CN109568574B - Application of sPD1 protein and/or sPD1 gene as immune adjuvant - Google Patents
Application of sPD1 protein and/or sPD1 gene as immune adjuvant Download PDFInfo
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- CN109568574B CN109568574B CN201710899157.6A CN201710899157A CN109568574B CN 109568574 B CN109568574 B CN 109568574B CN 201710899157 A CN201710899157 A CN 201710899157A CN 109568574 B CN109568574 B CN 109568574B
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Abstract
The invention belongs to the field of biological medicines, and particularly relates to application of sPD1 protein and/or sPD1 gene as an immunologic adjuvant in preparation of recombinant HPV vaccine. In the application, the sPD1 protein contains an amino acid sequence shown as SEQ ID NO. 3, or an amino acid sequence with the same function obtained by deleting, inserting or replacing the amino acid sequence shown as SEQ ID NO. 3; the sPD1 gene contains a nucleotide sequence shown as SEQ ID NO. 1 or a nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown as SEQ ID NO. 1. The sPD1 used as an immunologic adjuvant enhances the capacity of the HPV vaccine to stimulate the organism to produce antibodies, induce the organism to produce humoral immune response, stimulate lymphocytes to secrete IFN-gamma and stimulate the organism to produce cellular immune response.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of sPD1 protein and/or sPD1 gene as an immunoadjuvant.
Background
An immunoadjuvant, an adjuvant for short, is an immunopotentiator that, when injected or pre-injected into the body with an antigen, enhances the body's immune response to the antigen or alters the type of immune response. Adjuvants exert an immunopotentiating effect mainly through the following mechanism of action: 1) the degradation and elimination of the antigen are delayed, the half-life period of the antigen is improved, and the immune system is stimulated more effectively; 2) stimulating the monocyte-phagocyte system, enhancing its ability to process and present antigens; 3) stimulating the proliferation and differentiation of lymphocytes, and improving the antibody titer of the primary and secondary immune responses of the organism; 4) change the type of antibody production and produce delayed type allergy. Adjuvants can be divided into two major groups: an immunogenic substance, such as Mycobacterium tuberculosis and Bordetella pertussis; and the other is not immunogenic per se, such as aluminum hydroxide, mineral oil emulsion, surfactant and the like.
With the development of basic theories of life science, particularly the continuous development of immunology, virology, infectious disease and genetic engineering technologies, the research of modern vaccinology is changing day by day, the scientific research is deep, the development of novel vaccines is carried out, and novel immunologic adjuvants are urgently needed. Compared with the traditional vaccine, the novel vaccine mainly consists of genetic engineering recombinant antigen or chemically synthesized polypeptide, has the problems of antigen presentation, antigen expression, weak vaccine immunoreactivity and the like, and also urgently needs the research of a novel immunologic adjuvant. The novel immunologic adjuvant is required to be high in safety, break the immune tolerance of the organism and stimulate stronger humoral and cellular immune responses, and is more suitable for novel vaccines such as mucosal vaccines, DNA vaccines, RNA vaccines and tumor therapeutic vaccines. The currently developed novel immune adjuvants, such as cytokine interleukins 1, 2 and 12, CpG oligodeoxynucleotide (CpG-ODN), liposome adjuvant and heat shock protein adjuvant, are all used for directly enhancing the immune response of the organism; however, many viral diseases and tumors are now immune suppressive, resulting in the activation of T lymphocytes, which is suppressed by the activation of immune suppressive signaling pathways, and thus immune tolerance, no response or weak immune response to virus-infected cells.
Conventional adjuvants, such as Freund's adjuvant, are currently most widely used in experimental animals, but are not suitable for human use; the aluminum salt adjuvant is the only approved and safe human adjuvant at present, but fails to stimulate the generation of effective immune response when being used together with a novel polypeptide vaccine, and has limited application process in the novel vaccine. The existing novel immunologic adjuvant which is produced by the synergy with the novel vaccine directly enhances the immune response of the organism from the positive direction, and has little effect on viral diseases and tumors which are generated by dominating the immunosuppressive signal path in the organism and have the inhibited T cell activation.
