CN115058453A - Construction method of adhesion protein multi-epitope recombinant adenovirus delivery system - Google Patents

Abstract

A construction method of an adhesion protein multi-epitope recombinant adenovirus delivery system belongs to the technical field of biological engineering. The invention aims to provide a construction method for preparing an adhesion protein multi-epitope recombinant adenovirus delivery system by combining a protective B cell epitope with a series Th cell epitope box. The invention selects protective B cell epitope of staphylococcus aureus mainly adhered to related protein, obtains tandem B cell epitope box by short and flexible intermolecular connection sequence peptide tandem, obtains tandem Th cell epitope box by the same method, and co-expresses B cell epitope box and Th epitope box by IRES mediation. The invention prevents and treats staphylococcus aureus infection by an immunological means, provides a foundation for the construction of a polygene delivery system, and beneficially explores the prevention of staphylococcus aureus infection so as to solve the scientific problem that the related diseases caused by staphylococcus aureus can not be treated by only depending on antibiotics.

Description

Construction method of adhesion protein multi-epitope recombinant adenovirus delivery system

Technical Field

The invention belongs to the technical field of biological engineering.

Background

Staphylococcus aureus (s. aureus) infection is closely associated with the occurrence of local pyogenic infection, and the continuous emergence of drug-resistant strains, particularly the rapid spread of methicillin-resistant strains, results in the inability to rely solely on antibiotics to treat the associated diseases caused by the bacteria. Currently, the treatment of staphylococcus aureus infection can be generally carried out by using antibacterial drugs such as macrolides and quinolones, but due to the long-term abuse of antibiotics, a plurality of drug-resistant new strains are promoted, particularly, the rapid spread of MRSA causes the infection caused by the treatment of staphylococcus aureus only by the antibiotics to become unreliable, and the phenomenon that new drugs cannot catch up with drug-resistant bacteria appears, so that MRSA is listed as the first of three most difficult infectious diseases in the world, and a complete solution is not provided so far, so that the infection of the bacteria is hoped to be prevented and treated by vaccines.

Staphylococcus aureus lacks pilus structure and relies on surface proteins to adhere to host cells and subsequently invade host tissues and organs. The greater the ability of staphylococcus aureus to adhere to tissue, the greater the chances of survival, and so blocking the initial stages of s.aureus infection, especially preventing s.aureus from adhering to cell and mucosal surfaces, may be the most effective way to prevent infection. The major adhesion-related proteins of staphylococcus aureus are the coagulation factor a (ClfA), fibronectin (FnBPA), collagen binding protein (Cna) and elastin binding protein (Efb). The single antigen has low protection rate, the vaccine which tries the combination of a plurality of antigens has good protection effect, the combined vaccine which adopts protective antigens and a plurality of molecules can obviously improve the protection of the vaccine, but simultaneously can not avoid the tissue damage caused by the virulence factors of the staphylococcus aureus, help the expansion of infection and offset indirect immune defense, and bring serious toxic and side effects to human body. By referring to the vaccine research experience of diseases such as virus, tumor and the like, the multi-epitope vaccine is a safe and effective anti-infection vaccine form. It has been found that the effects produced by the numerous epitopes of antigenic molecules are not identical: there are protective epitopes, inhibitory epitopes and pathogenic epitopes. Therefore, in designing vaccines, there should be a greater preference for functionally defined epitopes, specifically enhancing the intensity of the immune response, while excluding the effects of non-protective as well as inhibitory epitopes.

Adenovirus has good application prospect as a transfer vector of recombinant vaccine and gene therapy.

Disclosure of Invention

The invention aims to provide a construction method for preparing an adhesion protein multi-epitope recombinant adenovirus delivery system by combining a protective B cell epitope with a tandem Th cell epitope box.

The invention selects protective B cell epitope of staphylococcus aureus mainly adhered to related protein, obtains tandem B cell epitope box by short and flexible intermolecular connection sequence peptide tandem, obtains tandem Th cell epitope box by the same method, and co-expresses B cell epitope box and Th epitope box by IRES mediation.

The sequence of the B cell epitope recombination fragment is SEQ ID number 1, and the sequence of the Th cell epitope box recombination fragment is SEQ ID number 2.

