WO2024037005A1

WO2024037005A1 - Human cytomegalovirus antigen epitope chimeric peptide and use thereof

Info

Publication number: WO2024037005A1
Application number: PCT/CN2023/088565
Authority: WO
Inventors: 王斌; 李俊; 江莎莎; 南福龙; 张现娟; 刘俸君; 鲁晓晴; 张万明; 高青
Original assignee: 青岛大学; 青岛万明赛伯药业有限公司
Priority date: 2022-08-16
Filing date: 2023-04-17
Publication date: 2024-02-22
Also published as: CN115894708A; CN115894708B

Abstract

The present invention provides a human cytomegalovirus antigen epitope chimeric peptide and a use thereof. An amino acid sequence of the human cytomegalovirus antigen epitope chimeric peptide is selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4. On the basis of optimized and screened HCMV overlapping T cell epitopes, Th cell epitopes and B cell epitopes, the present invention obtains four HCMV dominant antigen epitope chimeric peptides. Vaccines containing said epitope chimeric peptides have good immunogenicity and safety in mouse models, and can induce humoral and cellular immune responses in a short period of time.

Description

A kind of human cytomegalovirus epitope chimeric peptide and its application

Technical field

The invention belongs to the field of immune prevention and treatment. Specifically, the present invention relates to a human cytomegalovirus epitope chimeric peptide and its application.

Background technique

Human Cytomegalovirus (HCMV) infection is probably the most prevalent human infection. Although improved sanitation and less close contact between children and adults in developed countries have reduced the prevalence of CMV, almost 100% of adults in low- and middle-income countries are infected at a young age. Cytomegalovirus infects T cells and alters their responses, which is suspected of contributing to arteriosclerosis and immunosenescence and may promote cancer through non-regulatory effects. However, its most important medical significance is that it is the most common congenital infection in the world, most commonly causing hearing loss and, in some cases, microcephaly, mental retardation, hepatosplenomegaly, and thrombocytopenia Decreased purpura. Generally speaking, 1 in every 200 to 30 births Infection with maternally transmitted cytomegalovirus. The severity of fetal infection depends on maternal HCMV seropositivity or negativity. The prognosis for fetuses infected by seronegative pregnant women is the worst, but fetuses infected by seropositive mothers may also have serious consequences.

Additionally, cytomegalovirus is the most common infection in transplantation. Solid organ transplant patients receiving transplants from seropositive donors may develop disease, and seropositive transplant recipients may reactivate HCMV due to immunosuppression. These infections can lead to severe illness and transplant rejection. HCMV infection is the most common infectious complication of transplantation, whether solid organ or hematopoietic stem cell transplantation. In the case of solid organ transplantation, including kidney, liver, lung, etc., the most dangerous situation is when an HCMV-seronegative recipient receives an organ from an HCMV-seropositive donor. In this case, cytomegalovirus infection is almost certain, and illness is common. Taking kidney transplantation as an example, approximately one-third of seronegative kidney recipients will develop cytomegalovirus disease if antiviral prophylaxis is not taken. Interestingly, even seropositive recipients may develop CMV disease when receiving organ transplants from seronegative donors. Since seropositive recipients who received organs from seronegative donors had a lower incidence of CMV disease, this suggests that the problem in seropositive recipients is superinfection with new strains under the influence of immunosuppression rather than reactivation , but the situation after hematopoietic stem cell transplantation (HSCT) is different. In immunosuppression Reactivation under the influence seems to be even more dangerous. Since HCMV latency occurs not only in circulating T cells, but also in lymph nodes, endothelial cells, macrophages and other sites, it is not surprising that reactivation issues arise. Antiviral prophylaxis and/or treatment is routinely administered in transplant centers to prevent severe cytomegalovirus disease with considerable, but not complete, success. Vaccination has had some early success in reducing the severity of CMV disease.

Development of a cytomegalovirus vaccine began in the 1970s, when the toll the virus was taking on babies in the womb and transplant recipients became apparent. The two vaccine strains are attenuated from the laboratory-isolated viruses AD-169 and Towne. The AD169 attenuated strain was quickly abandoned, but the Towne attenuated strain continues to undergo extensive trials in solid organ transplant recipients and normal male and female volunteers. The Towne attenuated strain was highly protective against severe cytomegalovirus disease and graft rejection in kidney transplant recipients. However, there was no statistically significant difference in protection against HCMV infection. The Towne strain vaccine under study can protect humans against unattenuated CMV, but the immune protection obtained naturally is more potent than the immune protection obtained after the vaccine is injected. In addition, live-attenuated vaccines derived from weakened strains failed to protect women from CMV infection.

