CN114716516A

CN114716516A - Chicken DEC-205 specific binding phage display polypeptide VS and application thereof

Info

Publication number: CN114716516A
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: 2022-07-08
Anticipated expiration: 2042-05-10
Also published as: CN114716516B

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 ValSerProAlaTrpAspAlaArgThrArgSeraL. The phage display polypeptide VS specifically bound by the chicken DEC-205 has small relative molecular mass, and the synthesis, expression, modification and the like of the phage display polypeptide VS are easy to realize. The polypeptide VS can be specifically combined with chicken DEC-205 recombinant protein; the binding capacity of the polypeptide VS and 293t cells expressing chicken DEC-205 recombinant protein is strong; the polypeptide VS can be well combined with chicken bone marrow source DCs. The phage display polypeptide VS specifically combined with the chicken DEC-205 screened by the invention can be used together with various drug delivery carriers, and can be used as a vaccine adjuvant to prepare the chicken bone marrow-derived DCs targeted vaccine, so that the immune effect of the chicken bone marrow-derived DCs targeted vaccine can be improved.

Description

Chicken DEC-205 specific binding phage display polypeptide VS and application thereof

Technical Field

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

Background

Dendritic Cells (DCs) are professional antigen presenting cells, and the drug can be effectively delivered to corresponding parts through a dendritic cell-targeted biological immunotherapy, so that the dosage and administration frequency of the drug can be reduced, the treatment effect of the drug can be improved, and adverse reactions can be reduced. It has been shown in the prior art that human peripheral blood DCs targeting peptide is obtained by screening with phage display linear 12 peptide library technology, and better immune effect is obtained after fusion expression of the human peripheral blood DCs targeting peptide and antigen (Curiel T J, Morris C, Brumlink M, et al. peptides identified by recombinant vaccine to dendritic cells [ J ]. Journal of Immunology,2004,172(12): 7425-.

Targeting antigens to endocytic receptors on the surface of DCs is a new strategy to enhance the adaptive immune response. It has been shown that immunization with a prokaryotic Antigen (Newcastle disease Virus hemagglutinin-neuraminidase) fused to the single chain variable region of the anti-Chicken DEC-205 antibody resulted in higher neutralizing antibody titers (Shrestha A, Sadeyen J R, Lukosaite D, et al.Targeting Haemagglutin antibody of Avian Influenza Virus to Chicken Immune Cell Receptors Dec205 and CD11c inductive Differential antibodies-Potenuating Responses [ J ]. Vaccines,2021,9(7): 784.).

So far, no dominant polypeptide capable of specifically binding to chicken bone marrow-derived DCs receptor DEC-205 has been reported.

Disclosure of Invention

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

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

the amino acid sequence of the phage display polypeptide VS specifically combined with the chicken DEC-205 is ValSerProAlaTrpAspAlaArgThrArgSerAlaL.

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

In a preferred embodiment, the phage display polypeptide VS is obtained by designing signal peptides aiming at three C-type lectin-like domains of chicken bone marrow derived DCs receptor DEC-205, fusing and expressing the signal peptides with the three C-type lectin-like domains, human IgG1 Fc and his tag in 293t cells, identifying and purifying recombinant proteins of chicken DEC-205, and screening by using phage linear 12 peptide library technology.

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

In a preferred embodiment, the signal peptide has the nucleotide sequence of ATGGCTTCACCTCTCACAAGATTCTTGTCTCTG AACCTCCTGCTTCTCGGTGAATCAATTATCCTGGGTTCTGGCGAGGCT and the amino acid sequence of MetAlaSerProLeuThrArgPheLeuSerLeuAsnLeuLeuLeuLeuLeuLeuLeuLeuGluGluGluGluGluSereLeuGlyGlyGluAla.

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

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

The invention relates to application of a phage display polypeptide VS specifically bound by chicken DEC-205 in serving as a drug delivery carrier.

