Human FGFR3 (Fibroblast Growth Factor Receptor 3) antigen expression recombinant yeast vaccine as well as construction method and application thereof

Abstract

The invention provides a human FGFR3 (Fibroblast Growth Factor Receptor 3) antigen expression recombinant yeast vaccine as well as a construction method and application thereof. A specific primer is designed to amplify an FGFR3 gene, a pEGKT316-FGFR3 recombinant plasmid is constructed, a saccharomyces cerevisiae YEF473A strain is transformed, and the vaccine is prepared through induced expression and thermal inactivation. The vaccine can effectively activate DC cell maturation (CD80 + cell proportion reaches 78.7%) and promote Th1 type cell factor secretion (IL-12p70 reaches 5889pg / mL), and shows a remarkable tumor inhibition effect (plt; 0.05), and a novel specific treatment means is provided for postoperative immunotherapy of bladder cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for constructing a recombinant yeast vaccine based on fibroblast growth factor receptor 3 (FGFR3) antigen and its application. Background Art

[0002] Bladder cancer is a urinary system tumor with a high overall recurrence rate and poor metastasis prognosis. Its postoperative recurrence rate is as high as 70%, making it one of the most expensive malignant tumors to treat due to the need for repeated surgeries. After progressing to metastasis, the five-year survival rate of patients is less than 5%. To reduce the risk of tumor recurrence, progression, or metastasis, the NCCN guidelines for bladder cancer recommend drug perfusion therapy after transurethral resection of bladder tumor (TURBT) for non-muscle invasive bladder cancer to eliminate residual tumor cells. Currently, BCG (Bacillus Calmette-Guérin) is the preferred drug for postoperative perfusion therapy of bladder cancer. After being injected into the body, BCG can activate the immune system, causing a strong inflammatory response in the body to achieve the effect of promoting the immune system to clear tumor cells. Therefore, BCG perfusion therapy is actually an immunotherapy strategy. In addition, BCG can also infect bladder cancer cells, enter the cells, directly kill the cancer cells or indirectly guide immune cells to kill the cancer cells by expressing BCG antigens on the cell surface.

[0003] Since the immune response activated by BCG is not tumor cell-specific, and BCG is derived from a pathogenic bacterium (Mycobacterium bovis), which itself has an infective effect, BCG perfusion therapy is often accompanied by various serious toxic and side effects, including local side effects such as cystitis, hematuria, orchitis and epididymitis, and symptomatic granulomatous prostatitis, and systemic side effects such as joint pain, arthritis, persistent high fever and BCG sepsis. Aiming at the shortcoming that the immune response activated by BCG lacks specificity, exploring and developing a new type of perfusion drug that can induce the body to produce a bladder cancer antigen-specific immune response is of great significance for alleviating the pain of patients, further reducing the risk of tumor recurrence and progression, and improving the quality of life of bladder cancer patients. Summary of the Invention

[0004] The object of the present invention is to provide a recombinant yeast vaccine against human FGFR3 antigen, including:

[0005] a) A recombinant Saccharomyces cerevisiae strain expressing FGFR3-GST fusion protein;

[0006] b) The strain carries the pEGKT316-FGFR3 recombinant plasmid, which inserts the FGFR3 gene sequence shown in SEQ ID NO:3 through BamHI and EcoRI restriction sites.

[0007] Correspondingly, the present invention also provides a method for constructing a recombinant yeast vaccine, including the following steps:

[0008] A) Amplify the FGFR3 gene using the primer pair shown in SEQ ID NO:1 and SEQ ID NO:2;

[0009] B) Digest the amplification product with BamH I / EcoRI double enzymes and ligate it to the pEGKT316 vector;

[0010] C) Transform the recombinant plasmid into the YEF473A Saccharomyces cerevisiae strain;

[0011] D) Induce the expression of FGFR3 protein in the SRG-Ura medium;

[0012] SEQ ID NO:1: FGFR3-F1-BamH I forward primer, sequence: CGC GGATCC ATGGGCGCCCCTG, SEQ ID NO:2: FGFR3-R1-EcoRI reverse primer, sequence: CCG GAATTC TCACGTCCGCGAG; The underlined part is: the recognition site of the restriction endonuclease.