The staphylococcus aureus is NCTC 8325.

The main adhesion related proteins of the staphylococcus aureus are elastic binding protein, fibronectin, coagulation factor A and collagen binding protein, and the main adhesion related proteins of the staphylococcus aureus are sequences of Efb, FnBPA, ClfA and Cna.

The recombinant gene vector co-expressed by the B cell epitope box and the Th cell epitope box is adenovirus.

The construction process of the multi-epitope recombinant adenovirus, namely pAdEasy-1-pAdTrack-CMV-B epitope box-IRES-Th epitope box, comprises the following steps:

(1) protective B cell epitopes of Efb, FnBPA, ClfA and Cna obtained by identification are selected and connected in series through short and flexible intermolecular connecting sequence peptides;

(2) analyzing the tertiary structure which is closest to the natural state in theory to obtain a tandem B cell epitope box, and obtaining a tandem Th cell epitope box by the same method;

(3) combining the pAdTrack-CMV transfer plasmid map, the multiple cloning site and the enzyme cutting site contained in the insert fragment, firstly inserting an IRES fragment between the pAdTrack-CMV transfer vector SalI and NotI, and constructing a pAdTrack-CMV-IRES modified transfer empty vector;

(4) then inserting a Th cell epitope box fragment between the pAdT rack-CMV-IRES modified transfer empty vector XhoI and XbaI to construct a single-gene recombinant transfer vector of the pAdrack-CMV-IRES-Th epitope box;

(5) b cell epitope box fragments are inserted between single-gene recombinant transfer vectors BglII and SalI of the pAdTrack-CMV-IRES-Th epitope box to construct a pAdTrack-CMV-B epitope box-IRES-Th epitope box double-gene transfer vector;

(6) the constructed pAdTrack-CMV-B epitope box-IRES-Th epitope box recombinant transfer plasmid is linearized by single enzyme digestion at 37 ℃ for 2 h;

(7) co-transforming BJ5183 competent bacteria with pAdEasy-1 adenovirus skeleton plasmid calcium chloride method, screening LB agar plate containing Kana, selecting monoclonal extract plasmid, the Kana content is 50 μ g/ml;

(8) primarily screening pAdEasy-1-pAdTrack-CMV-B epitope box-IRES-Th epitope box homologous recombination clones according to the molecular weight of agarose electrophoresis;

(9) after transformation of DH 5. alpha. the plasmid was amplified and extracted for PCR and PacI single-enzyme restriction.

The invention has the beneficial effects that:

1. due to the long-term abuse of antibiotics, a variety of new strains resistant to antibiotics emerge, and particularly the rapid spread of MRSA makes the treatment of related diseases caused by staphylococcus aureus that rely solely on antibiotics increasingly unreliable, so the present study hopes to prevent and treat staphylococcus aureus infection by immunological means;

2. on the basis of 8 specific B, Th cell epitopes of the adhesion protein, two multi-epitope expression cassettes (a B expression cassette and a Th expression cassette) are respectively constructed through the connection of Linker among the epitopes, and a basis is provided for the construction of a multi-gene delivery system;

3. the research of preventing and treating staphylococcus aureus infection is carried out by a gene delivery mode of multi-epitope recombinant adenovirus, multi-epitope recombinant adenovirus (pAdEasy-1-pAdTrack-CMV-B epitope box-IRES-Th epitope box) is constructed, an in vitro cell adhesion blocking and in vivo mouse infection model is established, adhesion inhibition of multi-epitope adenovirus antiserum is detected, each epitope function of the recombinant adenovirus is analyzed, the immune mechanism of the recombinant adenovirus is researched, the infection prevention of staphylococcus aureus is beneficially explored, the scientific problem that the related diseases caused by staphylococcus aureus cannot be treated by antibiotics is solved, the important guiding significance is provided for preventing and treating staphylococcus aureus infection, and a new way is provided for developing medicaments with independent intellectual property rights in China.