Programmed death molecule 1 (PD 1) and its ligand (PDL) belong to the family B7 of costimulatory molecules. CD4+ T cells, CD8+ T cells, NKT cells, B cells and monocytes all inducibly express the PD1(CD279) molecule upon activation. PDL1 protein molecule is hardly expressed in normal tissues, but is generally present on the surfaces of various tumor cells of human breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer, bladder cancer, pancreatic cancer, glioma, melanoma and the like. PDL1 on the surface of tumor cells can induce tumor-specific CTL apoptosis, thereby inhibiting the immune response of organisms to tumors; whereas in lymphoid organs PDL1 on the surface of antigen presenting cells interacts with naive T lymphocytes to induce anergy of T lymphocytes. Therefore, PDL1 is an important molecule involved in tumor immune escape, and blocking PD1/PDL1 can inhibit tumor immune escape, improve the activation capability of initial T cells and the killing activity of CTL, and further improve the immune response of antigens.
The PD1 gene competes for PDL binding, thereby blocking the PD1/PDL signaling pathway. Inactivation of the PD1/PDL signaling pathway restores T cell proliferation, differentiation, and cytokine secretion as well as T cell effector functions, thereby enhancing the cellular immune response of the body. In addition to being involved in the above-mentioned cellular immune regulation, the PD1/PDL signaling pathway is also capable of regulating the production of antibodies by plasma cells. B cells can be activated by either antigenic peptides that directly recognize APC cells or by helper T cells (Tfh) in combination with co-stimulatory molecules. Therefore, the blocking of a PD1/PDL signal channel can promote the activation of B cells, the secretion of cytokines and the generation of specific IgG and IgM, and enhance the humoral immune response of the body; plays an important role in immunity against viral infection.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides application of an sPD1 protein and/or an sPD1 gene as an immunologic adjuvant, and corresponding recombinant protein vaccines, recombinant DNA vaccines and recombinant RNA vaccines, and aims to solve the technical problem that the existing HPV related diseases and tumors are prevented and the selection of a therapeutic vaccine X is limited.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides an application of sPD1 protein and/or sPD1 gene as an immunoadjuvant in preparing recombinant HPV vaccine; wherein,
the sPD1 protein contains an amino acid sequence shown as SEQ ID NO. 3, or an amino acid sequence which is obtained by deleting, inserting or replacing the amino acid sequence shown as SEQ ID NO. 3 and has the same function;
the sPD1 gene contains a nucleotide sequence shown as SEQ ID NO. 1 or a nucleotide sequence which is obtained by deleting, inserting or replacing the nucleotide sequence shown as SEQ ID NO. 1 and has the same function.
Further, the recombinant HPV vaccine comprises at least one of a recombinant DNA vaccine, a recombinant RNA vaccine and a recombinant protein vaccine.
Correspondingly, the invention provides a recombinant protein vaccine, which comprises PD1 protein and mE6E7 protein, wherein the sPD1 protein contains an amino acid sequence shown as SEQ ID NO. 3, or an amino acid sequence with the same function obtained by deleting, inserting or replacing the amino acid sequence shown as SEQ ID NO. 3; the mE6E7 protein contains an amino acid sequence shown as SEQ ID NO. 4, or an amino acid sequence with the same function obtained by deletion, insertion or substitution of the amino acid sequence shown as SEQ ID NO. 3.
Correspondingly, the invention provides a recombinant DNA vaccine, which comprises an sPD1 gene and an mE6E7 gene, wherein the sPD1 gene contains a nucleotide sequence shown as SEQ ID NO. 1, or a nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown as SEQ ID NO. 1; the mE6E7 gene contains a nucleotide sequence shown as SEQ ID NO. 2, or a nucleotide sequence with the same function obtained by deletion, insertion or substitution of the nucleotide sequence shown as SEQ ID NO. 1.
Accordingly, the present invention is a recombinant RNA vaccine that translates sPD1 protein and mE6E7 protein in the recombinant protein vaccine described above.
On the other hand, the invention also provides a recombinant plasmid, which contains pcDNA3.1(+) plasmid sequence and sPD1 gene sequence and mE6E7 gene sequence in the recombinant DNA vaccine.
In another aspect, the present invention also provides a genetically engineered host cell comprising the above recombinant plasmid.
On the other hand, the invention also provides a preparation method of the recombinant protein vaccine, which comprises the step of transfecting host cells by the recombinant plasmid.
Finally, the invention also provides a preparation method of the recombinant RNA vaccine, which comprises the following steps:
providing the recombinant plasmid;
transcribing the recombinant plasmid into RNA in vitro.
Further, the preparation process of the recombinant plasmid comprises the following steps:
artificially synthesizing an sPD1 gene sequence and an mE6E7 gene sequence, and connecting the sPD1 gene sequence and the mE6E7 gene sequence with pcDNA3.1(+) plasmid;
and (3) transfecting the connected product into a host cell for culturing to obtain a positive cloning recombinant expression vector.