[0013] Preferably, the pEGKT316 vector contains a GST tag coding sequence, and the nucleotide sequence of the FGFR3 gene is as shown in SEQ ID NO:3.

[0014] Preferably, the Saccharomyces cerevisiae strain is inactivated by heating in a water bath at 100 °C, and the cell concentration after inactivation is 1 - 10×10 8 CFU / mL.

[0015] Preferably, the dosage of the drug is 1×10 7 CFU each time, and it is administered once a week.

[0016] Correspondingly, the present invention also provides the application of the recombinant yeast vaccine. The therapeutic drug is administered through a bladder perfusion catheter, the perfusion fluid volume is 0.1 mL, and the retention time is 60 - 90 minutes.

[0017] Preferably, the vaccine further comprises a pharmaceutically acceptable carrier, and the carrier is selected from phosphate buffer solution, physiological saline or a stabilizer containing 0.1% human serum albumin.

[0018] Correspondingly, the present invention also provides the application of an inactivated FGFR3-expressing recombinant Saccharomyces cerevisiae cell in the preparation of a drug for immunotherapy of bladder cancer.

[0019] Preferably, the drug is administered by bladder perfusion, which can activate DC cells and increase the expression of CD80, CD5, MHC II molecules on their surface and cytokine secretion, thereby enhancing the body's ability to clear bladder tumor cells.

[0020] Correspondingly, the present invention also provides the application of the vaccine prepared by the above method in the postoperative perfusion treatment of bladder cancer.

[0021] Molecular genetic studies on bladder cancer have found that the fibroblast growth factor receptor FGFR3 protein is highly expressed in 80% of early bladder cancer clinical samples. As a classical eukaryotic model organism, Saccharomyces cerevisiae has the advantages of being easy to genetically modify and is a widely used eukaryotic protein expression system. Although Saccharomyces cerevisiae is not a human pathogen, it can bind to the mannan-fucose receptor and toll-like receptors (TLRs) of dendritic cells (DCs), causing DC cells to respond and having a natural vaccine adjuvant effect. Currently, there have been studies using recombinant yeast cells expressing specific antigens such as HIV Gag protein, HSV N53-Core protein, and influenza virus matrix protein as vaccines to kill infected virus antigen-expressing cells by activating the CTL immune response. Therefore, using recombinant Saccharomyces cerevisiae as an antigen protein carrier can effectively enhance the downstream immune response induced by antigens to activate DC cells and play an antigen clearance role.

[0022] The recombinant Saccharomyces cerevisiae strain expressing human FGFR3 constructed in the present invention can highly express the FGFR3 protein in a yeast liquid medium with raffinose-galactose as the carbon source. After being inactivated at 100°C, it becomes an inactivated yeast vaccine of FGFR3 antigen. Co-culturing the inactivated recombinant yeast cells expressing FGFR3 with DC cells in vitro, the recombinant yeast can be endocytosed by DC cells and induce the activation of DC cells, increasing the expression of cell surface proteins CD80, CD5, and MHC II on DC cells, as well as the production of cytokines TNF-α, IFN-γ, IL-8, IL-2, IL-10, and IL-1β. Bladder perfusion of the in situ mouse model of bladder cancer can significantly improve the ability of mice to clear bladder tumor cells. Brief Description of the Drawings

[0023] Figure 1 It is the circular structure diagram of the yeast shuttle plasmid pEGKT3016 (constructed based on pRS316, 6640 bp), marking key gene elements such as CEN, URA, Amp, and restriction enzyme sites, and is used for genetic engineering or molecular cloning research.

[0024] Figure 2 It is the schematic diagram of gene construction elements, including the CYC1-P promoter, GST tag, FGFR3 target gene, CYC1-T terminator, and GAL1-10 UAS regulatory sequence, and is used to describe specific DNA recombinant structures or expression vector designs.

[0025] Figure 3 Electrophoresis diagram of double digestion verification of pEGKT316-FGFR3 recombinant plasmid.

[0026] Figure 4 Verification of recombinant yeast FGFR3 protein expression.

[0027] Figure 5 Comparison diagram of the inhibitory effect of recombinant yeast-induced DC cells on bladder cancer in a mouse model of orthotopic bladder cancer.

