CN105254767A

CN105254767A - Protein D and HER2 fusion protein and preparing method and application thereof

Info

Publication number: CN105254767A
Application number: CN201510789201.9A
Authority: CN
Inventors: 李树刚; 张伟; 辛渝; 但国平; 柴新娟; 程丹凝; 潘玉竹; 曹莉君; 于廷和
Original assignee: CHONGQING KERUN BIOMEDICAL R&D Co Ltd
Current assignee: CHONGQING KERUN BIOMEDICAL R&D Co Ltd
Priority date: 2015-11-17
Filing date: 2015-11-17
Publication date: 2016-01-20

Abstract

The invention provides fusion protein. The fusion protein is formed by connecting adjuvant protein with the end N or the end C of a tumor-associated antigen through a linker or a direct connecting mode. The adjuvant protein contains Haemophilus influenzae (Hi) protein D or a segment of the Haemophilus influenzae (Hi) protein D; the tumor-associated antigen contains HER2 protein with the amino acid sequence being SEQ ID NO:3 or a segment of the HER2 protein. The invention further provides a preparing method for the fusion protein and application thereof. The prepared recombined protein can restrain proliferation of HER2 positive breast cancer cells in a Blab/c mice body, or delay the increase speed of breast tumors.

Description

Protein D and HER2 fusion Protein and preparation method and application thereof

Technical Field

The invention relates to the fields of biology and medicine, in particular to a fusion protein capable of preparing HER2/neu antigen positive tumor vaccine, and specifically relates to a fusion protein comprising Haemophilus influenzae (Hi) protein D (protein D, PD) and HER2/neu tumor antigen.

Background

For the treatment of tumors, there are currently surgical therapies, chemotherapies, radiation therapies, and the like. Surgical treatment is of no therapeutic interest for patients with multiple tissue metastases. Chemotherapy and radiation therapy can cause damage to normal cells due to poor specificity. Therefore, clinically, a drug with stronger specificity is needed, which specifically kills tumor cells without damaging normal cells. Tumor immunotherapy is a method of activating Cytotoxic T Lymphocytes (CTL) against tumor antigens in vivo, and killing tumor cells expressing the corresponding tumor antigens by the activated CTL, thereby having specificity. Tumor specific antigens or related antigens are key to the development of biologics with specific CTL activation.

HER2 is a tumor-associated antigen, whose coding gene is located at position 21 of the 17 th chromosome pair (17q21) and encodes a transmembrane receptor-like phosphorylated glycoprotein with a molecular weight of 185kD, and is also referred to as P185. P185 consists of 1255 amino acids, whose extracellular domain is a ligand binding domain consisting of 632 amino acids, this region being rich in cysteine; the transmembrane region is a strong hydrophobic region consisting of 22 amino acids, and serves as a membrane anchoring region to position the whole protein molecule on a cell membrane; the intracellular segment is made up of 580 amino acids and contains the tyrosine kinase activity domain (CoussensL, 1985).

In general, the HER2 gene is mainly expressed during human embryonic development and is involved in the growth and development of various tissues and organs. After human adult life, only in certain tissues, such as: low level expression is present in tissues such as kidney, liver, breast tissue, ovary, pancreas, stomach, etc. In human tumors, HER2/neu molecular expression is altered due to overexpression of the gene product, with HER2 amplification in 20% to 30% of invasive breast cancer patients. Furthermore, overexpression is found in ovarian cancer (17-37%), non-small cell lung cancer (27-56%), interstitial cancer (37%), bladder cancer (36%), esophageal cancer (60-73%), salivary gland cancer (32-62%), primary renal cell cancer (10%), pancreatic cancer (> 50%) (SlamonDJ, 1989; SchneiderPM, 1989; WeinerDB, 1990; YokotaJ, 1988; HouL, 1992; Scheurled, 2000; Seliger B, 2000; Lipponen P, 1994; TagliabueE, 2000). It can be seen that overexpression of HER2 plays a positive role in tumor transformation, and is one of the etiological factors of tumors. In addition, the high expression of HER2 protein, which occurs in the early stages of breast cancer development, also increases the metastatic capacity of tumor cells, and the high expression of HER2 by driving multiple metastasis-related mechanisms including an increase: cell migration, in vitro invasiveness, collagenase type IV activity, influence the synthesis of certain adhesion molecules such as epithelial cadherins, etc. to promote metastasis (Blume-JensenP, 2001). Therefore, HER2 is one of the important targets for tumor therapy vaccines.