The next important advance was the purification of the HCMV surface protein, called glycoprotein B or gB, Because it is homologous to the glycoproteins of other herpes viruses. When it was combined with the MF59 oil-in-water adjuvant, the body produced good levels of neutralizing antibodies after three injections over a six-month period. The regimen was tested twice alongside placebo in young women who were naturally exposed to CMV. In both cases, subjects were more effective against CMV infection than before, but antibodies and The effects wear off quickly. The booster shot did restore antibody levels. Furthermore, when the gB subunit protein was combined with the AS01 adjuvant, which stimulates toll-like receptor (TLR) 4, it elicited higher and longer-lasting anti-gB antibody levels in humans, but the effectiveness of adjuvanted vaccines has never been tested test. Notably, an investigational gB subunit vaccine conferred significant protection against cytomegalovirus disease in solid organ transplant patients, suggesting the importance of antibodies in this setting. The fact that gB is a trimeric fusion protein suggests that a more immunogenic prefusion form may exist, but this has not been confirmed.

There are several unanswered questions about the feasibility of a CMV vaccine, but there are some clear answers. Cytomegalovirus is acquired through contact with saliva, sexual secretions, and transplantation. In principle, there are four groups that can benefit from HCMV protection: seronegative women of childbearing age, seropositive women of childbearing age, solid organ (SO) recipients donated by HCMV seropositive individuals, and serum hematopoietic stem cell (HSC) recipients. The incidence of cytomegaloviremia was highest in both transplant populations. but However, antiviral prophylaxis is expensive and not completely effective, and cannot be continued indefinitely. Ideally, cytomegalovirus vaccine should be given before transplantation, but should be continued after transplantation for HSC trial patients who have acquired a new immune system. Although not 100% certain, it appears that HCMV antibodies are required in SO trial recipients and that HSCT recipients are required to boost T cell immunity to HCMV.

In regions such as North America, Europe, and Asia, many women do not have CMV antibodies during pregnancy, so CMV vaccination is reasonable. If vaccine-induced protection lasts long enough, it would provide women with strong indirect protection against intrauterine infection of the fetus in most pregnant women. Because mother-to-child transmission cannot be blocked, prevention through vaccination is necessary.

Contents of the invention

In view of the above-mentioned shortcomings of the existing technology, the object of the present invention is to provide a novel human cytomegalovirus chimeric polypeptide vaccine, which is a polypeptide vaccine based on epitope peptides of proteins encoded by human cytomegalovirus and can be used for Prevention and/or treatment of HCMV viremia in pregnant women and organ transplant patients. Based on the above purpose, the present invention first predicts HCMV antigenic epitopes through bioinformatics software, and optimizes and screens 4 HCMV dominant antigens from the predicted epitopes. epitope, and then expressed in a prokaryotic system to obtain a chimeric peptide vaccine based on the dominant epitope peptide of the protein encoded by human cytomegalovirus.

In one aspect, the invention provides a human cytomegalovirus epitope chimeric peptide, which contains or consists of two, three or four human cytomegalovirus epitope peptides, wherein the antigenic epitope peptide The amino acid sequence is selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

According to one embodiment of the present invention, the human cytomegalovirus epitope chimeric peptide includes or consists of four human cytomegalovirus epitope peptides, wherein the amino acid sequences of the antigenic epitope peptides are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.

On the other hand, the present invention also provides a pharmaceutical composition for preventing and/or treating human cytomegalovirus infection or its related diseases, which contains the human cytomegalovirus epitope chimeric peptide according to the present invention, and a pharmaceutical composition Acceptable carriers, diluents, excipients and/or adjuvants.

According to one embodiment of the invention, the pharmaceutical composition is a vaccine.

According to one embodiment of the present invention, the pharmaceutically acceptable carrier is a pET28a vector or a pET28a-SUMO vector. In a preferred embodiment of the invention, when the antigenic epitope When the amino acid sequence of the peptide is SEQ ID NO.1, SEQ ID NO.2 and/or SEQ ID NO.4, the pharmaceutically acceptable carrier is the pET28a vector; when the amino acid sequence of the epitope peptide is SEQ In case of ID NO.3, the pharmaceutically acceptable carrier is pET28a-SUMO vector

According to one embodiment of the present invention, the pharmaceutical composition is an intramuscular injection.

