTTCTCCTGCTTGGGATGCGAGGACTAGGAGTGCT.

As a preferred embodiment, the phage display polypeptide VS is obtained by designing signal peptides for three C-type lectin-like domains of chicken bone marrow derived DCs receptor DEC-205, expressing in 293t cells fused with three C-type lectin-like domains, human IgG1Fc and his tag, identifying and purifying chicken DEC-205 recombinant proteins, and screening by phage linear 12 peptide library technology.

As a preferred embodiment, the three C-type lectin-like domains are CTLD-4, CTLD-5 and CTLD-6, respectively.

As a preferred embodiment, the nucleotide sequence of the signal peptide is ATGGCTTCACCTCTCACAAGATTCTTGTCTCTG AACCTCCTGCTTCTCGGTGAATCAATTATCCTGGGTTCTGGCGAGGCT, the amino acid sequence is MetAlaaSerProLeuThrArgPheLeuSerLeuAsnLeuLeuLeuLeuGlyGluSerIleIleLeuGlySerGlyGluAla a. The invention relates to a method for producing a fibre-reinforced plastic composite.

As a preferred embodiment, the phage display polypeptide VS is capable of specifically binding to chicken DEC-205 recombinant protein.

As a preferred embodiment, the phage display polypeptide VS is capable of binding to chicken bone marrow-derived DCs.

The use of the phage display polypeptide VS specifically bound by chicken DEC-205 of the invention as a drug delivery vehicle.

The invention relates to application of phage display polypeptide VS specifically combined with chicken DEC-205 in targeting vaccine adjuvant of chicken bone marrow-derived DCs.

The beneficial effects of the invention are as follows:

the phage display polypeptide VS (ValSerProAlaTrpAspAlaArgThrArgSerAla) specifically combined with the chicken DEC-205 screened by the invention has small relative molecular mass, and is easy to realize synthesis, expression, modification and the like. Experiments prove that the phage display polypeptide VS specifically combined with the chicken DEC-205 can be specifically combined with the chicken DEC-205 recombinant protein; the phage display polypeptide VS specifically combined with the chicken DEC-205 has stronger combination capability with 293t cells expressing the chicken DEC-205 recombinant protein; the phage display polypeptide VS specifically bound by the chicken DEC-205 can be well bound with chicken bone marrow-derived DCs.

The phage display polypeptide VS specifically combined with the chicken DEC-205 screened by the invention can be combined with various drug delivery vectors, can be used as a vaccine adjuvant to prepare chicken bone marrow-derived DCs targeting vaccine, and can improve the immunization effect of chicken bone marrow-derived DCs targeting vaccine.

Drawings

FIG. 1 shows the results of the digestion of pcDNA3.1-DEC 205-Fc-his recombinant expression plasmids. In the figure, 1: a Marker;2: nheI-NotI double restriction plasmid; 3: pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid.

FIG. 2 shows the results of the cleavage assay of pcDNA3.1-Fc-his recombinant plasmids. In the figure, 1: nheI-NotI double restriction plasmid; 2: pcDNA3.1-Fc-his recombinant plasmid; 3: and (5) Marker.

FIG. 3 shows the result of western blot identification of cell lysates and cell supernatants (ECL color development) after 24h, 48h of recombinant plasmid transfection

FIG. 4 shows the result of western blot identification of cell supernatants after recombinant plasmid transfection (DAB color development).

FIG. 5 shows the result of western blot identification (ECL color development) of samples after 48h transfection of recombinant plasmids with different volumes of transfection reagent. In the figure, fc is human IgG1Fc, the protein size is about 30kD, DEC-Fc is the protein expressed by fusion of 3C-type lectin-like domains CTLD-4, CTLD-5 and CTLD-6 of chicken DEC205 and human IgG1Fc, and the protein size is about 90kD.

FIG. 6 shows the SDS-PAGE identification of purified recombinant protein of chicken DEC 205. In the figure, 1 is a recombinant protein sample expressed by pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid before purification, 2-4 is a recombinant protein sample expressed by purified pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid which is eluted sequentially, and M is Marker.

FIG. 7 is a schematic of a three-round screening assay for recovered phage titers. In the figure, a: a first round of screening; b: a second round of screening; c: and a third round of screening.

FIG. 8 shows the results of the detection of affinity of the affinity peptide of the chicken DEC-205 receptor.

FIG. 9 shows the binding effect of biotin-labeled short peptides to 293t cells.