Drawings

FIG. 1 is a schematic drawing of pGEMTEZ-B epitope;

FIG. 2 is a schematic representation of the co-expression pattern of a B epitope cassette and a Th epitope cassette mediated by IRES;

FIG. 3 is a schematic diagram of the construction process of the adenovirus expression vector and the plasmid map and Multiple Cloning Sites (MCS);

FIG. 4 is a schematic diagram of pShuttle-CMV-Th epitope.

Detailed Description

The invention selects protective B cell epitope of staphylococcus aureus mainly adhered to related protein, obtains tandem B cell epitope box by short and flexible intermolecular connection sequence peptide tandem, obtains tandem Th cell epitope box by the same method, and co-expresses B cell epitope box and Th epitope box by IRES mediation.

The sequence of the B epitope recombination fragment is SEQ ID number 1, and the sequence of the Th cell epitope box recombination fragment is SEQ ID number 2.

The staphylococcus aureus is NCTC 8325.

The main adhesion related proteins of the staphylococcus aureus are elastic binding protein, fibronectin, coagulation factor A and collagen binding protein, and the main adhesion related proteins of the staphylococcus aureus are sequences of Efb, FnBPA, ClfA and Cna.

The recombinant gene vector co-expressed by the B epitope box and the Th epitope box is adenovirus.

The construction process of the multi-epitope recombinant adenovirus, namely pAdEasy-1-pAdTrack-CMV-B epitope box-IRES-Th epitope box, comprises the following steps:

(1) protective B cell epitopes of Efb, FnBPA, ClfA and Cna obtained by identification are selected and connected in series through short and flexible intermolecular connecting sequence peptides;

(2) analyzing the tertiary structure which is closest to the natural state in theory to obtain a tandem B cell epitope box, and obtaining a tandem Th cell epitope box by the same method;

(3) combining the pAdTrack-CMV transfer plasmid map, the multiple cloning site and the enzyme cutting site contained in the insert fragment, firstly inserting an IRES fragment between the SalI and NotI of the pAdTrack-CMV transfer vector, and constructing a pAdTrack-CMV-IRES modified transfer empty vector;

(4) then inserting a Th epitope box fragment between the pAdT rack-CMV-IRES modified transfer empty vector XhoI and XbaI to construct a single-gene recombinant transfer vector of the pAdrack-CMV-IRES-Th epitope box;

(5) inserting a B epitope box fragment between BglII and SalI of the single-gene recombinant transfer vector of the pAdTrack-CMV-IRES-Th epitope box to construct a pAdTrack-CMV-B epitope box-IRES-Th epitope box double-gene transfer vector;

(6) the constructed pAdTrack-CMV-B epitope box-IRES-Th epitope box recombinant transfer plasmid is linearized by single enzyme digestion at 37 ℃ for 2 h;

(7) co-transforming BJ5183 competent bacteria with pAdEasy-1 adenovirus skeleton plasmid calcium chloride method, screening LB agar plate containing Kana, selecting monoclonal extract plasmid, the Kana content is 50 μ g/ml;

(8) primarily screening pAdEasy-1-pAdTrack-CMV-B epitope box-IRES-Th epitope box homologous recombination clones according to the molecular weight of agarose electrophoresis;

(9) after transformation of DH 5. alpha. the plasmid was amplified and extracted for PCR and PacI single-enzyme restriction.

The invention is described in detail below:

the amino acid sequence of main adhesion related proteins Efb, FnBPA, ClfA and Cna of staphylococcus aureus is shown in SEQ ID No. 3-6. Protective B cell epitopes of Efb, FnBPA, ClfA and Cna obtained by identification are selected and connected in series through short and flexible intermolecular connecting sequence peptides (beneficial to the space conformation of polypeptide chains and the original epitope specificity is kept), a three-level structure which is closest to a natural state in theory is analyzed, and a series B cell epitope box is obtained, wherein the schematic diagram is shown in figure 1; obtaining tandem Th cell epitope boxes by the same method; the use of an IRES-mediated co-expression pattern of the B and Th epitope cassettes, as schematically illustrated in figure 2; the adenovirus expression vector construction process and pAdTrack-CMV transfer plasmid map, Multiple Cloning Site (MCS) and the enzyme cutting site contained in the insert itself are shown in FIG. 3. Firstly, inserting an IRES fragment between the pAdTrack-CMV transfer vector SalI and NotI to construct a pAdTrack-CMV-IRES modified transfer empty vector; secondly, inserting a Th epitope box fragment between the pAdTrack-CMV-IRES modified transfer empty vector XhoI and XbaI to construct a pAdTrack-CMV-IRES-Th epitope box, wherein the schematic diagram is shown in figure 4, inserting a B epitope box fragment between BglII and SalI to construct a pAdTrack-CMV-B epitope box-IRES single-gene recombinant transfer vector; thirdly, inserting a B epitope box fragment between the pAdTrack-CMV-IRES-Th epitope box single-gene recombination transfer vector BglII and SalI to construct a pAdTrack-CMV-B epitope box-IRES-Th epitope box double-gene transfer vector, wherein the schematic diagram is shown in figure 3. The constructed recombinant transfer plasmids of pAdTrack-CMV-IRES, pAdTrack-CMV-IRES-Th epitope box, pAdTrack-CMV-B epitope box-IRES and pAdTrack-CMV-B epitope box-IRES-Th epitope box are linearized by PmeI at 37 ℃ for 2 h; respectively cotransforming BJ5183 competent bacteria with pAdEasy-1 adenovirus backbone plasmid calcium chloride method, and screening single clone extraction plasmid on LB agar plate containing Kana (50 μ g/ml) as shown in FIG. 3; primarily screening pAdEasy-1-pAdTrack-CMV-IRES (abbreviated as pAd-IRES), pAdEasy-1-pAdTrack-CMV-IRES-Th epitope box (abbreviated as pAd-IRES-Th epitope box), pAdEasy-1-pAdTrack-CMV-B epitope box-IRES (abbreviated as pAd-B epitope box-IRES) and pAdEasy-1-pAdTrack-CMV-B epitope box-IRES-Th epitope box (abbreviated as pAd-B epitope box-IRES-Th epitope box) homologous recombinant clones according to the molecular weight of agarose electrophoresis; after transformation of DH 5. alpha. the plasmid was amplified and extracted for PCR and PacI single-enzyme restriction.

SEQIDNO. 3：

Efb (GenBank: AJ306909.1)

Mknkliakslltiaaigittttiastadasegygprekkpvsinhniveyndgtfkyqsrpkfnstpkyikfkhdynilefndgtfeygarpqfnkpaa ktdatikkeq kliqaqnlvrefekthtvsahrkaqkavnlvsfeykvkkmvlqeridnvlkqglvr

SEQIDNO. 4：

FnBPA (GenBank：KJ001293.1)

Ggqnsgnqsfeedteedkpkyeqggnivdidfdsvpqiqgqnngdqsfeedteedkpkyeqggniididfdsvphihgfnkhteiieedtnkdkpn yqfgghssvd feedtlpqvsghnegqqtieedttppivpp

SEQIDNO. 5：

ClfA (GenBank: KU351167.1)

tstakvppimagdqvlangvidsdgnviytftdyvntkddvkatltmpayinpenvkktgnvtlatgigsttanktvlvdyekygkfynlsikgtidqi dktnntyrqt iyvnpsgdnviapvltgnlkpntdsnalidqqntsikvykvdnaadlsesyfvnpenfedvt

SEQIDNO. 6

Cna (GenBank: KU376312.1)

Vsltvkgeqvgqavitpdgatitfndkveklsdvsgfaefevqgrn ltqtntsddk vatitsgnkstnvtvhkseagtssvfyyktgem。

B epitope recombinant fragment

lltiaaigiaspgsgnqsfeedspgsvppimagdqspgsvkgeqvgqa

Th epitope recombinant fragment

Ktdatikkeq aspg yqfgghssvd spgs dktnntyrqt spgs ltqtntsddk。

The corresponding DNA sequences of the amino acid sequences of the B epitope recombinant fragment and the Th epitope recombinant fragment listed above are respectively: SEQ ID number 1 and SEQ ID number 2.