The sPD1 protein and/or the sPD1 gene provided by the invention are/is used as an immunologic adjuvant in the preparation of recombinant HPV vaccine, and the sPD1 is used as the immunologic adjuvant to enhance the capacity of the HPV vaccine for stimulating the organism to generate antibody, inducing the organism to generate humoral immune response, stimulating lymphocytes to secrete IFN-gamma and stimulating the organism to generate cellular immune response. The invention starts from blocking an immunosuppressive signal path, and develops a novel immunologic adjuvant, thereby enhancing the immunogenicity of the vaccine, overcoming the immunologic escape of viruses and virus infected cells, inducing the cellular immunity of target cells, and improving the safety and specificity of the vaccine. Combines vaccine and immune regulation, and particularly provides sPD1 as an immune adjuvant to be combined with HPV E6E7 gene to prepare recombinant HPV E6E7 vaccine, including recombinant HPV protein vaccine, recombinant HPV DNA vaccine and recombinant HPVRNA vaccine. In addition, the present invention provides a recombinant plasmid: pcDNA3.1(+) -sPD1-mE6E7, which is used for preparing recombinant RNA vaccine aiming at developing more efficient preventive and therapeutic vaccines for HPV related diseases and tumors.
Drawings
FIG. 1 is an electrophoresis chart showing the expression of mE6E7 protein and sPD1-mE6E7 protein in example 1 of the present invention;
FIG. 2 is a graph comparing the titer of E6-specific antibodies in serum of immunized mice in example 3 of the present invention;
FIG. 3 is a graph showing the comparison of IFN-. gamma.secretion levels of lymphocytes from immunized mice in example 4 of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In one aspect, the embodiment of the invention provides an application of sPD1 protein and/or sPD1 gene as an immune adjuvant in preparing recombinant HPV vaccine; wherein, the sPD1 protein contains an amino acid sequence shown as SEQ ID NO. 3, or an amino acid sequence which is obtained by deleting, inserting or replacing the amino acid sequence shown as SEQ ID NO. 3 and has the same function; the sPD1 gene contains a nucleotide sequence shown as SEQ ID NO. 1 or a nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown as SEQ ID NO. 1.
The sPD1 used as an immunologic adjuvant enhances the capacity of the HPV vaccine to stimulate the organism to produce antibodies, induce the organism to produce humoral immune response, stimulate lymphocytes to secrete IFN-gamma and stimulate the organism to produce cellular immune response. The invention starts from blocking an immunosuppressive signal path, and develops a novel immunologic adjuvant, thereby enhancing the immunogenicity of the vaccine, overcoming the immunologic escape of viruses and virus infected cells, inducing the cellular immunity of target cells, and improving the safety and specificity of the vaccine. The vaccine is combined with immune regulation, and the recombinant HPV vaccine prepared by combining the sPD1 as an immune adjuvant and the HPV E6E7 gene comprises at least one of recombinant DNA vaccine, recombinant RNA vaccine and recombinant protein vaccine.
Accordingly, the embodiment of the invention provides a recombinant protein vaccine, which comprises PD1 protein and mE6E7 protein, wherein the sPD1 protein contains an amino acid sequence shown as SEQ ID NO. 3, or an amino acid sequence with the same function obtained by deleting, inserting or replacing the amino acid sequence shown as SEQ ID NO. 3; the mE6E7 protein contains an amino acid sequence shown as SEQ ID NO. 4, or an amino acid sequence with the same function obtained by deletion, insertion or substitution of the amino acid sequence shown as SEQ ID NO. 3. In another embodiment of the invention, a recombinant DNA vaccine is provided, which comprises an sPD1 gene and an mE6E7 gene, wherein the sPD1 gene contains a nucleotide sequence shown as SEQ ID NO. 1, or a nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown as SEQ ID NO. 1; the mE6E7 gene contains a nucleotide sequence shown as SEQ ID NO. 2, or a nucleotide sequence with the same function obtained by deletion, insertion or substitution of the nucleotide sequence shown as SEQ ID NO. 1. Namely, the nucleotide sequence of the recombinant DNA vaccine encodes PD1 protein and mE6E7 protein in the recombinant protein vaccine. In another embodiment of the present invention, there is provided a recombinant RNA vaccine that translates the sPD1 protein and mE6E7 protein in the above-described recombinant protein vaccines of the embodiments.