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

The invention has the beneficial effects that:

the phage display polypeptide VS (ValSerProAlaTrpAspAlaArgThrArgSerAlaLa) 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 bound by the chicken DEC-205 can be specifically bound with the chicken DEC-205 recombinant protein; the phage display polypeptide VS specifically bound with the chicken DEC-205 has stronger binding capacity with 293t cells expressing recombinant proteins of the chicken DEC-205; 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 used together with various drug delivery carriers, and can be used as a vaccine adjuvant to prepare the chicken bone marrow-derived DCs targeted vaccine, so that the immune effect of the chicken bone marrow-derived DCs targeted vaccine can be improved.

Drawings

FIG. 1 shows the results of enzyme digestion of pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid. In the figure, 1: marker; 2: NheI-NotI double enzyme digestion plasmid; 3: pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid.

FIG. 2 shows the results of enzyme digestion identification of pcDNA3.1-Fc-his recombinant plasmid. In the figure, 1: NheI-NotI double enzyme digestion plasmid; 2: pcDNA3.1-Fc-his recombinant plasmid; 3: and (5) Marker.

FIG. 3 shows the results of western blot analysis (ECL color) of cell lysates and cell supernatants obtained after transfection of recombinant plasmids for 24h and 48h

FIG. 4 shows the result of western blot identification (DAB coloration) of cell supernatants after transfection with recombinant plasmids.

FIG. 5 shows the results of western blot analysis (ECL staining) of samples transfected with different volumes of transfection reagents for 48 h. In the figure, Fc is human IgG1 Fc and the protein size is about 30kD, DEC-Fc is the protein expressed by fusing 3C-type lectin-like domains CTLD-4, CTLD-5 and CTLD-6 of chicken DEC205 with human IgG1 Fc, and the protein size is about 90 kD.

FIG. 6 shows SDS-PAGE identification of chicken DEC205 recombinant protein after purification. In the figure, 1 is a recombinant protein sample expressed by pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid before purification, 2-4 are recombinant protein samples expressed by purified pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid after sequential elution, respectively, and M is Marker.

FIG. 7 is a schematic representation of the assay for recovery phage titer for three rounds of screening. In the figure, a: a first screening round; b: second screening; c: and (4) third screening.

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

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

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

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

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

A signal peptide is designed according to 3C-type lectin-like structural domains (CTLD-4, CTLD-5 and CTLD-6) with binding action of chicken DEC205 disclosed by an EMBL database (accession number AJ574899), namely the nucleotide sequence of a mouse CD8 alpha signal peptide is ATGGCTTCACCTCTCACAAGATTCTTGTCTCTG AACCTCCTGCTTCTCGGTGAATCAATTATCCTGGGTTCTGGCGAGGCT, the amino acid sequence of the signal peptide is MASPLTRFLSLNLLLLGESIILGSGEA(MetAlaSerProLeuThrArgPheLeuSerLeuAsnLeuLeuLeuLeuGlyGluSerIleIleLeuGlySerGlyGluAla), the signal peptide, human IgG1 Fc and a his tag are subjected to fusion expression, codons are optimized, a DEC 205-Fc-his target gene is synthesized, the target gene is cloned into a pcDNA3.1 expression vector to obtain a pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid, and the NheI-NotI double-enzymatic digestion pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid is shown in figure 1 to obtain a target fragment with the size of about 2228 bp.

Human IgG1 Fc and his label are fused and expressed, codon is optimized, Fc-his target gene is synthesized and cloned into pcDNA3.1 expression vector to obtain pcDNA3.1-Fc-his recombinant plasmid, and NheI-NotI double enzyme digestion pcDNA3.1-Fc-his recombinant plasmid as shown in FIG. 2 to obtain 842bp gene segment.

Example 2 expression identification and recovery of chicken DEC205 recombinant protein

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

And (3) recovering a large amount of the cell lysate and the cell supernatant, purifying the recombinant protein expressed by the pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid by using a nickel column (2, 3 and 4 are three tube samples which are eluted in sequence respectively), and detecting by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) to show that the target protein of about 90kD appears in both the sample 2 and the sample 3, as shown in figure 6.

