CN117257926A - Recombinant subunit glioma vaccine and application thereof - Google Patents

Abstract

The invention discloses a recombinant subunit glioma vaccine and application thereof, and relates to the field of biological vaccines. The invention provides a recombinant subunit glioma vaccine, which comprises recombinant protein, tris-HCI buffer solution, a glychol stabilizer and NaCl; wherein, the recombinant protein comprises HCMV IE1 protein and HCMV IE1 mutant protein, the amino acid sequence of the IE1 protein is shown as SEQ ID NO.1, and the amino acid sequence of the IE1 mutant protein is shown as SEQ ID NO. 2. The invention also provides application of the vaccine in preparing tumor immunotherapy medicaments. The recombinant subunit glioma vaccine and the application thereof are adopted, the chemical synthesis difficulty is low, the high-purity product can be directly synthesized, the cost is reduced, and the therapeutic recombinant protein tumor vaccine with good application potential is provided.

Description

Recombinant subunit glioma vaccine and application thereof

Technical Field

The invention relates to the technical field of biological vaccines, in particular to a recombinant subunit glioma vaccine and application thereof.

Background

Gliomas are one of the common intracranial malignancies, accounting for 40% -60% of intracranial tumors. At present, the clinical treatment mainly comprises surgical excision, radiotherapy and chemotherapy, and is assisted by temozolomide first-line treatment drug treatment. The diffuse infiltration is difficult to be completely resected by surgery, and is one of the main obstacles for failure of treatment and tumor recurrence. Even though current research treatments have made great progress, the overall prognosis for diagnosed patients is poor, with median survival still less than 15 months. Human Cytomegalovirus (HCMV) is a ubiquitous, species-specific beta herpes virus, which, after primary infection, establishes a lifetime latency, which encodes one of two major viral gene products, the Immediate early (IE 1) protein, which is a key regulator of viral replication and host cell proliferation, playing a vital role in the pathogenesis of many diseases. It has been demonstrated that HCMV has an important relationship with gliomas, that the prognosis of HCMV seropositive patients is poor, and that IE1 protein expression of HCMV is found in tumor samples of glioma patients, and that IE1 is able to promote malignant progression of gliomas.

Therapeutic tumor vaccines are an effective and economical way of treating tumors, and various tumor vaccines are currently marketed, mainly for targeting tumor antigens, and concentrate the power of the human immune system on the elimination of tumor cells, thereby causing systemic regression of tumors and prolonging the survival time of patients. Existing immunotherapy (e.g., immune checkpoint inhibitors as well as chimeric antigen T cells, DC cells, etc.) can target intracellular antigens other than tumor-specific surface antigens, even elicit new tumor-specific T cell immunity. The application of tumor vaccines in the field of tumor treatment has now seen remarkable therapeutic effects. However, immune checkpoint inhibitor therapy has some problems in treatment, the first being immune adverse reactions that are prone to specific organs, the second being the presence of delayed drug toxic reactions, and the third being the choice of drug dosage; and studies have shown that immune checkpoint inhibitors are not effective against all grades of glioma. The chimeric antigen cell therapy has a series of problems of difficult clinical treatment implementation, complicated preparation and the like. Thus, further exploration is needed to find an effective, safe, mass-producible therapeutic tumor vaccine.

Disclosure of Invention

The invention aims to provide a recombinant subunit glioma vaccine and application thereof, has small chemical synthesis difficulty, can be directly synthesized to obtain a high-purity product, has low cost and has good application potential.

In order to achieve the aim, the invention provides a recombinant subunit glioma vaccine which comprises recombinant protein, tris-HCI buffer solution, glychol stabilizer and NaCl;

wherein the recombinant protein comprises HCMV IE1 protein and HCMV IE1 mutant protein.

Preferably, the amino acid sequence of the HCMV IE1 protein is shown as SEQ ID NO.1, and the amino acid sequence of the HCMV IE1 mutant protein is shown as SEQ ID NO. 2.