On the other hand, the embodiment of the invention also provides a recombinant plasmid, which contains a pcDNA3.1(+) plasmid sequence, and an sPD1 gene sequence and an mE6E7 gene sequence in the recombinant DNA vaccine. The recombinant plasmid can be used for preparing recombinant RNA vaccines, and aims to develop more efficient preventive and therapeutic vaccines for HPV related diseases and tumors.
In another aspect, the embodiments of the present invention further provide a genetically engineered host cell, which comprises the recombinant plasmid.
In another aspect, the present invention also provides a method for preparing a recombinant protein vaccine, comprising the step of transfecting host cells with the recombinant plasmid. Specifically, expression identification is carried out after the recombinant plasmid is transfected into 293T cells, and the specific operation method is as follows: transfecting endotoxin-free pcDNA3.1(+) -sPD1-mE6E7 plasmid to 293T cells with the growth density of 80% according to a conventional laboratory molecular cloning method, culturing the cells in a 10% FBS DEME culture medium at the temperature of 5% CO2 and 37 ℃ for 48 hours, separating and collecting cell culture supernatant and cells, analyzing and identifying the expression of sPD1-mE6E7 fusion protein in the cells and the cell culture supernatant by using an E6 monoclonal antibody by using Western Blot, performing SDS-PAGE electrophoresis and the Western Blot method according to a molecular cloning experimental guideline, wherein the concentration of a concentrated gel is 5%, the concentration of a separation gel is 12%, the constant pressure of the concentrated gel is 80V, and the constant pressure of the separation gel is 120V for about 2 hours; carrying out constant pressure of 100V in a membrane conversion buffer solution system, carrying out membrane conversion for 90 minutes, sequentially adding an E6 monoclonal antibody after sealing, washing, and adding a horse radish peroxidase-labeled goat anti-mouse IgG antibody for Western Blot identification; the sPD1-mE6E7 fusion protein is obtained by screening, and the amino acid sequence of the sPD1-mE6E7 fusion protein is shown as SEQ ID NO:4, respectively. The sPD1 protein is used as an immunologic adjuvant to be combined with an HPV protein vaccine (E6E7 antigen) to obtain a recombinant HPV protein vaccine.
Finally, the invention also provides a preparation method of the recombinant RNA vaccine, which comprises the following steps:
s01: providing the recombinant plasmid;
s02: the above recombinant plasmid was transcribed into RNA in vitro.
Further, the preparation process of the recombinant plasmid comprises the following steps: artificially synthesizing an sPD1 gene sequence and an mE6E7 gene sequence, and connecting the sPD1 gene sequence and the mE6E7 gene sequence with pcDNA3.1(+) plasmid; and (3) transfecting the connected product into a host cell for culturing to obtain a positive cloning recombinant expression vector. The host cell can be cultured, and the positive clone recombinant expression vector pcDNA3.1(+) -sPD1-mE6E7 plasmid can be obtained through enzyme digestion identification. Specifically, when the sPD1 gene is fused with the HPV E6E7 gene and then is combined with pcDNA3.1(+) plasmid to construct a recombinant expression vector, the ratio range of the dosage of the pcDNA3.1(+) plasmid to the dosage of the virus DNA vaccine can be 1: 1-10: 1; in one embodiment, 1: 1. The recombinant plasmid pcDNA3.1(+) -sPD1-mE6E7 is used for preparing recombinant RNA vaccine aiming at developing more efficient preventive and therapeutic vaccines for HPV related diseases and tumors.
The invention is shown in SEQ ID NO: 1-4 are as follows:
SEQ ID NO:1:
5`-CCCGCCGCCACCATGTTTCAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTATGCACAGAGCTGCAAACAACTATACATGATATAATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTGCTTTTCGGGATTTATGCATAGTATATAGAGATGGGAGTCCATATGCTGTAGGAGATAAATGTTTAAAGTTTTATTCTAAAATTAGTGAGTATAGACATTATTGTTATAGTTTGTATGGAACAACATTAGAACAGCAATACAACAAACCGTTGTGTGATTTGTTAATTAGGTGTATTAACGGACAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGATTCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCCAGCTGTAAATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACGGATATGGACAATTAAGTGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAGCCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGGGACCCATCTGTTCCCAGAAACCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAA-3';
SEQ ID NO:2:
5`-CCCGCCGCCACCATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAATTTCAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTATGCACAGAGCTGCAAACAACTATACATGATATAATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTGCTTTTCGGGATTTATGCATAGTATATAGAGATGGGAGTCCATATGCTGTAGGAGATAAATGTTTAAAGTTTTATTCTAAAATTAGTGAGTATAGACATTATTGTTATAGTTTGTATGGAACAACATTAGAACAGCAATACAACAAACCGTTGTGTGATTTGTTAATTAGGTGTATTAACGGACAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGATTCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCCAGCTGTAAATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACGGATATGGACAATTAAGTGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAGCCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGGGACCCATCTGTTCCCAGAAACCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAA-3';
SEQ ID NO:3:
MFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGSPYAVGDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINGQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL;
SEQ ID NO:4:
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGSPYAVGDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINGQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL。
the invention is described in further detail with reference to a part of the test results, which are described in detail below with reference to specific examples.