[0028] Figure 6 Analysis diagram of survival rate in the inhibitory effect of recombinant yeast-induced DC cells on bladder cancer in a mouse model of orthotopic bladder cancer. Specific implementation mode

[0029] The following further describes in detail the preferred embodiments of the present invention with reference to the accompanying drawings:

[0030] The following embodiments facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all purchased from conventional biochemical reagent companies unless otherwise specified. All gene operations in this embodiment comply with the "Regulations on the Administration of Biosafety in Laboratories of Pathogenic Microorganisms" (Decree No. 424 of the State Council), and the biosafety level of the recombinant yeast is BSL-1 after inactivation treatment.

[0031] Example 1

[0032] I. Construction of human FGFR3 recombinant expression plasmid

[0033] 1. Amplification of FGFR3 gene

[0034] (1) Primer design: Primers with BamHI and EcoRI restriction enzyme sites are used

[0035] FGFR3-F1-BamH I: 5'-CGCGGATCCATGGGCGCCCCTG-3';

[0036] FGFR3-R1-EcoRI: 5'-CCGGAATTCTCACGTCCGCGAG-3';

[0037] (2) Preparation of cDNA template:

[0038] ① Culture T24 cells to 70% confluence (DMEM medium containing 10% fetal bovine serum);

[0039] ② Collect cells, extract total RNA, and synthesize a cDNA library by reverse transcription PCR;

[0040] (3) PCR amplification:

[0041] System: cDNA template + the above primers. Parameter: Amplify the FGFR3 gene sequence (2439bp) according to the conventional PCR procedure;

[0042] Verification: After recovering the product, sequence it to determine the correctness of the sequence.

[0043] 2. Construction of pEGKT316-FGFR3 recombinant plasmid

[0044] pEGKT316 is an expression vector plasmid in the Saccharomyces cerevisiae system, which can enable the target gene to be highly expressed in yeast cells under galactose induction. Double digest the amplified FGFR3 gene fragment and the plasmid vector pEGKT316 with the restriction endonucleases BamH I and EcoRI. After purifying the digested products, catalyze the ligation of the FGFR3 gene digested fragment and the pEGKT316 digested vector with T4 ligase. The ligation product is transformed into competent DH5α Escherichia coli, screened for ampicillin-resistant clones, verified by restriction digestion of the extracted Escherichia coli plasmid, and sequenced to obtain the correctly ligated pEGKT316-FGFR3 recombinant expression plasmid (9061bp). As Figure 1 and Figure 2 shown.

[0045] II. Yeast transformation and expression verification

[0046] After reviving the Saccharomyces cerevisiae strain YEF473A (MATa his3-Δ200 leu2-Δ1 lys2-801 trp1-Δ63 ura3-52) on the YPD solid medium, culture the yeast cells in the YPD liquid medium until the OD600 value of the bacterial liquid reaches 0.8 - 1.0. Collect the yeast cells, and through the lithium acetate / PEG chemical transformation method with salmon sperm ssDNA as the medium, transform the pEGKT316-FGFR3 recombinant plasmid into the yeast cells. Screen positive recombinant yeast clones on the selective SC-Ura solid medium. Extract the yeast cell protein and confirm the expression of FGFR3 by western blot.

[0047] III. Inactivated vaccine preparation

[0048] Culture the FGFR3-expressing recombinant yeast in the SRG-Ura liquid medium using raffinose and galactose to replace sucrose to induce the high-level expression of the FGFR3 fragment. After 24 hours, collect the recombinant yeast and inactivate it by heating in a water bath at 100°C to prepare an inactivated yeast vaccine of the FGFR3 antigen.

[0049] Example 2

[0050] I. Construction and verification of recombinant plasmid

[0051] 1. Verification of plasmid digestion

[0052] ① The recombinant plasmid pEGKT316 - FGFR3 was digested with the restriction endonucleases BamHI and EcoRI.

[0053] ② The digestion products were analyzed by agarose gel electrophoresis to confirm the presence of two nucleic acid bands: 6640 bp (vector pEGKT316) and 2439 bp (FGFR3 gene) Figure 3 ). The 6640 bp vector backbone band was the same size as the original pEGKT316 plasmid, and the 2439 bp FGFR3 gene insertion fragment band confirmed the successful construction of the recombinant plasmid.