The invention also provides a preparation method of the vaccine, which comprises the following steps:

a. protein sequence analysis; performing transmembrane region analysis, signal peptide analysis, hydrophobicity analysis, disorder analysis and antigenicity analysis on the selected protein sequence;

b. gene synthesis and vector construction; designing primers according to the target gene sequence and cloning mode, amplifying a sufficient amount of PCR products by PCR, connecting the vector and the target gene by using ligase after enzyme digestion, and then converting;

c. protein expression.

Preferably, the specific steps of the conversion in step b are:

1) The DNA fragment to be transformed is added to a tube containing competent cells (50. Mu.l of competent cells require 25ng of DNA) in a volume of not more than 5% of the competent cells, the contents are gently mixed by rotating several times, and ice-bath is performed for 30min;

2) Placing the centrifuge tube mixture into circulating water heated to 42 ℃, and carrying out heat shock for 90 seconds without shaking;

3) Transferring the tube into ice bath to cool the cells for 1-2min;

4) 200 μl of SOC liquid medium is added to each tube, the medium is heated to 37 ℃ by water bath, then the tube is transferred to a shaking table set at 37 ℃ for culturing for 45min at 220rpm, so that cells are revived and the resistance marker gene encoded by the plasmid is expressed;

5) Transfer appropriate volumes (up to 200 μl per 90mm plate) of transformed competent cells to LB medium containing the corresponding antibiotics;

6) Inverting the plate, culturing at 37 ℃ for 12-16 hours, generating bacterial plaques, randomly picking a plurality of bacterial colonies when the bacterial colonies grow on the plate, carrying out bacterial colony PCR verification, detecting the transformant, and carrying out sequencing verification on positive clones.

Preferably, the specific steps of protein expression in step c are:

1) Competent transformation and positive clone screening;

2) Small expression and identification of positive clones;

3) Protein is expressed in a large quantity and detected;

4) Purifying inclusion body protein;

5) Renaturation of inclusion body proteins;

6) Purifying renaturation protein;

7) And (5) enzyme cutting.

The invention also provides application of the vaccine in preparation of tumor immunotherapy medicaments.

The recombinant subunit glioma vaccine and the application thereof have the advantages and positive effects that:

1. the recombinant subunit glioma vaccine of the invention can induce strong humoral immunity and cellular immune response.

2. The recombinant subunit glioma vaccine provided by the invention uses a conventional adjuvant, thereby bypassing the production and safety problems of a novel adjuvant and having good safety and immune protection.

3. After the recombinant subunit glioma vaccine is expressed in a large amount by a prokaryotic expression system, the recombinant subunit glioma vaccine is detected by SDS-PAGE through thallus crushing supernatant and precipitation, and the recombinant protein is prepared by highly purifying protein through a high performance liquid chromatography technology and diluting the protein with a diluent. The finished product can be stored for a long time at-80 ℃ and has the characteristics of good stability, stable production, easy amplification of yield, low cost and the like.

The technical scheme of the invention is further described in detail through the drawings and the embodiments.

Drawings

FIG. 1 shows the verification of the expression of IE1 and IE1-mut recombinant proteins in the invention, wherein a is the detection of the SDS-PAGE of IE1 protein expression, b is the detection of the Western Blot of IE1 protein expression, c is the detection of the SDS-PAGE of IE1-mut protein expression, and d is the detection of the Western Blot of IE1-mut protein expression;

FIG. 2 shows the humoral immunity detection induced by the recombinant proteins IE1 and IE1-mut in the invention, wherein a is the detection result of specific antibody IgM, and b is the detection result of specific antibody IgG;

FIG. 3 shows the cell immunity detection induced by the IE1 and IE1-mut recombinant proteins in the invention, wherein a is the detection result of specific CTL in spleen, and b is the detection result of specific CTL in lymph node;

FIG. 4 shows the therapeutic effect of IE1 and IE1mut recombinant proteins on glioma in the present invention.

Detailed Description

The technical scheme of the invention is further described below through the attached drawings and the embodiments.

Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.

Unless otherwise defined, the experimental materials used in the examples below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1 Synthesis preparation of IE1 protein and IE1mut protein

The recombinant protein is prepared and expressed by a prokaryotic expression system for efficiently preparing protein polypeptide according to the amino acid sequences of IE1 protein and IE1 mutant protein, and is obtained after transferring a plasmid vector into escherichia coli for culture and induction, crushing thalli to collect protein and highly purifying.