Example 1
The recombinant expression vectors pcDNA3.1(+) -mE6E7 and pcDNA3.1(+) -sPD1-mE6E7 are transfected into 293T cells and expressed and identified.
First, the endotoxin-free pcDNA3.1(+) -mE6E7 or pcDNA3.1(+) -sPD1-mE6E7 recombinant plasmid was transfected into 293T cells that had grown to a density of 80% in 5% CO by the conventional method of laboratory molecular cloning2Culturing in 10% FBS DEME medium at 37 deg.C for 48 hr, and separatingCell culture supernatants and cells were isolated and collected. Then, using E6 monoclonal antibody (purchased commercially and branded as Abcam), adopting WesternBlot analysis and identifying the expression of mE6E7 protein and sPD1-mE6E7 fusion protein in cells and cell culture supernatant, performing SDS-PAGE electrophoresis and Western Blot method according to molecular cloning experimental guidelines, wherein the concentration of concentrated gel is 5%, the concentration of separation gel is 12%, the concentration of concentrated gel is 80V at constant pressure, and the separation gel runs for about 2 hours at constant pressure of 120V; carrying out constant pressure of 100V in a membrane conversion buffer solution system, carrying out membrane conversion for 90 minutes, sequentially adding an E6 monoclonal antibody after sealing, washing, and adding a horse radish peroxidase-labeled goat anti-mouse IgG antibody for Western Blot identification; the screening resulted in the fusion protein mE6E7 and sPD1-mE6E7, the results of which are shown in FIG. 1.
FIG. 1 is the electrophoresis chart of mE6E7 protein and sPD1-mE6E7 fusion protein. In FIG. 1, the left lane is the electrophoresis diagram of the protein expressed by the pcDNA3.1(+) -mE6E7 plasmid after transfecting 293T cell, and the right lane is the electrophoresis diagram of the protein expressed by the pcDNA3.1(+) -sPD1-mE6E7 plasmid after transfecting 293T cell, and the amino acid sequences of the mE6E7 protein and the sPD1-mE6E7 fusion protein are respectively shown in SEQ ID NO: 3. SEQ ID NO:4, respectively.
Example 2
The effect of PD1 on HPV E6E7RNA vaccine immunoadjuvant was verified.
The pcDNA3.1(+) -sPD1-mE6E7 plasmid containing the sPD1 gene coding sequence and the pcDNA3.1(+) -mE6E7 plasmid not containing the sPD1 coding sequence are constructed according to the method described in the specification, the two plasmids are transcribed into RNA in vitro to obtain the corresponding RNA vaccines, and the specific inoculation scheme for the mice is shown in Table 1:
TABLE 1
The HPVME6E7RNA vaccine group and the sPD1+ mE6E7RNA vaccine group are all four immunization injection needles, the vaccine is injected through muscle firstly, then the NEPA GENE animal electrotransfer instrument is used for electrical transfer stimulation, under the voltage of 80V, the pulse is carried out for 6 times, the pulse time interval of each time is 25ms, the pulse frequency is 2Hz, and the immunization efficiency of the RNA vaccine is improved through the electrical transfer stimulation of the animal electrotransfer instrument. The four needles were injected at week 0, week 2, week 4 and week 6, week 7, and the mice were sacrificed to detect specific cellular and humoral immune responses.
Example 3
Comparing the humoral immune response detection after the in vivo immunization of the mice of the example 2, the method specifically comprises the following steps:
1) serum preparation: about 100. mu.L of blood was collected from the orbit of the mouse at weeks 3, 5 and 7 of the immunized mouse, the blood sample was allowed to stand at room temperature for 1 hour, serum was centrifuged, and the serum sample was stored at-20 ℃ for measurement.