Epitopes (also known as epitopes) of antigens are the fundamental units for specific binding of T cell antigen receptors (TCRs) and B cell antigen receptors (BCRs) and antibodies. Epitopes can be divided into T cell epitopes and B cell epitopes by differentiation of binding to TCR and BCR. Because the epitope vaccine lacks three-dimensional structure, has small molecules, poor immunogenicity and short half-life, the epitope vaccine cannot independently induce immune response, namely has no immunogenicity, but can obtain immunogenicity and induce immune response after being crosslinked or combined with carriers such as macromolecular protein or non-antigenic polylysine and the like.

The haemophilus influenzae PD protein is a highly conserved surface lipoprotein that plays an important role in the process of Hi causing respiratory tract infections as a characteristic virulence determinant of Hi. The haemophilus influenzae PD protein was first confirmed in studies of Hi binding to human IgD myeloma protein 4490. Sequence analysis of the coding region DNA of the Haemophilus influenzae PD protein showed that the mature protein of the Haemophilus influenzae PD protein is encoded by 1092bp open reading frame, consists of 346 amino acids and has a relative molecular mass of 42000. Studies have shown that there are only small variations in PD protein among each Hi strain. Sequence analysis at the nucleic acid and amino acid levels showed that each strain had a high degree of conservation of PD and that the gene substitutions were evenly distributed throughout the gene. Analysis of Hi isolated from different stages of infection in patients with chronic bronchitis revealed only minor mutations in the gene for the Haemophilus influenzae PD protein of each strain. Western blot analysis using 3 different murine anti-PD monoclonal antibodies revealed that the PD protein was present in all 127 strains of Hi, with a number of PD molecules per bacterial strain of about 2800. These studies all confirm that the haemophilus influenzae PD protein is an outer membrane protein located on the surface of the bacterial body, has high antigen conservation, and is a potential vaccine candidate protein.

The haemophilus influenzae PD protein is a bacterial virulence factor with diacylglycerol phosphate enzyme activity that causes the release of phosphorylcholine from host epithelial cells. The streptococcus pneumoniae polysaccharide conjugate vaccine taking the PD protein as a carrier protein can prevent the streptococcus pneumoniae otitis. PD protein is by far the first non-typeable haemophilus influenzae antigen capable of inducing a protective response in the human body and is also the most potent candidate carrier protein for polysaccharide conjugate vaccines.

The haemophilus influenzae PD protein is one of novel conjugate vaccine protein carriers, a ten-valent pneumonia conjugate vaccine Synflorix developed by glatiramer in 2009 is marketed in europe, and this vaccine links pneumococcal capsular polysaccharides of three serotypes 1, 5 and 7F to haemophilus influenzae PD protein, and clinical data indicate its good immunogenicity and safety.

Two of the major causative bacteria of acute otitis media disease are pneumococci and acapsular haemophilus influenzae, both of which are considered to be lower respiratory tract infectionsThe main factor of dyeing. Although vaccines of pneumococcal capsular polysaccharides have been used for many years, protection against children is poor. Later, CRM for the heptavalent pneumococcal polysaccharide vaccine₁₉₇The combined vaccine as a carrier can play a role in protecting children and preventing invasive diseases and acute otitis media caused by pneumococcus. And the disadvantage is CRM₁₉₇This carrier is also used in conjunction with Hib and meningitis vaccines, and therefore the same carrier may have a negative impact on vaccine immunity. Based on the reasons and the advantages of the haemophilus influenzae PD protein, such as membrane localization, sequence conservation, wide distribution, pathogenicity and good preclinical experimental results, the haemophilus influenzae PD protein becomes a carrier protein of the pneumococcal decavalent polysaccharide vaccine and can prevent the double infection of pneumococcus and non-capsular Hib. Meanwhile, the early work in the laboratory proves that the Hi protein is used as a carrier adjuvant protein, and the exogenous epitope carried by the Hi protein can induce animals to generate good immune response after the addition of or without the addition of an adjuvant.

Disclosure of Invention

The invention aims to provide a fusion protein which can be used for preparing a HER2 antigen positive tumor therapeutic vaccine.

The primary aim of tumor therapeutic vaccine design is to break the immune tolerance of the organism caused by tumor cells, and due to the influence of the immune tolerance of the organism, the immune system of the organism cannot recognize non-self components, and the immune system cannot recognize tumor-related antigens, so that the immune suppression is finally caused. Research reports show that the HER2 protein with the immune complete sequence cannot break immune tolerance and stimulate immune response, and the applicant finds that the HER2 protein with the complete sequence contains epitopes causing the immune tolerance in the research, so that in the design of a tumor therapeutic vaccine, a intercepted partial sequence is selected to replace a full-length sequence to be more beneficial to break the immune tolerance, such as an extracellular region, a transmembrane region, an intracellular region or a plurality of Tcell epitopes and Bcell epitopes of HER 2.