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

First round of screening (a in fig. 7): mu.l protein G-agarose medium was washed twice with 1ml TBS + 0.1% Tween (TBST) solution, resuspended in 1ml blocking buffer and allowed to react for 60min at 4 ℃. 1.5X 10 with TBST¹¹Each phage particle (corresponding to 10. mu.l of the original library) and 300ng of the above chicken DEC205 recombinant protein were diluted to a final volume of 200. mu.l and allowed to react for 20min at room temperature. After blocking reaction, protein G-agarose medium was precipitated, washed 4 times with 1ml TBST, phage-recombinant protein mixture was added to washed protein G-agarose medium, mixed gently, left to react at room temperature for 15min and not mixed. Washed 10 times with 1ml TBST, resuspended in precipitation medium in 1ml, 0.2M Glycine-HCl (pH 2.2) +1mg/ml BSA, and the bound phage eluted and allowed to act for 10min at room temperature. The mixture was centrifuged for 1min, and the supernatant was transferred to a centrifuge tube and the eluate was neutralized with 150. mu.l of 1M Tris-HCl (pH 9.1). The eluent titer was determined on LB/IPTG/Xgal plates. And (3) respectively mixing the eluent with an E.coli ER2738 culture solution (A600 nm is approximately equal to 0.5) in a conical flask, violently shaking for 4.5 hours, preparing a phage amplification solution by a polyethylene glycol NaCl secondary precipitation method, and measuring the titer. Second round of screening (b in fig. 7): the first round of screening steps was repeated by replacing 100. mu.l of protein G-agarose medium 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 by replacing 100. mu.l of protein A-agarose medium 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 phage were sequenced, and 42 samples lost 12 peptides (the infectivity was much higher than that of phage expressing exogenous peptide).

TABLE 1

Number of screening rounds	Input quantity (pfu)	Recovery volume (pfu)	Recovery rate	Binding medium
					1	1.5×10¹¹	1.26×10⁶	8.4×10^-6	protein G-agarose
2	5×10¹¹	2×10⁶	4×10^-6	protein A-agarose
					3	2×10¹⁰	2.2×10⁴	1.1×10^-6	protein G-agarose

EXAMPLE 3 Synthesis of chicken DEC-205 receptor affinity peptide

The short peptide VS (vspadadartrsa) with the highest repetition rate in the third round of screening was used as the phage display polypeptide VS of the invention. Meanwhile, random disordered short peptide PS (PSPLLRSNFFSI) is used as a reference, Fmoc method is adopted for synthesis, biotin (FITC) is used for labeling the short peptide, and HPLC purification (purity)>95%) and mass spectrometry (done by wuhanhaoded bio). The biotin-labeled short peptide powder is pre-dissolved in DMSO, then PBS is added until the final concentration of the DMSO is 30 percent, the molar mass of the short peptide VS is 1542.4g/mol, the molar mass of the short peptide PS is 1603.7g/mol, and finally the mixture is respectively diluted into a solution with the working concentration of about 0.465 mu g/mu l, wherein each 100 mu l of the solution contains about 3X 10 mu g/mu l^-8mol short peptide, and storing at-20 ℃ for later use.

Example 4 detection of affinity of chicken DEC-205 receptor affinity peptides

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

The recombinant plasmids obtained in example 1 were transfected into 293t cells, respectively, according to the method described in the X-tremeGENE HP DNA Transfection Reagent. After 48h of transfection, the cells were recovered, washed once with PBS, pre-plated with polylysine (0.1mg/ml) in 96-well plates for half an hour at room temperature, air-dried, and the cells were plated at 2X 10⁵Wells, plated, room temperature for half an hour, PBS washed to remove nonadherent cells, untransfected cells as blank control. Fixing with 95% ethanol at room temperature for 10min, washing with PBS three times, allowing 0.2% Triton-100 to act at room temperature for 10min, washing with PBS three times, allowing 3% BSA-PBS to act at 37 deg.C for 1min, and washing with PBS three times. Adding 3X 10 respectively^-8And (3) performing reaction on the biotin-labeled short peptide VS and the short peptide PS at room temperature for 3 hours, and washing the PBST for 3 times. Adding HRP-labeled streptavidin (diluted at 1:2000 (v/v)) for acting at 37 ℃ for 1h, washing with PBST for 3 times, adding TMB developing solution, acting at 37 ℃ for 15min, adding stop solution, and detecting OD450 with an enzyme-labeling instrument. Each set 3 parallel wells and the experiment was repeated three times.