Wherein, the amino acid sequence of the IE1 recombinant protein (SEQ ID NO. 1) is as follows:

MESSAKRKMDPDNPDEGPSSKVPRPETPVTKATTFLQTMLRKEVNSQ

LSLGDPLFPELAEESLKTFEQVTEDCNENPEKDVLAELVKQIKVRVDMVRH

RIKEHMLKKYTQTEEKFTGAFNMMGGCLQNALDILDKVHEPFEEMKCIGL

TMQSMYENYIVPEDKREMWMACIKELHDVSKGAANKLGGALQAKARAK

KDELRRKMMYMCYRNIEFFTKNSAFPKTTNGCSQAMAALQNLPQCSPDEI

MAYAQKIFKILDEERDKVLTHIDHIFMDILTTCVETMCNEYKVTSDACMMT

MYGGISLLSEFCRVLCCYVLEETSVMLAKRPLITKPEVISVMKRRIEEICMK

VFAQYILGADPLRVCSPSVDDLRAIAEESDEEEAIVAYTLATAGVSSSDSLVS

PPESPVPATIPLSSVIVAENSDQEESEQSDEEEEEGAQEEREDTVSVKSEPVSE

IEEVAPEEEEDGAEEPTASGGKSTHPMVTRSKADQ。

the amino acid sequence of the IE1 mutant recombinant protein (SEQ ID No. 2) is as follows:

MESSAKRKMDPDNPDEGPSSKVPRPETPVTKATTFLQTMLRKEVNSQ

LSLGDPLFPELAEESLKTFEQVTEDCNENPEKDVLAELVKQIKVRVDMVRH

RIKEHMLKKYTQTEEKFTGAFNMMGGCLQNALDILDKVHEPFEEMKCIGL

TMQSMYENYIVPEDKREMWMACIKELHDVSKGAANKLGGALQAKARAK

KDELRRKMMYMCYRNIEFFTKNSAFPATTNGCSQAMAALQALPQCSPDEI

MAYAQKIFKILDEERDKVLTHIDHIFMDILTTCVETMCAEYKVTSDACMAT

MYGGISLLSEFCAVLCCYVLEETSVMLAKRPLITKPEVISVMKRRIEEICMK

VFAQYILGADPLRVCSPSVDDLRAIAEESDEEEAIVAYTLATAGVSSSDSLVS

PPESPVPATIPLSSVIVAENSDQEESEQSDEEEEEGAQEEREDTVSVRSEPVSE

IEEVAPEEEEDGAEEPTASGGKSTHPMVTRSKADQ。

the specific steps of vaccine preparation are as follows:

1. protein sequence analysis. And (3) performing transmembrane region analysis, signal peptide analysis, hydrophobicity analysis, disorder analysis and antigenicity analysis on the selected protein sequence.

2. And (5) gene synthesis and vector construction. Primers are designed according to the target gene sequence and cloning mode, and PCR products with enough quantity are amplified by PCR. The vector and the target gene are subjected to enzyme digestion and then are connected by using a ligase. Then transformation is performed.

The specific steps of the transformation are as follows:

1) The DNA fragment to be transformed was added to the tube containing competent cells (50. Mu.l competent cells required 25ng DNA) in a volume of not more than 5% of the competent cells, and the contents were gently mixed by rotating several times and ice-cooled for 30min.

2) The centrifuge tube mixture was placed in circulating water heated to 42 ℃ and heat-shocked for 90s without shaking.

3) The tube was quickly transferred to an ice bath and the cells were allowed to cool for 1-2 min.

4) 200 μl of SOC liquid medium was added to each tube, the medium was warmed to 37℃with a water bath, then the tube was transferred to a shaking table set at 37℃and cultured at 220rpm for 45min, the cells were resuscitated and plasmid-encoded resistance marker genes were expressed.

5) Appropriate volumes (up to 200. Mu.l per 90mm plate) of transformed susceptor cells were transferred to LB medium containing the corresponding antibiotics.