The recombinant adenovirus packaging and amplification method comprises the following specific steps:

carrying out PacI linearization on the constructed pAd-IRES, pAd-IRES-Th epitope box, pAd-B epitope box-IRES and pAd-B epitope box-IRES-Th epitope box homologous recombination adenovirus plasmids and then recovering a large fragment; transfecting QBI-293A cells respectively to package to generate first generation recombinant adenovirus; the first generation recombinant adenovirus crude extract is subjected to two rounds of infection and amplification in QBI-293A cells to obtain Ad-IRES, Ad-IRES-Th epitope boxes, Ad-B epitope boxes-IRES and Ad-B epitope boxes-IRES-Th epitope boxes with higher titer, and the third generation recombinant adenovirus is stored at-80 ℃ for later use; respectively infecting QBI-293A cells with Ad-IRES, Ad-IRES-Th epitope box, Ad-B epitope box-IRES and Ad-B epitope box-IRES-Th epitope box adenoviruses for 48 h, centrifuging at 1000r/min for 5min, collecting virus infected cells and virus-free QBI-293A cell contrast, washing with PBS for 2 times, extracting QBI-293A cell total RNA of each group according to the instruction of a total RNA extraction kit, and performing RT-PCR;

the transcription of IRES, B epitope box, Th epitope box and internal reference beta-actin gene in QBI-293A cell is respectively identified. QBI-293A cells infected with different recombinant adenoviruses are washed twice by PBS (0.01M) with pH 7.4 and then are resuspended in 20ul PBS; adding an isovolumetric 2 Xsample buffer solution, carrying out water bath at 95 ℃ for 5min, centrifuging at 16,000 g for 10 min, and taking 15 mul for SDS-PAGE. After the electrophoresis is finished, sticking the nitrocellulose membrane with a PAGE (polyacrylamide gel electrophoresis) glue of a sample to be detected, and carrying out constant-voltage 100V transfer electrophoresis for 1 h; taking down a nitrocellulose membrane, soaking the nitrocellulose membrane in 10ml TBS, rinsing for 15 min, transferring the nitrocellulose membrane into 5% skimmed milk-TBS, sealing for 1h at 37 ℃, washing the TBS, adding a labeled antibody, and incubating for 1h at 37 ℃; TBST is washed for three times, 15 min/time, substrate color developing liquid is added for color development for 15 min, and the reaction is stopped by flushing with running water.

The steps for analyzing the functions of each epitope of the multi-epitope recombinant adenovirus are as follows:

detecting specific antibodies in serum by using an rEfb/rFnBPA/rClfA/rCna, whole bacterial protein and multi-epitope recombinant adenovirus coating ELISA plate, diluting the serum antibodies in a multiple proportion for detection, and investigating whether the antibodies generated by induction can recognize the multi-epitope recombinant adenovirus, the rEfb/rFnBPA/rClfA/rCna protein and the whole bacterial protein. Detecting immunoreactivity of rEfb/rFnBPA/rClfA/rCna and multi-epitope recombinant adenovirus antibody serum by Western-blot; immunizing a mouse by using the multi-epitope recombinant adenovirus, separating splenic lymphocytes of the immunized mouse, and respectively adding the Th epitope peptide, the multi-epitope recombinant adenovirus and rEfb/rFnBPA/rClfA/rCna. Flow cytometry is used for analyzing change of spleen lymphocyte subpopulation, lymphocyte proliferation experiment is used for detecting specific proliferation of spleen lymphocytes, ELISA is used for detecting secretion of splenic lymphocyte cytokine, RT-PCR is used for detecting expression levels of IL-4 and IFN-gamma mRNA of spleen lymphocytes, and ELISA is used for detecting generation of specific antibody IgG.

Screening and in vitro and in vivo identification of epitope and Th epitope

Preparing 4 monoclonal antibody strains of the ClfA, Efb, FnBPA and Cna on the basis of expression and purification of four antigens by taking staphylococcus aureus adhesion-related proteins Efb, FnBPA, ClfA and Cna as research objects, and verifying the adhesion blocking effect of the monoclonal antibodies; screening 4B cell epitopes with specific protective effects on ClfA, Efb, FnBPA and Cna by using a random phage display library, and clarifying the specific protective effect of the screened B cell epitopes;