In order to achieve the purpose, the invention provides the following technical scheme:

a fusion protein formed by connecting the adjuvant protein with the N terminal or the C terminal of a tumor-associated antigen through a Linker (Linker) or a direct connection mode; wherein,

the adjuvant protein comprises haemophilus influenzae (haemophilus influenzae, Hi) protein D or a fragment of the haemophilus influenzae protein D;

the tumor-associated antigen comprises an amino acid sequence of SEQ ID NO: 3 or a fragment thereof;

preferably, the tumor-associated antigen is formed by combining an extracellular region fragment of a human HER2 antigen and a peptide fragment of a HER2 epitope, and the amino acid sequence of the extracellular region fragment of the HER2 antigen is represented by SEQ ID NO: 4, the amino acid sequence of the HER2 epitope peptide fragment is SEQ ID NO: 5.

preferably, the amino acid sequence of the linker is-GGGGSGGGGSGGGGS-; preferably, the adjuvant protein is linked to the N-terminus of the tumor associated antigen by direct linkage.

In one embodiment according to the invention, the amino acid sequence of the fusion protein is seq id no: 6.

the present invention also provides an expression vector constructed by linking a nucleotide sequence encoding the fusion protein according to claim 1 or 2 to a commercialized vector selected from one or more of pET-28a-c (+), pET29a, pET-30a-c (+), pET39b (+), pET-40b (+), pET-41a (+) and pET-43.1a (+) and optionally a sequence encoding a cleavage recognition site sequence and/or a sequence encoding a protein tag; preferably, the nucleotide sequence is seq id no: 7.

in one embodiment according to the present invention, the expression vector further comprises a coding nucleic acid sequence corresponding to the sequential arrangement of the restriction enzyme recognition site, the histidine tag and the enterokinase site at the 5' end of the nucleotide sequence, preferably, the sequential arrangement of the restriction enzyme recognition site, the histidine tag and the enterokinase site corresponds to the coding nucleic acid sequence of seq id no: 8.

in another embodiment according to the present invention, the expression vector further comprises a stop codon sequence and a cleavage recognition site sequence located at the 3' end of the nucleotide sequence; preferably, the stop codon sequence and the enzyme cutting recognition site sequence are SEQ ID NO: 9; more preferably, the nucleotide sequence encoding the fusion protein is seq id no: 10.

the invention also provides a host cell containing the expression vector, wherein the host cell is a bacterium or a fungus; preferably, the host cell is escherichia coli; more preferably, the E.coli is selected from BL21(DE3), BL21(DE3) PlysS or TB 1.

The invention further provides a preparation method of the fusion protein, which comprises the steps of inducing the host cell to express the fusion protein through IPTG (Merk, USA), collecting inclusion bodies, extracting the fusion protein in the inclusion bodies, and purifying to obtain the fusion protein;

preferably, the purification is achieved by:

the fusion protein was purified by passing through a chelate column.

In one embodiment of the present invention, the purification step further comprises:

the fusion protein is cut by enterokinase, then the cut fusion protein is loaded on the cut target protein and loaded on Ni2+ -ChelatingSepharose Fastflow, and then the fusion protein is eluted by imidazole with the concentration of 50 mM.

The fusion protein provided by the invention can be used for preparing a HER2 antigen positive tumor vaccine, and the vaccine comprises the fusion protein; preferably, the vaccine is a HER2 antigen positive tumor therapeutic vaccine; preferably, the antigen positive tumor is a breast tumor.

Further, the vaccine also comprises an adjuvant, wherein the adjuvant is selected from one or more of GM-CSF and GM-CSF + CpG; preferably, the vaccine is in the form of injection.

The preparation method and the application of the protein-HER 2 fusion protein therapeutic vaccine comprise the following steps:

taking the construction of a recombinant pET-30a (+) prokaryotic expression vector as an example: firstly, constructing a pET-30a (+) prokaryotic expression vector; ② a recombinant prokaryotic expression vector containing a nucleic acid sequence of PD protein 19-129aa encoding Hi, a nucleic acid sequence of HER2 protein 41-560aa, a nucleic acid sequence of HER2 protein 774-790aa and a nucleic acid sequence of Linker; providing an Escherichia coli for expression, transforming a recombinant prokaryotic expression vector containing a PD protein 19-129aa nucleic acid sequence encoding Hi, a nucleic acid sequence of HER2 protein 41-560aa, a nucleic acid sequence of HER2 protein 774-790aa and a nucleic acid sequence of Linker into the Escherichia coli for expression, and stably expressing a protein D and HER2 fusion protein after transformation; and providing a method for purifying the protein and HER2 fusion protein.

The percentage contents in the product and the method are mass volume percentage contents.