In another aspect, the present invention also provides the use of the human cytomegalovirus epitope chimeric peptide or pharmaceutical composition according to the present invention in the preparation of medicaments for preventing and/or treating human cytomegalovirus infection or its related diseases.

According to one embodiment of the present invention, the disease related to human cytomegalovirus infection is human cytomegalovirus viremia in pregnant women.

According to one embodiment of the present invention, the disease related to human cytomegalovirus infection is human cytomegalovirus viremia in organ transplant patients.

The present invention provides the dominant antigenic epitope of the protein encoded by human cytomegalovirus, and expresses the chimeric peptide based on the dominant antigenic epitope of the protein encoded by human cytomegalovirus in a prokaryotic system. As a new type of human cytomegalovirus vaccine, it covers the causes of PP65, PP150, IE1 proteins for effective cellular immunity and gB and gH proteins for effective humoral immunity. It has good immunogenicity in both mouse models and clinical population samples, and can induce the body to produce strong cellular and humoral immune responses in a short period of time. answer. Experimental results showed that 14 days after three immunizations, both killer T cells and helper T cells could be significantly activated. The preparation method of the vaccine is fast and simple, and can achieve large-scale production in a short period of time, so that it can be used to prevent and/or treat latent infection with human cytomegalovirus and diseases caused by infection.

Description of drawings

Below, the embodiments of the present invention are described in detail with reference to the accompanying drawings, wherein:

Figures 1A to 1D show the gene expression vector construction diagram of the four dominant antigen epitopes of human cytomegalovirus obtained in the present invention, wherein Figure 1A is the gene expression vector construction diagram of the dominant antigen epitope 1; Figure 1B is the dominant antigen epitope The construction diagram of the gene expression vector of 2; Figure 1C is the construction diagram of the gene expression vector of the dominant antigen epitope 3; Figure 1D is the construction diagram of the gene expression vector of the dominant antigen epitope 4.

Figures 2A to 2D show the colony PCR verification results of amplification of the four dominant epitope genes of human cytomegalovirus obtained in the present invention.

Figures 3A to 3E show the results of expression and purification of the dominant antigenic epitope 1 protein of human cytomegalovirus obtained in the present invention.

Figures 4A to 4D show the results of expression and purification of the dominant epitope 2 protein of human cytomegalovirus obtained in the present invention.

Figures 5A-5C show the results of expression and purification of the dominant antigenic epitope 3 protein of human cytomegalovirus obtained in the present invention.

Figures 6A to 6D show the results of expression and purification of the dominant epitope 4 protein of human cytomegalovirus obtained in the present invention.

Figure 7 shows the changes in mouse body weight within 42 days after the first vaccination of mice with the vaccine, where the arrow points to the time of vaccine injection.

Figure 8 shows the expression of IL2, IL4 and TNF-α in mouse spleen CD3+/CD4+ T cells after three injections of vaccine immunization (14 days/injection).

Figure 9 shows the expression of IL2, IFN-γ and TNF-α in mouse spleen CD3+/CD8+ T cells after three injections of vaccine immunization (14 days/injection).

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

Screening of dominant antigenic epitopes of human cytomegalovirus

1. Use bioinformatics methods to predict B cell, Th cell and CTL cell epitopes of five HCMV-encoded membrane proteins. The membrane protein sequence of HCMV was derived from the protein database of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih .gov/).

Specifically, five proteins, PP65, PP150, IE, gB and gH, were selected, and DNAstar, IEDB (score>0.5) and TMHMM software were used to predict B cell epitopes respectively; SYFPEITHI (score>20), IEDB (rank< 1) Use NetMHCII pan and NetMHCII software to predict Th cell antigen epitopes on HCMV membrane proteins; use IEDB, SYFPEITHI and NetMHCI software to predict CTL cell antigen epitopes on HCMV membrane proteins.

1.1 Screening of Th epitopes

Four softwares: SYFPEITHI, IEDB, NetMHCII pan and NetMHCII were used to screen and predict the Th epitopes of 21 membrane proteins and set virus-encoded proteins such as IE, gB, gH, PP65, PP150, etc., and select overlapping results. Six HLA alleles were selected, including HLA-DRB1*01:01, HLA-DRB1*03:01, HLA-DRB1*04:01, HLA-DRB1*07:01, HLA-DRB1*11:01 and HLA-DRB1*15:01. The screening criteria were set as SYFPEITHI score >20, IEDB score <1.00, NetMHC II pan score <10, NetMHC II score <10. The length of the ligand was set to 15, and other settings were default values.