FIG. 10 shows the binding effect of biotin-labeled short peptides on chicken bone marrow-derived DCs.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 construction of pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid

The signal peptide was designed according to 3C-type lectin-like domains (CTLD-4, CTLD-5, CTLD-6) published by EMBL database (accession number AJ 574899) with binding effect, namely the nucleotide sequence of mouse CD8 alpha signal peptide was ATGGCTTCACCTCTCACAAGATTCTTGTCTCTG AACCTCCTGCTTCTCGGTGAATCAATTATCCTGGGTTCTGGCGAGGCT, the amino acid sequence thereof was MASPLTRFLSLNLLLLGESIILGSGEA (MetAlaSerProLeuThrArgPheLeuSerLeuAsnLeuLeuLeuLeuGlyGluSerIleIleLeuGlySerGlyGluAla), the signal peptide, human IgG1Fc and his tag were fused for expression, codons were optimized, DEC 205-Fc-his target genes were synthesized, and the resultant was cloned into pcDNA3.1-DEC 205-Fc-his expression vector to obtain pcDNA3.1-DEC 205-his recombinant expression plasmid, and NheI-NotI double-digested pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid, as shown in FIG. 1, to obtain a target fragment of about 2228 bp.

The human IgG1Fc and his label are fused for expression, codons are optimized, fc-his target genes are synthesized, pcDNA3.1-Fc-his recombinant plasmids are obtained by cloning the target genes into pcDNA3.1 expression vectors, and NheI-NotI double-restriction pcDNA3.1-Fc-his recombinant plasmids are obtained, as shown in figure 2, and a 842bp gene fragment is obtained.

Example 2 identification and recovery of expression of recombinant protein of chicken DEC205

The recombinant plasmids obtained in example 1 were transiently transfected into 293t cells, respectively, and the transfection method was described in X-tremeGENE HP DNA Transfection Reagent. Cell lysate and cell supernatant after 24h and 48h transfection are respectively taken, and the target band of about 90kD appears in the cell lysate and the cell supernatant through western blot detection (shown in figure 3), the primary antibody is a his tag during detection, the molecular weight of the primary antibody is similar to that of the calculated target protein, and the 48h expression quantity is larger than 24h expression quantity. The target band of about 90kD appears in the cell supernatant as found by western blot detection (FIG. 4), and the primary antibody is diluted (1:300 (v/v)) prepared by immunizing mice with CTLD-4, CTLD-5 and CTLD-6 prokaryotic expression proteins. 2. Mu.g of the recombinant plasmid obtained in example 1 was mixed with 3. Mu.l and 5. Mu.l of the transfection reagent respectively, and after 48 hours of transfection, samples were collected and subjected to western blot detection, and the results are shown in FIG. 5, wherein the target protein was detected in both the cell supernatants and the cell lysates of the 3. Mu.l and 5. Mu.l transfection reagent groups, and the target protein expression level in the cell supernatants and the cell lysates of the 3. Mu.l transfection reagent groups was greater than 5. Mu.l transfection reagent groups.

And (3) recovering the cell lysate and the cell supernatant in a large amount, purifying recombinant proteins expressed by pcDNA3.1-DEC 205-Fc-his recombinant expression plasmids by using a nickel column (2, 3 and 4 are respectively three sequentially eluted samples), and detecting the target proteins of about 90kD in the sample 2 and the sample 3 by SDS-PAGE (shown in FIG. 6).

Example 2 screening of dominant binding peptides for recombinant proteins of chicken DEC-205 Using phage Linear 12 peptide library technology