immunizing a BALB/c mouse by using the purified protein, respectively establishing hybridoma cell strains secreting staphylococcus aureus Efb, FnBPA, Cna and ClfA by using a cell fusion technology, and obtaining a specific monoclonal antibody through ELISA screening and western-blot identification; observing the influence of the monoclonal antibody on the staphylococcus aureus adhesion host cell through immunofluorescence double labeling and a laser confocal microscope; the method comprises the steps of observing a host cell adsorption inhibition test and an adhesion dynamic test by an adhesion counting method for bacteria, carrying out three rounds of biological affinity panning on a phage display random 12 peptide library by using a purified monoclonal antibody as a screening ligand, enriching high-affinity phage, using phage subjected to panning and amplification in each round for sandwich ELISA detection, mixing and incubating the monoclonal antibody and phage clones with different concentrations, adding an ELISA plate coated with an antigen, and calculating the inhibition rate of ELISA reaction by using an irrelevant antigen coating as a negative control. And (3) determining the sequence of the phage exogenous DNA, deducing the amino acid sequence, and finding out the core sequence for analysis. Immunizing BALB/c mice with phage clones with core sequences, and respectively identifying B cell epitopes of ClfA, Efb, FnBPA and Cna proteins by ELISA and western-blot analysis; polypeptide synthesis and in vitro stimulation of rClfA/rEfb/rFnBPA/rCna protein sensitized BALB/c mice (H-2d) spleen lymphocytes, through lymphocyte proliferation experiments, flow cytometry analysis and CD4+ T lymphocyte proliferation experiments for identification. ELISA method analyzes the cell factor secreted by the epitope stimulation CD4+ T lymphocyte, and lymphocyte proliferation experiment analyzes the synergistic effect between epitopes. Lymphocyte proliferation experiments are used for detecting cellular immune responses, RT-PCR is used for detecting the cytokine expression of spleen lymphocytes, and flow cytometry is used for analyzing the relative quantity of spleen CD4+ T lymphocytes of polypeptide immune mice.

4 Th cell epitopes of ClfA, Efb, FnBPA and Cna are screened and identified by bioinformatics and immunology means, bioinformatics software prediction analysis and synthesis of Th epitope polypeptides, lymphocyte proliferation experiments and phenotype analysis are carried out, MHC restriction analysis, the polarization direction after ELISA detection of polypeptide stimulation lymphocyte proliferation, epitope peptide co-stimulation experiments, ELISA detection of the immunoreactivity of epitope peptide with mouse anti-ClfA/rEfb/rFnBPA/rCna and mouse anti-staphylococcus aureus serum, mouse lymphocyte proliferation experiments after polypeptide immunization, RT-PCR detection of spleen lymphokine after polypeptide immunization, relative quantity change of CD4+ T cells after epitope peptide immunization of mice, and the adhesion blocking function of the epitopes is researched from molecular level, further, the roles of ClfA, Efb, FnBPA and Cna in the pathogenic process of staphylococcus aureus are clear.

Adhesion inhibition and immune effect evaluation of polyepitope recombinant adenovirus

The immunoreactivity of the specific antibody of the multi-epitope recombinant adenovirus and rClfA/rEfb/rFnBPA/rCna is detected by adopting western-blot and ELISA. The function of inhibiting the adhesion of staphylococcus aureus and epithelial cells by the multi-epitope recombinant adenovirus antibody is detected, and the effect of B cell epitope is detected through the adhesion and adhesion inhibition experiment of the staphylococcus aureus and the epithelial cells. After the animal is immunized by the multi-epitope recombinant adenovirus, the change of the splenic lymphocyte subpopulation is analyzed by flow cytometry, the specific proliferation of splenic lymphocytes is detected by a lymphocyte proliferation experiment, the expression levels of IL-4 and IFN-gamma of splenic lymphocytes are detected by ELISA and RT-PCR, and the level of specific antibody IgG in serum is detected by ELISA.