2) Detection of E6, E7 antibody in serum:
in this example, an antibody detection kit (commercially available under the brand of Ssnta Cruz) for E6 or E7, which is prepared by coating purified human papillomavirus E6 or E7 protein as a detection antigen on an enzyme label plate, is used to detect the humoral immune response of the RNA vaccine induced by the level of the specific antibody of the experimental animal.
The specific operation steps are as follows:
a. adding 50 mu L of sample to be detected into each hole of the kit, setting 3 holes of negative control and positive control respectively, adding 50 mu L of negative control (or positive control) into each hole, and setting 3 holes of blank control;
b. adding 50 μ L of enzyme conjugate (horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody) to each well (except for blank control wells), mixing well, sealing, and incubating at 37 deg.C for 30 min;
c. washing the plate: discarding the liquid in the wells, filling each well with washing solution (20mM phosphate buffer solution containing 0.05% Tween 20), standing for 5 s, spin-drying, repeating for 3 times, and patting to dry;
d. adding color developing agent A solution (H) into each hole2O2) Mixing with developer B (tetramethylbenzidine TMB) 50 μ L, sealing, and incubating at 37 deg.C for 15 min;
e. stop solution (2M H) was added to each well2SO4Solution) 50 μ L, mix well;
f. reading by a microplate reader, taking the wavelength of 450nm, calibrating to zero by using blank holes, and then reading the OD value of each hole.
The experimental results are shown in FIG. 2, and FIG. 2 is a graph comparing the titer of E6 or E7 specific antibodies in the serum of immunized mice. The experimental results in fig. 2 show that the recombinant sPD1+ mE6E7RNA vaccine is more effective in stimulating the production of E6 and E7 antibodies and inducing stronger humoral immune response than the E6E7RNA vaccine alone.
Example 4
Comparing the cellular immune response assay after in vivo immunization of the mice of example 2, specifically comprising the steps of:
1) preparation of mouse lymphocytes:
a. after the mice immunized in the example 2 are killed, the mice are soaked in 75% alcohol for disinfection and then are operated under the condition of a sterile super clean workbench;
b. taking out the spleen of a mouse under the aseptic condition, putting the spleen into a DMEN serum-free culture medium, grinding and dispersing splenocytes, then transferring the spleen into a 15mL aseptic centrifuge tube, and centrifuging the spleen for 5min under the conditions of 4 ℃ and 1000 rpm;
c. the supernatant was discarded and 3mL (1.3 g Tris, NH) of 4 ℃ precooled erythrocyte lysate was added4Cl 3.55g, diluting to a constant volume of 500mL, adjusting pH to 7.2, autoclaving, storing at 4 ℃ for later use), cracking for 3min, and then centrifuging for 5min at 4 ℃ and 1000 rpm;
d. discarding the supernatant, after resuspending the cells, adding 10mL of culture medium to wash the cells, and centrifuging for 5min at 4 ℃ and 1000 rpm;
e. discarding supernatant, resuspending cells, adding 5mL complete culture medium containing 10% fetal calf serum, mixing cells, counting cells in cell counter, and adjusting cell concentration to 1 × 10 according to the obtained cell number7/mL;
f. Setting experiment group, positive control group and negative control group, each group has 3 multiple wells, and adding 50 μ L prepared lymphocytes into each well to obtain a total volume of 200 μ L (total cell number of each well is 5 × 10)5) (ii) a The experimental group was added with E6 or E7 protein at a final concentration of 1. mu.g/mL, the positive control was added with no protein, ConA (concanavalin A) at a final concentration of 1. mu.g/mL, the negative control was added with no protein, and the complete culture medium was added. CO at 37 deg.C2The culture was carried out in an incubator for 36 hours.
2) ELISA for detecting immune response level of spleen lymphocytes
The IFN-gamma detection kit (the cargo number: DKW12-2000) of Dake biotechnology limited company is used for detecting the secretion level of the cell factors of the splenocytes of the immune mice after being stimulated by specific antigens.