The invention relates to a therapeutic vaccine of protein and HER2 fusion protein. The vaccine comprises 19-129aa of PD protein, 41-560aa of HER2 protein and 774-790aa of HER2 protein, and can stimulate good immune response of organisms and break immune tolerance. The protein fusion protein of ProteinD and HER2 prepared by the invention can stimulate macrophages in vitro to secrete and express TNF-alpha in large quantity. Meanwhile, the protein D and HER2 fusion protein prepared by the invention is subjected to a pharmacodynamic experiment, and the fusion protein therapeutic vaccine formed by immunity injection is added or not added with an adjuvant, so that the proliferation of 4T1-HER2 cells in a breast cancer animal model of a Balb/c mouse can be inhibited within 50 days, the tumor growth inhibition rate reaches over 90 percent, and the cellular immunity activated by the protein D and HER2 fusion protein therapeutic vaccine is obviously higher than that of a PBS control group. Therefore, the developed therapeutic vaccine of the protein D and HER2 fusion protein has good immunotherapeutic effect on inhibiting the proliferation of 4T1-HER2 cells in Balb/c mouse breast cancer animal models under the condition of containing or not containing adjuvants. Wherein the 4T1-HER2 cell is constructed by transfecting cDNA of human HER2 with a mouse breast cancer 4T1 cell strain through retrovirus; wherein the 4T1-HER2 breast cancer animal model is an animal model established subcutaneously by injecting 4T1-HER2 into female BALB/C mice.

The invention has the beneficial effects that:

by screening the antigen epitope and carrying out tandem expression, the HER2 protein immune tolerance epitope is avoided, the self immune tolerance of a tumor patient is broken, and the anti-tumor immunity of an organism is activated.

The recombinant protein prepared by coupling expression of the antigen epitope and the PD protein and using the high immunogenicity of the PD protein as an adjuvant protein can improve the immunogenicity of the recombinant protein, promote antigen presentation and enhance the anti-tumor immune response of an organism.

The recombinant protein prepared by the invention can inhibit the proliferation of HER2 positive breast cancer cells in a Blab/c mouse body, or delay the growth rate of breast tumors.

The recombinant protein prepared by the invention has great application prospect in prevention and treatment of recurrence after human HER2 positive breast cancer operation and adjuvant therapy of chemotherapy and radiotherapy treatment, and can stimulate immune response of organisms and kill cancer cells by utilizing an autoimmune system.

The recombinant protein prepared by the invention has great application prospect in prevention and treatment of relapse after operation of human HER2 positive tumor, adjuvant therapy of chemotherapy and radiotherapy treatment, and can stimulate immune response of organisms and kill cancer cells by utilizing an autoimmune system.

The therapeutic vaccine of the protein and HER2 fusion protein prepared by the invention does not contain an adjuvant, and also can contain one or more of the following adjuvants: GM-CSF; ② CpG; ③ GM-CSF + CpG, etc.

The protein and HER2 fusion protein therapeutic vaccine prepared by the invention can be prepared into various clinically applicable formulations, including but not limited to injection formulations.

Drawings

FIG. 1 depicts the construction of pET30-a (+) -PD-HER2 expression vector;

FIG. 2 shows SDS-PAGE electrophoresis of induced expression of pET30-a (+) -PD-HER2/BL21(DE3) engineering bacteria;

FIG. 3PD-HER2 fusion protein vaccine in vitro cell viability curve;

FIG. 4 experimental tumor growth curves in PD-HER2 fusion protein vaccine mice;

FIG. 5PD-HER2 fusion protein vaccine in vivo experimental survival curves in mice.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the description of the specific embodiments is intended to illustrate and not to limit the invention.

Example 1Construction of recombinant PD-HER2 fusion protein therapeutic vaccine engineering bacteria

Construction of pET30-a (+) -PD-HER2/BL21(DE3) engineering bacteria

The Hi-derived PD protein (GenBank accession number: CAA84716) was selected to have a total length of 364 amino acids, as shown in SEQ ID NO: 1 is shown. Amino acids 19-127 of the PD protein are selected as adjuvant proteins, such as SEQ ID NO: 2, respectively. Tumor associated antigen human HER2(GenBank accession No.: AAA75493.1) has a full length of 1255 amino acids as shown in seq id no: 3, respectively. The HER2 antigen extracellular region fragment (41-560aa) and the HER2 epitope peptide fragment (774-788aa) are selected as vaccine molecule antigens. The amino acid sequence of the HER2 antigen extracellular region fragment (41-560aa) is shown as SEQ ID NO: 4, the HER2 epitope peptide fragment (774-788aa) is shown as SEQ ID NO: 5, respectively.