As shown in FIG. 8, the short peptide VS specifically bound to the cells of pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid set (D-F-h) but not to the cells of pcDNA3.1-Fc-his recombinant expression plasmid set (F-h), and the short peptide PS bound to neither the cells of pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid set (D-F-h) nor the cells of pcDNA3.1-Fc-his recombinant expression plasmid set (F-h), thus demonstrating that the short peptide VS specifically bound to the chicken DEC-205 recombinant protein.

2. Fluorescence microscope observation of fluorescent peptide binding Effect

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

As shown in FIG. 9, the short peptide VS bound 293t cells expressing DEC 205-Fc-his (recombinant protein expressed from pcDNA3.1-DEC 205-Fc-his recombinant expression plasmid), but bound 293t cells expressing Fc-his (recombinant protein expressed from pcDNA3.1-Fc-his recombinant plasmid) weakly, and bound short peptide PS to both cells weakly.

(2) Poly-lysine (0.1mg/ml) was pre-plated in a 24-well plate, and chicken bone marrow-derived DCs (cell number about 10) cultured until day 6 were obtained⁵) After being stimulated by lipopolysaccharide of 200ng/ml, the mature chicken bone marrow-derived DCs are formed, 50 mu g of biotin-labeled short peptide VS and short peptide PS are respectively added into each well, the mixture acts for 30min at room temperature, PBS is washed for three times, FITC-labeled streptavidin (1:500(v/v) is added for dilution), the mixture acts for 1h at room temperature in a dark place, and the fluorescent peptide binding effect is observed by a fluorescent microscope.

As shown in FIG. 10, the fluorescence intensity of chicken bone marrow-derived DCs in the short peptide VS group (VS-FITC) was higher than that in the short peptide PS group (PS-FITC), indicating that the short peptide VS (VS-FITC) could be well bound to the chicken bone marrow-derived DCs.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> agricultural science and academy of Jiangsu province

<120> chicken DEC-205 specific binding phage display polypeptide VS 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 valserpro alatratrpaaargthrragserra.

2. The chicken DEC-205 specifically binding phage display polypeptide VS according to claim 1, wherein the nucleotide sequence encoding the amino acid sequence of phage display polypeptide VS is GTTTCTCCTGCTTGGGATGCGAGGACTAGGAGTGCT.

3. The phage display polypeptide VS of claim 1, wherein said phage display polypeptide VS is obtained by designing signal peptide for three C-type lectin-like domains of chicken bone marrow derived DCs receptor DEC-205, fusing and expressing said signal peptide with three C-type lectin-like domains, human IgG1 Fc and his tag in 293t cells, identifying and purifying chicken DEC-205 recombinant protein, and screening with phage linear 12 peptide library technology.

4. The chicken DEC-205-specifically binding phage display polypeptide VS according to claim 3, wherein said three C-type lectin-like domains are CTLD-4, CTLD-5, and CTLD-6, respectively.

5. The chicken DEC-205 specific binding phage display polypeptide VS according to claim 3, wherein the nucleotide sequence of the signal peptide is ATGGCTTCACCTCTCACAAGATTCTTGTCTCTG AACCTCCTGCTTCTCGGTGAATCAATTATCCTGGGTTCTGGCGAGGCT, and the amino acid sequence thereof is metalaerp leuthr argphe leu serleu asn leu glu serel ileylleu serglu ala.

6. The chicken DEC-205 specific binding phage display polypeptide VS of claim 1, wherein said phage display polypeptide VS is capable of specifically binding to a chicken DEC-205 recombinant protein.

7. The chicken DEC-205 specifically binding phage display polypeptide VS according to claim 1, wherein said phage display polypeptide VS is capable of binding to chicken bone marrow-derived DCs.

8. Use of a chicken DEC-205 specifically binding phage display polypeptide VS as claimed in any one of claims 1-7 as a drug delivery vehicle.

9. Use of the chicken DEC-205 specifically bound phage-displayed polypeptide VS as claimed in any one of claims 1-7 as a chicken bone marrow-derived DCs targeted vaccine adjuvant.