6) The plate was inverted and incubated at 37℃for 12-16h to allow plaque to appear. And (3) after colonies grow out on the flat plate, randomly picking a plurality of colonies, carrying out colony PCR verification, and detecting transformants. Positive clones were then sequenced.

3. Protein expression.

1) Competent transformation and positive clone selection

Taking out competent cells from the ultralow temperature refrigerator, melting on ice, adding plasmid, gently blowing and sucking, mixing thoroughly, and standing on ice for 30min; heat-shocking the water bath kettle at 42 ℃ for 90s, and placing the water bath kettle on ice for 1-2min; adding 800 μl of preheated LB liquid medium, and culturing at 37deg.C and 158rpm for 50-60min; centrifuging at 6000rpm for 4min, removing part of supernatant, re-suspending the rest bacterial liquid, and coating onto Carna resistance LB plate; single colonies (positive clones) were seen by inverting the plate and incubating for 12-16h at 37 ℃.

2) Positive clone miniexpression and identification

Selecting single colony containing recombinant plasmid into 5mL LB liquid culture medium (kana resistance), culturing at 37 ℃ overnight, and preserving at-20 ℃; then, single colony containing recombinant plasmid is selected to 5mL LB liquid medium (kana resistance) and shake cultured at 37 ℃ until OD ₆₀₀ About 0.6; taking 800ul of bacterial liquid as a control group, adding IPTG inducer (final concentration is 0.1 mM) into the residual bacterial liquid, and carrying out shake culture for 4 hours at 37 ℃; two sets of bacterial solutions were centrifuged at 0.15mL and 12000g for 2min, and the bacterial pellet was resuspended and lysed at 40. Mu.L of 1×loading buffer, and detected by 10. Mu.LSDS-PAGE.

3) Protein mass expression and detection

Inoculating 100 μl of strain stored at-20deg.C into 100mL LB liquid medium (kana resistance), shake culturing for 16 hr; inoculating 100mL of bacterial liquid into 2000mL of LB liquid culture medium, and performing amplification culture at 37 ℃ until reaching OD ₆₀₀ About 0.6, reducing the incubation temperature to 30 ℃; adding IPTG inducer to a final concentration of 0.1mM, and continuing shake culture at 30 ℃ for 8 hours; centrifuging at 8000rpm for 3min to collect thallus, re-suspending in 50mL pre-cooled NTA-0 buffer, adding lysozyme (final concentration 0.lmg/mL), and ice-bathing for 30min; ultrasonically crushing thalli, wherein parameters are set to be power of 200W, work for 3s, pause for 4s and time of 25-30min; centrifuging at 16000rpm for 50min at 4deg.C, separating supernatant and precipitate, and collecting supernatant and precipitate; 10ul of the supernatant and the precipitate were taken, respectively, and SDS-PAGE was performed, and the remaining supernatant and precipitate were kept at 4℃for further use.

4) Inclusion body protein purification

The pellet was resuspended in 50mL STET buffer and DTT was added to a final concentration of 1mM; ultrasonic promoting the dissolution of the hybrid protein, wherein parameters are set to be power 200W, work 3s, pause 3s and time 10min; centrifuging at 1000rpm and 4 ℃ for 10min, and removing supernatant; repeating the three steps until the supernatant is transparent; suspending the sediment by PBS, performing ultrasound, and setting parameters to be 200W in power, 3s in work, 3s in pause and 10min in time; centrifuging at 16000rpm and 4deg.C for 10min, and removing supernatant; the inclusion bodies were resuspended in 3mL 6M guanidine hydrochloride, and DTT was added to a final concentration of 5mM; shaking for 4 hours at 220rpm and 37 ℃ until the inclusion bodies are completely dissolved; centrifuging at 10000rpm at 4deg.C for 10min, and collecting supernatant. 10ul of the protein solution was taken for SDS-PAGE detection.

5) Inclusion body protein renaturation

Diluting the protein solution with 2 times of 3M guanidine hydrochloride, dropwise adding the diluted protein solution into 200mL renaturation solution (pH 8.0) by a syringe at the temperature of 4 ℃, regulating the rotating speed to the maximum, and stirring for 24h; reducing the rotating speed and stirring for 24 hours; taking a protein solution in a dialysis bag, and concentrating the protein solution to 50-100 mL by using PEG 20000; dialyzing with NTA-0 buffer solution at 4deg.C for 48 hr; taking a protein solution, and concentrating the protein solution to 10-20mL by using PEG 20000; dialysis was performed with NTA-0 buffer at 4℃for 48h.