The recombinant PD-HER2 fusion protein therapeutic vaccine molecule is that amino acids 19-127 of PD protein are connected to the N end of HER2 antigen extracellular region segment (41-560aa) through Linker sequence-GGGGSGGGGSGGS-, and then HER2 epitope peptide segment (774-788aa) is directly connected to the C end of HER2 antigen extracellular region segment (41-560 aa). The designed PD-HER2 fusion protein has an amino acid sequence shown as SEQ ID NO: 6, and the corresponding coding nucleic acid sequence is shown as SEQ ID NO: shown at 7.

For convenient purification, a nucleic acid sequence corresponding to 6 × His tag and enterokinase site (HHHHHHDDDDK) and an NdeI recognition site, such as SEQ ID NO: 8, are sequentially added at the 5' end of the PD-HER2 fusion protein nucleic acid sequence (SEQ ID NO: 7)CATATGCACCACCACCACCACCACGACGACGACGATAAA, wherein the underline indicates the NdeI recognition site, and CACCACCACCACCACCACGACGACGACGATAAA-HHHHHHDDDDK corresponds to the coding nucleic acid sequence. Meanwhile, two stop codon sequences and a NotI recognition sequence are sequentially added at the 3' end of the PD-HER2 fusion protein nucleic acid sequence (SEQ ID NO: 7), such as SEQ ID NO: 9TAATGAGCGGCCGC, the two stop codon sequences underlined, GCGGCCGC is the NotI recognition sequence. The designed PD-HER2 fusion protein nucleotide sequence is entrusted to Nanjing Kingsry Biotech Co., Ltd for whole gene synthesis (the nucleotide sequence is SEQ ID NO: 10). The expression vector pET30-a (+) -PD-HER2 was constructed by subcloning NdeI/NotI sites (TAKARA, Dalian) into an expression vector selected from the commercial vector pET-30a-c (+) (Merk, USA), and the construction scheme is shown in FIG. 1.

The constructed expression vector is transferred into an escherichia coli expression host bacterium BL21(DE3) by a chemical transfection method, and a recombinant expression engineering bacterium pET30-a (+) -PD-HER2/BL21(DE3) is obtained by screening LB + kan solid plates.

2. Induced screening of engineering bacteria

The above pET30-a (+) -PD-HER2/BL21(DE3) engineered bacteria were randomly picked up and inoculated into 10ml LB medium (100ug/ml kan), cultured in 100ml Erlenmeyer flask, and shake-cultured overnight at 37 ℃ and 220 rpm. The next day, the mother solution of overnight culture was transferred to 40ml of LB liquid medium (100ug/ml kan) at a ratio of 1%, and cultured in a 250ml Erlenmeyer flask at 37 ℃ and 220rpm2.5h to OD₆₀₀The value is about 0.6-1.0. Add 1mMIPTG to the final concentration and induce 4h at 30 ℃.

SDS-PAGE, the results are shown in FIG. 2, lanes 1-3: pET30-a (+) -PD-HER2/BL21(DE3) No. 1-3 engineering bacteria induces 4h total protein; comparison: total protein was not induced; lane M: protein molecular weight Marker; lanes S1, S2: 1. breaking the bacteria of the engineering bacteria No. 2 and supernatant; lanes P1, P2: 1. and (3) breaking the bacteria of the engineering bacteria No. 2 and precipitating. Compared with a control, the specific expression of 3 engineering bacteria at the theoretical molecular weight of 74kD is realized. 2 strains of the induced expression thalli are subjected to bacterium breaking analysis, and all the strains are expressed by inclusion bodies.

Example 2Fermentation of recombinant PD-HER2 fusion protein engineering bacteria

The pET30-a (+) -PD-HER2/BL21(DE3) engineered bacterium constructed in the above example 1 was streaked on LB plate (kan100mg/L), and cultured in a 37 ℃ incubator (Boxun, Shanghai) for about 16-18 hours until a single colony grew. A single colony of the engineering bacteria is selected and inoculated into 20ml LB culture medium (kan100mg/L), and cultured for 8h at 37 ℃ and 230 rpm. 0.1% was transferred to 250ml LB (kan100mg/L), 1L Erlenmeyer flask, cultured at 37 ℃ and 230rpm for 13 hours. Culturing 4 bottles in parallel, preparing 1000ml of bacterial liquid, inoculating 5% of the bacterial liquid into a tank-loading culture medium of a fermentation tank NLF-2220L, adjusting the pH to 7.0 by using ammonia water before inoculation, and controlling the temperature to be 36 ℃ in the fermentation process. The pH value and dissolved oxygen of the culture medium are controlled by feeding ammonia water and increasing the stirring speed and ventilation, and the dissolved oxygen is more than 30 percent. Carbon source depletion, OD in the culture Medium after about 5h₆₀₀After reaching 20, the culture was continued by starting feeding the medium (40% glycerol + 20% yeast powder) at a rate of 240mL/h/20L of the medium, OD₆₀₀35, feeding the feed culture medium at the speed of 60mL/h/20L of culture medium to start induction, feeding isopropyl- β -D-thiogalactoside (IPTG, Merk) as an inducer, keeping the feeding speed at a final concentration of 1 mM., maintaining DO above 30%, inducing for 4h, and discharging.