1.2 Prediction results of Th epitopes of 21 membrane proteins and other virus-encoded proteins

Four types of software were used to screen the Th epitopes of 21 membrane proteins and virus-encoded proteins such as IE1, IE2, PP65, and PP150 set by references: SYFPEITHI, IEDB, NetMHCII pan, and NetMHCII, and use them to simultaneously predict and select overlapping results. Select 6 HLA alleles, including HLA-DRB1*01:01, HLA-DRB1*03:01, HLA-DRB1*04:01, HLA-DRB1*07:01, HLA-DRB1*11:01, HLA -DRB1*15:01. The screening criteria were set as SYFPEITHI score >20, IEDB score <1.00, NetMHC II pan score <10, and NetMHC II score <10. The length of the ligand was set to 15, and other settings were default values.

1.3 Screening of B epitopes

Use the software TMHMM, ABCpred, IEDB and DNAstar to predict B cell epitopes. Considering that extramembrane proteins are the precursor conditions for B cell epitopes, TMHMM software was used to predict the non-transmembrane regions of 17 HCMV virus-encoded membrane glycoproteins as segments for screening epitopes. ABCpred is used as the second HCMV virus-encoded B cell epitope prediction software. The parameters are set as follows: the peptide length is 16 amino acids, and the threshold value is greater than or equal to 0.8 points. The obtained peptide is used as the prediction result of this software. The result is Benchmarks, combined with other software to screen, exclude those related to Peptides predicted by other software do not overlap. Prediction is carried out through the Bepipred method in the online software IEDB. The parameters are the default parameters. The obtained results are compared with the results predicted by the ABCpred software, and the peptides with consistent prediction results of the two software are retained. Use the Protean module in DNAstar for prediction. The selected parameters include β-turn and random coil in the secondary structure, hydrophilicity, flexibility, surface accessibility and antigen index. Select parameters that satisfy the above four conditions at the same time. The peptide segment of the parameters is used as the prediction result of this software. This result is then compared with the results obtained by the above three softwares, and the epitopes that do not overlap with this software are further eliminated to obtain the final predicted screening epitope, which is regarded as the dominant antigen epitope of HCMV virus-encoded B cells.

2. Further screen the overlapping portions of B cell epitopes, Th cell epitopes and CTL cell epitopes of the five proteins PP65, PP150, IE1, gB and gH.

The epitopes of 5 proteins, namely PP150, PP65, IE, gH and gB, which can be recognized by B cells, CTL cells and Th cells at the same time are used as dominant antigenic epitopes 1, 2, 3 and 4, and their amino acid sequences are represented by SEQ. As shown in ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, the obtained nucleotide sequences of overlapping dominant antigenic epitopes 1, 2, 3 and 4 are as shown in SEQ ID NO. 5. As shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, the corresponding chimeric protein can be obtained in the E. coli prokaryotic expression system.

Three online softwares, SYFPEITHI, IEDB and NetMHC, were used to predict the potential CD8+ T cell epitopes of the protein. In this example, HLA-A*01:01, HLA-A*02:01, and HLA-A*03 were selected: 01, HLA-A*11:01, HLA-A*24:02, HLA-A*26:01, a total of 6 MHC class I molecular dominant gene loci with coverage reaching 57.6% of Chinese people. The screening criteria were set as: SYFPEITHI score >20, IEDB score <0.1, and NetMHC score <10. The peptide length of each epitope was set to 9 amino acids, and other settings were default values.

Example 2

Expression of dominant antigenic epitopes of human cytomegalovirus

1. Vector construction and amplification

1.1 Vector construction

As shown in Figure 1A to Figure 1D, gene expression vectors containing the four dominant antigen epitope genes obtained in Example 1 were constructed.

1.2 PCR amplification

Use the PCR amplification reaction system shown in Table 1 and the primers shown in Table 2, and perform PCR amplification under the PCR amplification reaction conditions shown in Table 3.

Table 1 PCR amplification reaction system

Table 2 Primers

Table 3 PCR amplification reaction conditions

The electrophoresis detection and recovery steps of the amplified fragments were described in the kit instructions (DNA recovery kit, Tiangen Biochemical Technology Co., Ltd.).