First round screening (a in fig. 7): 100 μl protein G-sepharose medium plus 1ml TBS+0.1%Tween (TBST) solution was washed twice, resuspended in 1ml blocking buffer and allowed to act at 4deg.C for 60min. 1.5X10 with TBST ¹¹ Each phage particle (corresponding to 10. Mu.l of original library) and 300ng of the chicken DEC205 recombinant protein were diluted to a final volume of 200. Mu.l and allowed to act at room temperature for 20min. After blocking the reaction, protein G-agarose medium was precipitated, washed 4 times with 1ml TBST, and phage-recombinant protein mixture was added to the washed protein G-agarose medium, gently mixed, allowed to act at room temperature for 15min and mixed from time to time. Washing with 1ml TBST 10 timesThe precipitation medium was resuspended in 1ml, 0.2M Glycine-HCl (pH 2.2) +1mg/ml BSA, and the bound phage eluted and allowed to react at room temperature for 10min. The mixture was centrifuged for 1min, transferred to a centrifuge tube, and the eluate was neutralized with 150. Mu.l of 1M Tris-HCl (pH 9.1). The elution droplet size was determined on LB/IPTG/Xgal plates. The eluate was vigorously shaken in an Er2738 culture solution (A600 nm. Apprxeq.0.5) for 4.5h with an E.coli ER2738 culture solution, and the phage amplification solution was prepared by a polyethylene glycol NaCl secondary precipitation method to measure the titer. Second round of screening (b in fig. 7): the first round of screening steps was repeated with 100. Mu.l of protein G-agarose medium replaced with 100. Mu.l of protein A-agarose medium. Third round of screening (c in fig. 7): the first round of screening steps was repeated with 100. Mu.l of protein A-agarose medium replaced with 100. Mu.l of protein G-agarose medium. Plaques were randomly picked on LB/IPTG/Xgal plates, M13 phage single-stranded DNA was extracted with the kit and sent to the company for sequencing.

The number of screening rounds, the amount of input and the amount of recovery are shown in the table. In the third round of screening plaque sequencing results, 120 total samples were sequenced, of which 42 lost 12 peptide phage (much more infectious than phage expressing the foreign peptide).

TABLE 1

Number of screening rounds	Input (pfu)	Recovery (pfu)	Recovery rate	Binding medium
					1	1.5×10 ¹¹	1.26×10 ⁶	8.4×10 ^-6	protein G-agarose
2	5×10 ¹¹	2×10 ⁶	4×10 ^-6	protein A-agarose
					3	2×10 ¹⁰	2.2×10 ⁴	1.1×10 ^-6	protein G-agarose

EXAMPLE 3 Synthesis of chicken DEC-205 receptor affinity peptides

The phage display polypeptide VS of the invention was taken as the short peptide VS (VSPAWDARTRSA) with the highest repetition rate in the third round of screening. Meanwhile, random disordered short peptide PS (PSPLLRSNFFSI) is used as a reference, fmoc method is adopted to synthesize the short peptide respectively, biotin (FITC) is used for marking the short peptide, and HPLC purification (purity)>95%) and mass spectrometry (done by the wunhan de biosome company). The biotin-labeled oligopeptide powder was pre-dissolved in DMSO, PBS was added to a final concentration of 30% in DMSO, the molar mass of the oligopeptide VS was 1542.4g/mol, the molar mass of the oligopeptide PS was 1603.7g/mol, and finally diluted to a working concentration of about 0.465. Mu.g/. Mu.l, each 100. Mu.l containing about 3X 10 ^-8 And (3) mol short peptide, and preserving at-20 ℃ for standby.

Example 4 detection of affinity of the affinity peptide for the chicken DEC-205 receptor

1. ELISA (enzyme-linked immunosorbent assay) method for determining affinity

The recombinant plasmids obtained in example 1 were transfected into 293t cells, respectivelyThe method is shown in the specification of X-tremeGENE HP DNA Transfection Reagent. Cells were recovered 48h after transfection, washed once with PBS, pre-plated with polylysine (0.1 mg/ml) in 96-well plates, air-dried at room temperature for half an hour, and the cells were incubated at 2X 10 ⁵ Well, plating, half an hour at room temperature, PBS washes off non-adherent cells, non-transfected cells as a blank. The mixture was fixed with 95% ethanol at room temperature for 10min, washed with PBS three times, with 0.2% Triton-100 at room temperature for 10min, washed with PBS three times, with 3% BSA-PBS at 37℃for 1min, and washed with PBS three times. Respectively adding 3×10 ^-8 mol of biotin-labeled short peptide VS and short peptide PS were allowed to act for 3h at room temperature, and PBST was washed 3 times. HRP-labeled streptavidin (1:2000 (v/v) diluted) was added and the mixture was allowed to react at 37℃for 1h, PBST was washed 3 times, TMB chromogenic solution was added and allowed to react at 37℃for 15min, stop solution was added, and OD450 was detected by an ELISA. Each set 3 parallel wells and the experiment was repeated three times.

As a result, as shown in FIG. 8, the short peptide VS was able to specifically bind to pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid group (D-F-h) cells, but not to pcDNA3.1-Fc-his recombinant plasmid group (F-h) cells, and the short peptide PS was able to specifically bind to chicken DEC-205 recombinant protein by not binding to pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid group (D-F-h) and pcDNA3.1-Fc-his recombinant plasmid group (F-h) cells.