The specific operation steps are as follows:
a. sample adding: adding 100 μ L of IFN- γ standard in gradient dilution (500,250,125,62.5,31.25,15.625,7.5825,0pg/mL) to each well of the kit, adding 100 μ L of lymphocyte culture supernatant to each well of the sample wells, and setting blank wells, positive and negative control wells;
b. adding a detection antibody: adding 50 mu L of biotin-labeled antibody into each hole, uniformly mixing, covering a sealing plate membrane, and incubating for 90 minutes at 37 ℃;
c. washing the plate: the well contents were removed and 300. mu.L of wash solution (20mM phosphate buffer, 0.05% Tween 20) was added to each well; standing for 1 min, discarding the liquid in the hole, repeating for 4 times, and finally patting the liquid in the hole;
d. adding an enzyme: adding 100 mu L of horseradish peroxidase-labeled streptomycin into each well, covering a sealing plate membrane, and incubating for 30 minutes at 37 ℃;
e. washing the plate: repeating the step c;
f. color development: adding 100 mu L of color development liquid TMB (tetramethylbenzidine) into each hole, incubating for 5-30 minutes at 37 ℃ in a dark place, and judging to stop reaction according to the color depth in the holes;
g. and (3) terminating the reaction: add 100. mu.L of stop solution (2M H) to each well2SO4Solution) to terminate the reaction;
h. reading a plate: after the reaction was terminated, the OD value of each well was measured in terms of reading at a detection wavelength of 450nm within 10 minutes.
The experimental results are shown in FIG. 3, and FIG. 3 is a graph comparing the IFN-. gamma.secretion content of lymphocytes from immunized mice. The results in FIG. 3 show that: after the splenic lymphocytes are stimulated by the E6 protein, the lymphocytes of the mice immunized by the recombinant HPV RNA vaccine of the embodiment have stronger capacity of secreting IFN-gamma, and the recombinant HPV RNA vaccine is also proved to be capable of powerfully stimulating the cellular immune response of experimental animals and playing the capacity of killing HPV infected cells.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Shenzhen advanced technology research institute of Chinese academy of sciences
Application of <120> sPD1 protein and/or sPD1 gene as immune adjuvant
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 885
<212> DNA
<213> Artificial sequence (Artificial)
<400> 1
cccgccgcca ccatgtttca ggacccacag gagcgaccca gaaagttacc acagttatgc 60
acagagctgc aaacaactat acatgatata atattagaat gtgtgtactg caagcaacag 120
ttactgcgac gtgaggtata tgactttgct tttcgggatt tatgcatagt atatagagat 180
gggagtccat atgctgtagg agataaatgt ttaaagtttt attctaaaat tagtgagtat 240
agacattatt gttatagttt gtatggaaca acattagaac agcaatacaa caaaccgttg 300
tgtgatttgt taattaggtg tattaacgga caaaagccac tgtgtcctga agaaaagcaa 360
agacatctgg acaaaaagca aagattccat aatataaggg gtcggtggac cggtcgatgt 420
atgtcttgtt gcagatcatc aagaacacgt agagaaaccc agctgtaaat gcatggagat 480
acacctacat tgcatgaata tatgttagat ttgcaaccag agacaactga tctctacgga 540
tatggacaat taagtgacag ctcagaggag gaggatgaaa tagatggtcc agctggacaa 600
gcagaaccgg acagagccca ttacaatatt gtaacctttt gttgcaagtg tgactctacg 660
cttcggttgt gcgtacaaag cacacacgta gacattcgta ctttggaaga cctgttaatg 720
ggcacactag gaattgtggg acccatctgt tcccagaaac catttttttt tttttttttt 780
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 840
tttttttttt tttttttttt tttttttttt tttttttttt tttaa 885
<210> 2
<211> 1383
<212> DNA
<213> Artificial sequence (Artificial)
<400> 2
cccgccgcca ccatgcagat cccacaggcg ccctggccag tcgtctgggc ggtgctacaa 60
ctgggctggc ggccaggatg gttcttagac tccccagaca ggccctggaa cccccccacc 120
ttctccccag ccctgctcgt ggtgaccgaa ggggacaacg ccaccttcac ctgcagcttc 180
tccaacacat cggagagctt cgtgctaaac tggtaccgca tgagccccag caaccagacg 240
gacaagctgg ccgccttccc cgaggaccgc agccagcccg gccaggactg ccgcttccgt 300
gtcacacaac tgcccaacgg gcgtgacttc cacatgagcg tggtcagggc ccggcgcaat 360
gacagcggca cctacctctg tggggccatc tccctggccc ccaaggcgca gatcaaagag 420
agcctgcggg cagagctcag ggtgacagag agaagggcag aagtgcccac agcccacccc 480
agcccctcac ccaggccagc cggccagttc caatttcagg acccacagga gcgacccaga 540
aagttaccac agttatgcac agagctgcaa acaactatac atgatataat attagaatgt 600
gtgtactgca agcaacagtt actgcgacgt gaggtatatg actttgcttt tcgggattta 660
tgcatagtat atagagatgg gagtccatat gctgtaggag ataaatgttt aaagttttat 720
tctaaaatta gtgagtatag acattattgt tatagtttgt atggaacaac attagaacag 780