6) Renaturation protein purification

Filtering the dialyzed protein solution with a 0.22um filter for later use; preparing a Ni-NTA column and loading a protein solution at a flow rate of 1 mL/min; washing the column with NTA-0 buffer (pH 8.0) until the effluent is free of protein (G250 detection solution does not change color); eluting with 20mM, 60mM, 200mM and 500mM imidazole respectively, and collecting eluate in sections until G250 detection solution is not discolored; washing column materials with 3 times of column volume of deionized water, and sealing the column with 20% ethanol; the collected eluate was concentrated by dialysis, and 10ul of the eluate was subjected to SDS-PAGE electrophoresis.

7) Enzyme cutting

The prepared recombinant protein and thrombin are digested in a mass ratio of 1:1000, namely, 1mg of target protein is added into 2U of enzyme (2000U/mg), and when the recombinant protein and thrombin are used, the enzyme is prepared into 100U/mL by using normal saline, and buffer solution is effectively digested: 20mM Tris-HCl, 150mM NaCl, pH8.0. Cutting at 20-37 deg.c for 0.3-16 hr. And then purifying and dialyzing and concentrating by column affinity to obtain the target protein. 10ul of the sample was subjected to SDS-PAGE.

Example 2 verification of expression of IE1 and IE1-mut recombinant proteins

And (3) processing the purified protein, preparing a sample, running gel, detecting the molecular weight and confirming IE1 protein, wherein the verification result is shown in figure 1.

As can be seen from FIG. 1, we successfully expressed recombinant proteins IE1 and IE1-mut using the prokaryotic system.

Example 3IE1 and IE1-mut recombinant protein-induced humoral immunity detection

In the case of equivalent amounts of IE1 recombinant protein (30 μg/dose), MF59 adjuvant analogues were prepared at 50 μl in combination with phosphate buffer (phosphate buffer saline, PBS) to a volume of 100 μl/dose, and were intramuscular injected into 6-8 week old female mice of C57BL/6 strain, each with 0.1mL intramuscular thigh, with 6-8 animals per group, for a total of three groups (PBS blank, IE1 mut). The same dosage form is injected into the same part every 14 days, 3 needles are continuously immunized, the blood is collected from the orbit on the 14 th day after the last immunization, the blood is stood at room temperature for solidification, and is centrifuged for 10min at the temperature of 2-8 ℃ at 4000rpm, and the serum of the mice is taken to detect the generation condition and titer of specific antibodies IgM and IgG by adopting an indirect ELISA method. The detection results are shown in FIG. 2.

As can be seen from FIG. 2, both IE1 and IE1-mut have good immunogenicity, and can excite the organism to generate strong specific humoral immune response.

Example 4IE1 and IE1-mut recombinant protein-induced cellular immunodetection

In the case of equivalent amounts of IE1 recombinant protein (30 μg/dose), MF59 adjuvant analogues were prepared at 50 μl in combination with phosphate buffer (phosphate buffer saline, PBS) to a volume of 100 μl/dose, and were intramuscular injected into 6-8 week old female mice of C57BL/6 strain, each with 0.1mL intramuscular thigh, with 6-8 animals per group, for a total of three groups (PBS blank, IE1 mut). After 3 needles are continuously immunized every 14 days by injecting one dose of the same dosage form at the same position, spleens (spleens) and draining lymph nodes (cLN) of immunized mice are aseptically extracted from the 14 th day after the last immunization to prepare single-cell suspension, after red blood cells are removed by using red blood cell lysate, cells are stained by using corresponding flow antibodies, and then lymphocytes, mainly cytotoxic T Cells (CTL) are detected on the machine. The results are shown in FIG. 3.

As can be seen from FIG. 3, after three immunizations IE1 and IE1-mut, the body stimulated the immune organ to generate a powerful cellular immune response, in particular a specific cytotoxic T cell immune response. Provides a powerful guarantee for IE1 and IE1-mut as anti-tumor vaccine.