2. Fluorescent peptide binding effect observed by fluorescent microscope

(1) 293t cells were plated in 24-well plates, and the recombinant plasmids obtained in example 1 were transiently transfected into 293t cells, respectively, as described in X-tremeGENE HP DNA Transfection Reagent. After 48h of transfection, the solution was discarded, washed twice with PBS, fixed with 95% ethanol at room temperature for 10min, washed three times with PBS, washed three times with 0.2% Triton-100 at room temperature for 10min, washed three times with PBS, washed three times with 3% BSA-PBS at 37℃for 30min, washed three times with PBS, and added 10% respectively ^-6 mol biotin-labeled short peptide VS and short peptide PS are reacted for 1h at room temperature, PBS is used for three times, FITC-labeled streptavidin (1:500 (v/v) is added for dilution), and the reaction is carried out for 1h at room temperature in a dark place, and fluorescent peptide binding effect is observed by a fluorescent microscope.

As a result, as shown in FIG. 9, the short peptide VS was able to bind to 293t cells expressing DEC 205-Fc-his (recombinant protein expressed by pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid), whereas it was able to bind to 293t cells expressing Fc-his (recombinant protein expressed by pcDNA3.1-Fc-his recombinant plasmid) less, and the short peptide PS was able to bind to both cells less.

(2) Polylysine (0.1 mg/ml) was pre-plated in 24-well plates and chicken bone marrow-derived DCs (cell number approximately 10) were cultured until day 6 ⁵ ) After stimulation, 200ng/ml lipopolysaccharide becomes mature chicken bone marrow-derived DCs, 50 μg of biotin-labeled short peptide VS and short peptide PS are respectively added into each well, the mixture is subjected to room temperature action for 30min, PBS is used for three times, FITC-labeled streptavidin (1:500 (v/v) is added for dilution), the mixture is subjected to light-shielding room temperature action for 1h, and fluorescent peptide binding effect is observed by a fluorescent microscope.

As shown in FIG. 10, the fluorescence intensity of the short peptide VS group (VS-FITC) chicken bone marrow-derived DCs was higher than that of the short peptide PS group (PS-FITC), which indicated that short peptide VS (VS-FITC) was able to bind well to chicken bone marrow-derived DCs.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> academy of agricultural sciences in Jiangsu province

<120> phage display polypeptide VS specifically bound by chicken DEC-205 and uses thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Val Ser Pro Ala Trp Asp Ala Arg Thr Arg Ser Ala

1 5 10

Claims

1. A phage display polypeptide VS specifically binding to chicken DEC-205, characterized in that its amino acid sequence is valserproalatrpacppaaragthrargserala.

2. The phage display polypeptide VS to which chicken DEC-205 specifically binds according to claim 1, characterized in that the nucleotide sequence encoding the phage display polypeptide VS amino acid sequence is GTTTCTCCTGCTTGGGATGCGAGGACTAGGAGTGCT.

3. The phage display polypeptide VS specifically binding to chicken DEC-205 according to claim 1, characterized in that it is obtained by designing signal peptide for three C-type lectin-like domains of chicken bone marrow derived DCs receptor DEC-205, fusion-expressing in 293t cells with three C-type lectin-like domains, human IgG1Fc and his tag, identifying and purifying chicken DEC-205 recombinant protein, and screening using phage linear 12 peptide library technology.

4. A phage display polypeptide VS which specifically binds to chicken DEC-205 according to claim 3, characterized in that the three C-type lectin-like domains are CTLD-4, CTLD-5 and CTLD-6, respectively.

5. A phage display polypeptide VS which specifically binds to chicken DEC-205 according to claim 3, characterized in that the nucleotide sequence of the signal peptide is ATGGCTTCACCTCTCACAAGATTCT TGTCTCTGAACCTCCTGCTTCTCGGTGAATCAATTATCCTGGGTTCTGGCG AGGCT and the amino acid sequence thereof is MetAlaSerProLeuThrArgPheLeuSerLeuAsnLeuLeuLeu LeuGlyGluSerIleIleLeuGlySerGlyGluAla.

6. Use of a phage display polypeptide VS specifically binding to chicken DEC-205 as claimed in any of claims 1-5 for the preparation of a drug delivery vector.

7. Use of a phage display polypeptide VS specifically binding to chicken DEC-205 as claimed in any of claims 1-5 for the preparation of a chicken bone marrow derived DCs targeting vaccine adjuvant.