caatacaaca aaccgttgtg tgatttgtta attaggtgta ttaacggaca aaagccactg 840
tgtcctgaag aaaagcaaag acatctggac aaaaagcaaa gattccataa tataaggggt 900
cggtggaccg gtcgatgtat gtcttgttgc agatcatcaa gaacacgtag agaaacccag 960
ctgtaaatgc atggagatac acctacattg catgaatata tgttagattt gcaaccagag 1020
acaactgatc tctacggata tggacaatta agtgacagct cagaggagga ggatgaaata 1080
gatggtccag ctggacaagc agaaccggac agagcccatt acaatattgt aaccttttgt 1140
tgcaagtgtg actctacgct tcggttgtgc gtacaaagca cacacgtaga cattcgtact 1200
ttggaagacc tgttaatggg cacactagga attgtgggac ccatctgttc ccagaaacca 1260
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1320
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1380
taa 1383
<210> 3
<211> 151
<212> PRT
<213> Artificial sequence (Artificial)
<400> 3
Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu Cys
1 5 10 15
Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr
20 25 30
Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg
35 40 45
Asp Leu Cys Ile Val Tyr Arg Asp Gly Ser Pro Tyr Ala Val Gly Asp
50 55 60
Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys
65 70 75 80
Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu
85 90 95
Cys Asp Leu Leu Ile Arg Cys Ile Asn Gly Gln Lys Pro Leu Cys Pro
100 105 110
Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile
115 120 125
Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg
130 135 140
Thr Arg Arg Glu Thr Gln Leu
145 150
<210> 4
<211> 317
<212> PRT
<213> Artificial sequence (Artificial)
<400> 4
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Phe Gln Asp Pro Gln Glu Arg Pro Arg
165 170 175
Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile
180 185 190
Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val
195 200 205
Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Ser
210 215 220
Pro Tyr Ala Val Gly Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser
225 230 235 240
Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln
245 250 255
Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Gly
260 265 270
Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys
275 280 285
Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met Ser
290 295 300
Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu
305 310 315
Claims (9)
- The application of sPD1 protein and/or sPD1 gene as immunological adjuvant in preparing recombinant HPV vaccine; wherein,the sPD1 protein is an amino acid sequence shown as SEQ ID NO. 3;the sPD1 gene is the nucleotide sequence shown as SEQ ID NO. 1;the recombinant HPV vaccine comprises mE6E7 protein, and the mE6E7 protein is an amino acid sequence shown as SEQ ID NO. 4.
- 2. The use of claim 1, wherein the recombinant HPV vaccine comprises at least one of a recombinant DNA vaccine, a recombinant RNA vaccine, a recombinant protein vaccine.
- 3. A recombinant protein vaccine comprising sPD1 protein and mE6E7 protein,the sPD1 protein is an amino acid sequence shown as SEQ ID NO. 3;the mE6E7 protein is an amino acid sequence shown as SEQ ID NO. 4.
- 4. A recombinant DNA vaccine comprising an sPD1 gene and an mE6E7 gene,the sPD1 gene is the nucleotide sequence shown as SEQ ID NO. 1;the mE6E7 gene is a nucleotide sequence shown as SEQ ID NO. 2.
- 5. A recombinant plasmid which contains pcDNA3.1(+) plasmid sequence and sPD1 gene sequence and mE6E7 gene sequence in the recombinant DNA vaccine of claim 4.
- 6. A genetically engineered host cell comprising the recombinant plasmid of claim 5.
- 7. A method for producing a recombinant protein vaccine, comprising the step of transfecting a host cell with the recombinant plasmid of claim 5.
- 8. A preparation method of a recombinant RNA vaccine is characterized by comprising the following steps:providing the recombinant plasmid of claim 5;transcribing the recombinant plasmid into RNA in vitro.
- 9. The method of claim 8, wherein the recombinant plasmid is prepared by the following steps:artificially synthesizing an sPD1 gene sequence and an mE6E7 gene sequence, and connecting the sPD1 gene sequence and the mE6E7 gene sequence with pcDNA3.1(+) plasmid;and (3) transfecting the connected product into a host cell for culturing to obtain a positive cloning recombinant expression vector.
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An HPV-E6/E7 immunotherapy plus PD-1 checkpoint inhibition results in tumor regression and reduction in PD-L1 expression;Rice AE等;《Cancer Gene Ther》;20151231;第22卷(第9期);454-462 * |
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