Sequence listing

<110> Jilin university

<120> construction method of adhesion protein multi-epitope recombinant adenovirus delivery system

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 144

<212> DNA

<213> Staphylococcus aureus (Staphylococcus aureus)

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ctgctgacca tcgccgccat cggcatcgcc agccccggca gcggcaacca gagcttcgag 60

gaggacagcc ccggcagcgt gccccccatc atggccggcg accagagccc cggcagcgtg 120

aagggcgagc aggtgggcca ggcc 144

<210> 2

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<213> Staphylococcus aureus (Staphylococcus aureus)

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aagaccgacg ccaccatcaa gaaggagcag gccagccccg gctaccagtt cggcggccac 60

agcagcgtgg acagccccgg cagcgacaag accaacaaca cctacaggca gaccagcccc 120

ggcagcctga cccagaccaa caccagcgac gacaag 156

Claims (6)

1. A construction method of an adhesion protein multi-epitope recombinant adenovirus delivery system is characterized in that: protective B cell epitope of staphylococcus aureus mainly adhered to related protein is selected, a tandem B cell epitope box is obtained by connecting short and flexible intermolecular connecting sequence peptides in series, a tandem Th cell epitope box is obtained by the same method, and IRES is adopted to mediate the co-expression of the B cell epitope box and the Th epitope box.

2. The method for constructing an adhesion protein polyepitope recombinant adenovirus delivery system according to claim 1, wherein: the sequence of the B cell epitope recombination fragment is SEQ ID number 1, and the sequence of the Th cell epitope box recombination fragment is SEQ ID number 2.

3. The method for constructing an adhesion protein polyepitope recombinant adenovirus delivery system according to claim 1, wherein: the staphylococcus aureus is NCTC 8325.

4. The method for constructing an adhesion protein polyepitope recombinant adenovirus delivery system according to claim 1, wherein: the main adhesion related proteins of staphylococcus aureus are elastic binding protein, fibronectin, coagulation factor A and collagen binding protein, and the main adhesion related proteins of staphylococcus aureus are sequences of Efb, FnBPA, ClfA and Cna.

5. The method for constructing an adhesion protein polyepitope recombinant adenovirus delivery system according to claim 1, wherein: the recombinant gene vector co-expressed by the B cell epitope box and the Th cell epitope box is adenovirus.

6. The method for constructing an adhesion protein polyepitope recombinant adenovirus delivery system according to claim 4, wherein: the construction process of the multi-epitope recombinant adenovirus, namely pAdEasy-1-pAdTrack-CMV-B epitope box-IRES-Th epitope box, comprises the following steps:

(1) protective B cell epitopes of Efb, FnBPA, ClfA and Cna obtained by identification are selected and connected in series through short and flexible intermolecular connecting sequence peptides;

(2) analyzing the tertiary structure which is closest to the natural state in theory to obtain a tandem B cell epitope box, and obtaining a tandem Th cell epitope box by the same method;

(3) combining the pAdTrack-CMV transfer plasmid map, the multiple cloning site and the enzyme cutting site contained in the insert fragment, firstly inserting an IRES fragment between the pAdTrack-CMV transfer vector SalI and NotI, and constructing a pAdTrack-CMV-IRES modified transfer empty vector;

(4) then inserting a Th cell epitope box fragment between the pAdT rack-CMV-IRES modified transfer empty vector XhoI and XbaI to construct a single-gene recombinant transfer vector of the pAdrack-CMV-IRES-Th epitope box;

(5) b cell epitope box fragments are inserted between single-gene recombinant transfer vectors BglII and SalI of the pAdTrack-CMV-IRES-Th epitope box to construct a pAdTrack-CMV-B epitope box-IRES-Th epitope box double-gene transfer vector;

(6) the constructed pAdTrack-CMV-B epitope box-IRES-Th epitope box recombinant transfer plasmid is linearized by single enzyme digestion at 37 ℃ for 2 h;

(7) co-transforming BJ5183 competent bacteria with pAdEasy-1 adenovirus skeleton plasmid calcium chloride method, screening LB agar plate containing Kana, selecting monoclonal extract plasmid, the Kana content is 50 μ g/ml;

(8) primarily screening pAdEasy-1-pAdTrack-CMV-B epitope box-IRES-Th epitope box homologous recombination clones according to the molecular weight of agarose electrophoresis;

(9) after transformation of DH 5. alpha. the plasmid was amplified and extracted for PCR and PacI single-enzyme restriction.

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