The thalli is collected by centrifugation, and suspended in a bacteria breaking buffer (20mmTris-HCl, 5mM EDTA, PH8.0), and the ratio of the thalli to the bacteria breaking buffer is as follows: 1g of cells: 10ml of buffer. After the suspension was sufficiently suspended, the suspension was crushed by a high-pressure homogenizer (model AH-PILOT manufactured by ATSENGINEERINGINC) under a pressure of 80MPa, and the cells were disrupted by circulation three times, and the inclusion bodies and the disrupted cells were separated by a high-speed refrigerated centrifuge (Hitachi, CR21 GIII). The centrifugation condition is 8000g at 10 ℃, and the centrifugation is carried out for 15 min. The precipitate was collected and the supernatant discarded.

Example 3 isolation and purification of recombinant PD-HER2 fusion protein

1. Inclusion body washing

(1) First washing

The precipitate collected in example 2 was taken and contained as Inclusion Bodies (IB) as a main component. Washed with wash buffer (20mM Tris-HCl, 2M urea (guggai, shanghai), 1% TritonX-100(sigma, USA), 5mM EDTA (guggai, shanghai), 0.2M NaCl (guggai, shanghai), pH 8.0). IB/washing buffer (1 g/20 ml), magnetic stirring at room temperature for 30min, and centrifuging at 8000g for 10min to remove supernatant.

(2) Second washing

The inclusion bodies collected after the first washing were precipitated and washed with a washing buffer (20mM Tris-HCl, 2M urea, 5mM EDTA, 0.2M NaCl, pH 8.0). IB/washing buffer (1 g/20 ml), magnetic stirring at room temperature for 30min, and centrifuging at 8000g for 10min to remove supernatant.

(3) Third washing

The inclusion bodies collected after the third washing were precipitated and washed with a washing buffer (20mM Tris-HCl, 5mM EDTA, 0.2M NaCl, pH 8.0). IB/washing buffer (1 g/20 ml), magnetic stirring at room temperature for 30min, and centrifuging at 8000g for 10min to remove supernatant.

2. Inclusion body solubilization and chromatographic renaturation

(1) Dissolution of IB

Dissolving buffer solution: 20mM Tris-HClpH8.0+8M Urea (national medicine, Shanghai) +5mM DTT (sigma, USA). IB (g): (1 g): (ml): (1 g): (10 ml), and the mixture was dissolved at room temperature for 6 hours with stirring by a magnetic stirrer.

(2) Renaturation

100ml of the inclusion body dissolved sample is taken, added into 900ml of dilution renaturation buffer (1M urea, 5% glycerol, 0.1M NaCl, 50M Tris-HCl, pH8.0) at the flow rate of 0.2ml/min for dilution renaturation, the buffer is stirred at a low rotation speed (60rpm/min) by a magnetic stirrer, the sample is stirred for about 4 hours after the addition is finished, then the mixture is placed into a chromatography cabinet at 4 ℃ for overnight renaturation (12 hours), and the concentration of a denaturant in the sample after dilution renaturation is between 1.5 and 2M.

(3) Purification by chelate chromatography

Column specification and treatment: Φ 3.5cm × H10cm (chelating fillers 80ml, GE, USA); the column was buffered with 0.1M nickel sulfate 4 column volumes and then the dH2O test water was used to wash the column 10 column volumes.

And (3) chelating chromatographic equilibrium: equilibration buffer (2.0M urea, 0.1M NaCl, 50mM Tris-HCl, pH8.0), flow rate 20ml/min, equilibration time 30 min.

Pumping into the chromatographic column by a peristaltic pump at a flow rate of 20 ml/min. After the sample injection is finished, the balance is continued for 20 min. Eluted with buffer (20mMPB, 0.2M NaCl, 5% glycerol, pH8.0) at a flow rate of 20 ml/min. Then eluted with a buffer (20mM PB, 0.2M NaCl +,% glycerol, 40mM imidazole, pH8.0) at a flow rate of 20 ml/min. Then eluted with a buffer (20mM PB, 0.2M NaCl, 5% glycerol, 200mM imidazole, pH8.0) at a flow rate of 20ml/min, and the desired peak was collected.

The collected fusion protein of interest was desalted and digested with 0.5U enterokinase (sigma, USA, 50mM Tris-HCl, 2mM CaCl2, 0.1% Tween-20, pH8.0) per 1mg of fusion protein at 6-8 ℃ for about 17 hours.