[0054] II. Preparation of recombinant yeast and protein expression

[0055] The proteins of recombinant yeast cells were extracted, and the expression of FGFR3 was detected by western blot. Since the expression product of FGFR3 carried a GST protein tag at the end after being inserted into the recombinant plasmid, a GST protein antibody was used to detect FGFR3. The detection results were as Figure 4 shown: Compared with the yeast transfected with the empty plasmid vector pEGKT316 (Vec), the expression of FGFR3 - GST could be detected in the proteins of the recombinant yeast transfected with the recombinant plasmid pEGKT316 - FGFR3. A specific band appeared at approximately 110 kDa in the experimental group (the theoretical molecular weight of FGFR3 - GST is 115 kDa), and no color - developed band was observed at this position in the control group, confirming the successful expression of the FGFR3 - GST fusion protein.

[0056] III. Experiment on the induction of DC cell maturation

[0057] 1. Experimental grouping

[0058] ① Blank control group: Immature DC cells were cultured routinely for 48 hours.

[0059] ② CD40L group: 1 μg / mL CD40L agonist was added.

[0060] ③ Control yeast group: Empty vector yeast was added at a ratio of DC:yeast = 1:10.

[0061] ④ Recombinant yeast group: Inactivated recombinant yeast was added at a ratio of DC:yeast = 1:10.

[0062] 2. Detection by flow cytometry

[0063] After culturing for 48 hours, the expression of DC surface markers (CD80, CD83, CD54, CD58, MHC I / II classes) was detected:

[0064] Results showed that: The proportion of positive cells such as CD80 (78.7%), CD83 (74.4%), and CD54 (99.9%) was significantly increased in the recombinant yeast group (Table 1).

[0065] Table 1 Expression of DC cell-induced maturation markers

[0066]

[0067] Results showed that: Compared with untreated DC cells (blank control group), the proportion of positive cells expressing CD80, CD83, CD54, and CD58 markers on the cell surface of DC treated with control yeast, recombinant yeast, and CD40L for 48 hours increased significantly; the proportion of positive cells such as CD80 (78.7%), CD83 (74.4%), and CD54 (99.9%) was significantly increased in the recombinant yeast group (Table 1). The proportion of positive cells expressing MHC class I and class II markers on the cell surface increased moderately. Moreover, the mean fluorescence intensity of positive cells expressing MHC class I and class II markers in the three groups of treated cells was significantly enhanced compared with the blank control group. It is suggested that co-culturing yeast with immature DC cells can effectively induce the latter to mature. Under the condition of a cell number ratio of DC:yeast = 1:10, the induction effect can be comparable to that of the agonist CD40L.

[0068] 3. Detection of cytokine secretion

[0069] The culture supernatant of the above four groups of cells after 48 hours of culture was collected, and the secretion levels of cytokines IL-8, IL-2, IL-4, IL-13, IL-5, IL-10, IL-1β, IL-12p70, TNF-α, and IFN-γ were detected (see Table 2). The secretion levels of IL-12p70 (5889 pg / mL), TNF-α (>10000 pg / mL), and IFN-γ (6457 pg / mL) in the recombinant yeast group were significantly higher than those in the control group. The results showed that the secretion levels of IL-12p70, TNF-α, IFN-γ, IL-8, and IL-2 in DC cells induced by control yeast and recombinant yeast were significantly higher than those in the blank control group and even DC cells induced by CD40L.

[0070] Table 2 Expression of cytokines in DC cells after induction

[0071]

[0072] IV. Treatment experiment on a mouse model of bladder cancer

[0073] 1. Model establishment

[0074] ① Use a 0.33 mm × 12.7 mm insulin needle to inject 1×106 One T24 cell expressing luciferase was injected into the bladder of BALB / c nude mice aged 10 - 12 weeks.

[0075] ② Seven days after culturing the mice, it was confirmed through the third-generation small animal in vivo optical platform whether the orthotopic tumors were successfully implanted.

[0076] 2. Group treatment

[0077] ① Control group: The bladder was perfused with untreated DC cells (1×10 5 ).