Example 5 detection of the Effect of IE1 and IE1-mut recombinant proteins on glioma treatment

In the case of equivalent amounts of IE1 recombinant protein (30 μg/dose), MF59 adjuvant analogues were prepared at 50 μl in combination with phosphate buffer (phosphate buffer saline, PBS) to a volume of 100 μl/dose, and were intramuscular injected into female tumor bearing mice of 6-8 week old C57BL/6 strain, each with 0.05mL intramuscular injection of two forelegs each, each with 6-8 total groups (PBS blank, IE1 experimental, IE1-mut experimental). The growth of intracranial tumors in tumor-bearing mice was observed after 3 needles of immunotherapy using a small animal in vivo imaging technique, and representative results were shown. The results are shown in FIG. 4.

As can be seen from fig. 4, the brain tumor of mice in the IE1 and IE1-mut groups is significantly smaller than the tumor size of the PBS control group after receiving different immunotherapeutic treatments in the tumor-bearing mice under the same conditions, indicating that our IE1 and IE1-mut proteins have better immunotherapeutic effects on gliomas. Therefore, the recombinant subunit glioma vaccine and the application thereof are adopted, the chemical synthesis difficulty is low, the high-purity product can be directly synthesized, the cost is reduced, and the therapeutic recombinant protein tumor vaccine with good application potential is provided.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.

Claims (6)

1. A recombinant subunit glioma vaccine characterized by: comprises recombinant protein, tris-HCI buffer solution, glychol stabilizer and NaCl;

wherein the recombinant protein comprises HCMV IE1 protein and HCMV IE1 mutant protein.

2. The vaccine of claim 1, wherein: the amino acid sequence of the HCMV IE1 protein is shown as SEQ ID NO.1, and the amino acid sequence of the HCMV IE1 mutant protein is shown as SEQ ID NO. 2.

3. A method of preparing a vaccine according to claim 1 or 2, comprising the steps of:

a. protein sequence analysis; performing transmembrane region analysis, signal peptide analysis, hydrophobicity analysis, disorder analysis and antigenicity analysis on the selected protein sequence;

b. gene synthesis and vector construction; designing primers according to the target gene sequence and cloning mode, amplifying a sufficient amount of PCR products by PCR, connecting the vector and the target gene by using ligase after enzyme digestion, and then converting;

c. protein expression.

4. A process according to claim 3, wherein the specific step of conversion in step b is:

1) The DNA fragment to be transformed is added to a tube containing competent cells (50. Mu.l of competent cells require 25ng of DNA) in a volume of not more than 5% of the competent cells, the contents are gently mixed by rotating several times, and ice-bath is performed for 30min;

2) Placing the centrifuge tube mixture into circulating water heated to 42 ℃, and carrying out heat shock for 90 seconds without shaking;

3) Transferring the tube into ice bath to cool the cells for 1-2min;

4) 200 μl of SOC liquid medium is added to each tube, the medium is heated to 37 ℃ by water bath, then the tube is transferred to a shaking table set at 37 ℃ for culturing for 45min at 220rpm, so that cells are revived and the resistance marker gene encoded by the plasmid is expressed;

5) Transfer appropriate volumes (up to 200 μl per 90mm plate) of transformed competent cells to LB medium containing the corresponding antibiotics;

6) Inverting the plate, culturing at 37 ℃ for 12-16 hours, generating bacterial plaques, randomly picking a plurality of bacterial colonies when the bacterial colonies grow on the plate, carrying out bacterial colony PCR verification, detecting the transformant, and carrying out sequencing verification on positive clones.

5. A method according to claim 3, wherein the specific steps of protein expression in step c are:

1) Competent transformation and positive clone screening;

2) Small expression and identification of positive clones;

3) Protein is expressed in a large quantity and detected;

4) Purifying inclusion body protein;

5) Renaturation of inclusion body proteins;

6) Purifying renaturation protein;

7) And (5) enzyme cutting.

6. Use of the vaccine of claim 1 or 2 in the manufacture of a medicament for tumour immunotherapy.