The enzyme-digested target protein is loaded on Ni²⁺-ChelatingSepharose Fastflow (GE, USA), and the target protein and fusion tag were simultaneously hung on the column. However, the binding force of the target protein hanging column is low, and the target protein is eluted by imidazole (Shanghai Biotech, batch number: B421BA0025) with the concentration of 50mM, and the peak of the eluted protein is collected. Then performing CMFF fine purificationObtaining the high-purity protein. The molecular weight of the product is about 72kD as detected by SDS-PAGE, and the purity is more than 95%.

Example 4 in vitro Activity assay of recombinant PD-HER2 fusion protein therapeutic vaccines

PMA stimulation THP-1 differentiation macrophage detection recombinant protein immunogenicity

1. The method uses macrophage differentiated from THP-1 cell (ATCC, USA) to measure the activity of recombinant PD-HER2 fusion protein, and can directly and truly reflect the immunogenicity of the recombinant protein in an in vitro environment.

2. Experimental methods

The activity of the recombinant PD-HER2 fusion protein is determined by adding phorbol ester (PMA, sigma) to stimulate THP-1 cells to differentiate into macrophages, utilizing the capability of the macrophages to take up and present antigens and detecting the content of TNF-alpha generated by the macrophages after adding the antigens through a TNF-alpha detection kit (MABTECH, the code of 3510-1H-6).

2.1PMA stimulation of THP-1 differentiation

2.1.1 harvesting THP-1 cells, centrifuging (300g, 5 min) to collect the cells, and discarding the supernatant;

2.1.2 resuspend cells in 1640 medium with 10% FBS and mix well;

2.1.3 under the mirror for cell count 1, and using the culture medium to adjust the cell density to 7X 105/ml;

2.1.4 setting the concentration of PMA stimulant at 100 ng/ml;

2.1.5 adding the uniformly mixed THP-1 cell sap containing PMA into a 24-hole cell culture plate at a volume of 500 mul/well; placing the cell culture plate at 37 ℃ and a 5% CO2 cell culture box for culturing for 37 h;

2.2 antigenic stimulation of macrophages

2.2.1 serum-free treatment for 24h, medium in the 24-well plate was aspirated and washed once with serum-free 1640 medium, followed by addition of 500. mu.l serum-free medium per well for further 24 h.

2.2.2 Add 500. mu.l of diluted antigen to the corresponding wells in groups as above, continue incubation for 24h, collect the supernatant and use the kit to detect TNF-. alpha.levels.

2.2.3 TNF-alpha ELISA assay

The coating was taken 28. mu.l and PBS was added to 7 ml.100. mu.l/well overnight at 4 ℃.

The plates were washed 2 times with PBS.

Blocking was performed with PBS37 containing 0.1% BSA for 1 hour.

Wash plate 5 times, PBS + 0.05% tween 20;

adding 1 μ g/ml standard, adding PBS containing 0.1% BSA, diluting to 10ng/ml, and making 7 2-fold dilutions; culture supernatants were aspirated from the cell culture plates at 100. mu.l/well. At 37 ℃ for 2 hours.

The plate was washed 5 times.

Adding mAbTNF 5-biotin: mu.l of PBS containing 0.1% BSA was taken up to 7ml.100 μ l/well. The reaction was carried out at 37 ℃ for 1 hour.

The plate was washed 5 times.

streptavidin-HRP 7. mu.l was added to PBS containing 0.1% BSA to 7ml, 100. mu.l/well. The reaction was carried out at 37 ℃ for 1 hour.

The plate was washed 5 times.

Color development: each well was filled with 100. mu.l of TMB substrate solution and acted upon at 26 ℃.

650nm dynamic plate reader (detection every 1min, moderate oscillation 3sec)

Calculation of the diastolic EC with Graphprism5.0 software₅₀And half-effect dilution factor, calculated as:

3. results of the experiment

The PD-HER2 fusion protein prepared by the invention has biological activitySexual EC₅₀8 μ g/ml, see FIG. 3.

EXAMPLE 5 recombinant PD-HER2 fusion protein therapeutic vaccine in mice tumor suppressor Activity assay

1. The method uses Balb/c mouse 4T1-HER2 transfer animal model to determine the tumor inhibition activity of the PD-HER2 fusion protein prepared by the invention. Wherein the 4T1-HER2 cell is constructed by transfecting cDNA carrying human HER2 with a retrovirus into a mouse breast cancer 4T1 cell line (ATCC, USA); wherein the 4T1-HER2 breast cancer animal model is an animal model established subcutaneously by injecting 4T1-HER2 into female BALB/C mice.