[0078] ② Experimental group: The bladder was perfused with DC cells induced by recombinant yeast (1×10 5 ).

[0079] 3. Efficacy evaluation

[0080] ① Tumor growth monitoring: The fluorescence intensity of the tumors was observed by in vivo imaging on the 7th, 14th, 21st, and 28th days. The growth of tumors in the experimental group was significantly inhibited ( Figure 5 ). On the 28th day of treatment, the tumor area was significantly smaller than that of the control group.

[0081] ② Survival rate analysis: Compared with the control group, DC cells induced by recombinant yeast could inhibit the growth rate of orthotopic tumors implanted in the bladders of mice and significantly prolong the survival period of mice in the orthotopic implantation model of bladder cancer (p < 0.05) ( Figure 6 ).

[0082] The above embodiments efficiently express the FGFR3 antigen through recombinant yeast, and verify its effect on inducing DC maturation and anti-bladder cancer, providing an experimental basis for vaccine development.

[0083] By adopting the above embodiments, the FGFR3 gene was amplified by designing specific primers, the pEGKT316-FGFR3 recombinant plasmid was constructed and transformed into the Saccharomyces cerevisiae YEF473A strain, and the vaccine was prepared through induced expression and heat inactivation. This vaccine can effectively activate the maturation of DC cells (the proportion of CD80+ cells reaches 78.7%), promote the secretion of Th1-type cytokines (IL-12p70 reaches 5889 pg / mL), and shows a significant tumor inhibitory effect in the orthotopic mouse model of bladder cancer (p < 0.05), providing a new specific treatment method for postoperative immunotherapy of bladder cancer.

[0084] The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. A recombinant yeast vaccine of human FGFR3 antigen, characterized in that: include: a) a recombinant Saccharomyces cerevisiae strain expressing FGFR3-GST fusion protein; b) The strain carries the pEGKT316-FGFR3 recombinant plasmid, which is cloned by BamHI and EcoRI The restriction site was inserted into the FGFR3 gene sequence shown in SEQ ID NO:

3.

2. The method for constructing a recombinant yeast vaccine according to claim 1, characterized in that: The following steps are involved: A) amplifying the FGFR3 gene using the primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2; B) The amplified product was double-digested with BamHI / EcoRI and then ligated into the pEGKT316 vector; C) transforming the recombinant plasmid into YEF473A Saccharomyces cerevisiae strain; D) Induction of FGFR3 protein expression in SRG-Ura medium; SEQ ID NO: 1: FGFR3-F1-BamHI forward primer, sequence: CGC GGATCC ATGGGCGCCCCTG, SEQ ID NO: 2: FGFR3-R1-EcoRI reverse primer, sequence: CCG GAATTC TCACGTCCGCGAG; the underlined part is: restriction endonuclease recognition site.

3. The method according to claim 2, characterized in that , the pEGKT316 vector contains a GST tag coding sequence, and the nucleotide sequence of the FGFR3 gene is shown in SEQ ID NO:

3.

4. The vaccine according to claim 1, characterized in that The brewer's yeast strain was inactivated by heating in a water bath at 100°C, and the concentration of the inactivated yeast was 1-10×10 8 CFU / mL.

5. Use of the vaccine according to claim 1 in the preparation of a drug for postoperative instillation treatment of bladder cancer, characterized in that The dosage of the drug is 1×10 7 CFU, administered once a week.

6. The use according to claim 5, characterized in that The therapeutic drug is administered through a bladder instillation catheter with an instillation volume of 0.1 and a retention time of 60-90 minutes.

7. The vaccine according to claim 1, characterized in that , which further comprises a pharmaceutically acceptable carrier selected from phosphate buffer, physiological saline or a stabilizer containing 0.1% human serum albumin.

8. Use of an inactivated FGFR3-expressing recombinant Saccharomyces cerevisiae cell in the preparation of a drug for bladder cancer immunotherapy.

9. The use according to claim 8, wherein the drug is administered by bladder instillation, can activate DC cells and increase the expression of CD80, CD5, MHC I molecules and cytokine secretion on their surfaces, thereby enhancing the body's ability to clear bladder tumor cells.

10. Use of the vaccine prepared by the method according to claim 2 in postoperative instillation therapy for bladder cancer.

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