2. Experimental methods

2.14T1-HER2Balb/c mouse animal model establishment

40 BALB/c mice were inoculated subcutaneously in situ with 1 × 10⁵Cell/tumor 4T1-HER2 suspension.

2.2 recombinant PD-HER2 fusion protein immunized animals

At the same time of tumor inoculation, the mice are divided into 4 groups and are treated by the drug, 10 mice are respectively treated by 2-3 times of immunization injection, and the interval between the two immunizations is 1 week, namely, the drug is respectively administered on 0, 7 and 14 days of tumor inoculation. The grouping is as follows:

control group: injecting PBS buffer;

② vaccine group: the dosage of the vaccine is 100 mu g/mouse;

③ adjuvant group: injection of an adjuvant;

vaccine + adjuvant group: the vaccine is mixed with 100 mug of adjuvant, shaken up and injected.

2.3 Experimental period

The experimental period was 50 natural days from the day of tumor inoculation. During the experiment, tumor volume and body weight of mice were measured 2 times per week, and tumor rate and tumor volume dynamic records were made, along with survival rate.

2.4 results of the experiment

The recombinant protein and Her2 fusion protein can inhibit the proliferation of 4T1-HER2 cells in a breast cancer animal model of Balb/c mice within 50 days, and the tumor growth inhibition rate reaches over 90 percent, as shown in figure 4. Therefore, the research and development of the therapeutic vaccine of the protein D and Her2 fusion protein has good immunotherapeutic effect on inhibiting the proliferation of 4T1-Her2 cells in a Balb/c mouse breast cancer animal model under the condition of containing or not containing an adjuvant, and can improve the survival rate of the Balb/c mouse breast cancer animal model, and the reference of figure 5 shows that the therapeutic vaccine of the protein D and Her2 fusion protein has good immunotherapeutic effect on inhibiting the proliferation of 4T1-Her2 cells in the.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.

Claims

1. A fusion protein, which is formed by connecting the adjuvant protein with the N-terminal or C-terminal of a tumor-associated antigen through a Linker (Linker) or a direct connection manner; wherein,

the tumor-associated antigen comprises an amino acid sequence of SEQ ID NO: 3 or a fragment thereof, HER2 protein; preferably, the tumor-associated antigen is formed by combining an extracellular region fragment of a human HER2 antigen and a peptide fragment of a HER2 epitope, and the amino acid sequence of the extracellular region fragment of the HER2 antigen is represented by SEQ ID NO: 4, the amino acid sequence of the HER2 epitope peptide fragment is SEQ ID NO: 5;

preferably, the amino acid sequence of the adjuvant protein is seq id no: 2;

2. The fusion protein of claim 1, wherein the amino acid sequence of the fusion protein is seq id no: 6.

3. an expression vector constructed by linking a nucleotide sequence encoding the fusion protein of claim 1 or 2 to a commercial vector selected from one or more of pET-28a-c (+), pET29a, pET-30a-c (+), pET39b (+), pET-40b (+), pET-41a (+) and pET-43.1a (+) and optionally adding a cleavage recognition site sequence and/or a sequence encoding a protein tag; preferably, the nucleotide sequence is seq id no: 7.

4. the expression vector of claim 3, further comprising a coding nucleic acid sequence corresponding to the sequential arrangement of the enzyme cleavage recognition site, the histidine tag and the enterokinase site at the 5' end of the nucleotide sequence, preferably the sequential arrangement of the enzyme cleavage recognition site, the histidine tag and the enterokinase site corresponds to the coding nucleic acid sequence of SEQ ID NO: 8.

5. the expression vector of claim 3 or 4, further comprising a stop codon sequence and a cleavage recognition site sequence located 3' of the nucleotide sequence; preferably, the stop codon sequence and the enzyme cutting recognition site sequence are SEQ ID NO: 9; more preferably, the nucleotide sequence encoding the fusion protein is seq id no: 12.

6. a host cell comprising the expression vector of any one of claims 3 to 5, wherein the host cell is a bacterium or a fungus; preferably, the host cell is escherichia coli; more preferably, the E.coli is selected from BL21(DE3), BL21(DE3) PlysS or TB 1.

7. A method for preparing the fusion protein of claim 1 or 2, wherein the host cell of claim 6 is induced by IPTG to express the fusion protein, the inclusion bodies are collected, the fusion protein in the inclusion bodies is extracted and purified to obtain the fusion protein;

preferably, the purification is achieved by:

the fusion protein was purified by passing through a chelate column.

8. The method of claim 7, wherein the purifying step further comprises:

9. A HER2 antigen positive tumor vaccine comprising the fusion protein of claim 1 or 2; preferably, the vaccine is a HER2 antigen positive tumor therapeutic vaccine; preferably, the antigen positive tumor is a breast tumor.

10. The vaccine of claim 9, further comprising an adjuvant selected from the group consisting of GM-CSF, CpG, and GM-CSF + CpG; preferably, the vaccine is in the form of injection.