1.3 Colony PCR verification

Take 100 μl of the transformed bacterial solution and spread it on LB solid medium containing ampicillin resistance, and culture it at 37°C overnight. Pick a single clone and shake it briefly. Take the bacterial liquid and use the reaction system shown in Table 1 and the primers shown in Table 2 to perform colony PCR experiments. The colony PCR verification results are shown in Figure 2A-Figure 2D. Figure 2A shows the colony PCR verification results of the dominant antigen epitope 1 gene amplification; Figure 2B shows the colony PCR verification results of the dominant antigen epitope 2 gene amplification; Figure 2C shows the colony PCR verification of the dominant antigen epitope 3 gene amplification Results; Figure 2D shows the colony PCR verification results of dominant epitope 4 gene amplification. As can be seen from Figure 2A to Figure 2D, the four vectors have been constructed and single clonal colonies were successfully selected.

2. Expression verification

2.1 Verification of expression of the dominant antigen epitope 1 protein (i.e. P1), including: SDS-PAGE detection of small-scale expression; SDS-PAGE detection of large-scale expression and bacteriostasis; SDS-PAGE detection of purified protein; SDS-PAGE detection of renatured purified protein ; Enzyme digestion.

2.2 Verification of expression of the dominant antigen epitope 2 protein (i.e. P2), including: small-amount expression SDS-PAGE detection; large-scale expression and bacteriolysis SDS-PAGE detection, purified protein SDS-PAGE detection, renaturation purification and enzyme-digested protein SDS -PAGE detection.

2.3 Verification of expression of the protein with dominant epitope 3 (i.e. P3), including: small-scale expression SDS-PAGE detection; SDS-PAGE detection of large-scale expression and bacteriolysis; SDS-PAGE detection of supernatant purification and enzyme-digested recombinant protein.

2.4 Verification of the expression of the dominant antigen epitope 4 protein (i.e. P4), including: SDS-PAGE detection of small-scale expression; SDS-PAGE detection of large-scale expression and bacteriostasis; SDS-PAGE detection of purified protein; SDS-refolding purification and enzymatic digestion of protein -PAGE detection.

Figures 3A to 3E show the results of expression and purification of the dominant antigenic epitope 1 protein of human cytomegalovirus obtained in the present invention, in which all arrows point to recombinant proteins.

Figure 3A shows the SDS-PAGE electrophoresis diagram of the whole bacterial protein of the positive strain in the small-scale expression SDS-PAGE test. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa), lane 1: host bacteria Whole bacterial protein, lane 2: IPTG induced whole bacterial protein;

Figure 3B shows the SDS-PAGE electrophoresis pattern of the disrupted supernatant and precipitated cells of heavily expressed bacteria in SDS-PAGE detection. Lane M: protein molecular weight scale (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa ), lane 1: induced expression of fragmented supernatant protein, lane 2: induced expression of fragmented insoluble protein;

Figure 3C shows the SDS-PAGE electrophoresis pattern of purified inclusion body protein in SDS-PAGE detection of purified protein. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa). Lane 1: purified inclusion body protein;

Figure 3D is the SDS-PAGE electrophoresis diagram of the eluted concentrated protein in the SDS-PAGE detection of renatured purified protein. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa), lane 1: wash deconcentrated protein;

Figure 3E is an SDS-PAGE electrophoresis diagram of purified protein after enzyme digestion. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa). Lane 1: purified protein after enzyme digestion. Lane 2: Purify the protein before enzyme digestion.

Figures 4A to 4D show the results of expression and purification of the human cytomegalovirus dominant epitope 2 protein obtained in the present invention, in which the arrows point to recombinant proteins.

Figure 4A shows the SDS-PAGE electrophoresis diagram of the whole bacterial protein of the positive strain in the small-scale expression SDS-PAGE test. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa), lane 1: host bacteria Whole bacterial protein, lane 2: IPTG induced whole bacterial protein;

Figure 4B shows the SDS-PAGE electrophoresis pattern of the disrupted supernatant and precipitated cells of heavily expressed bacteria in SDS-PAGE detection. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa ), lane 1: induced expression of fragmented supernatant protein, lane 2: induced expression of fragmented insoluble protein;

Figure 4C is an SDS-PAGE electrophoresis diagram of purified inclusion body protein in SDS-PAGE detection of purified protein. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa), lane 1: purified inclusion body. protein;

Figure 4D is the SDS-PAGE electrophoresis pattern of the eluted concentrated protein during renaturation purification and enzyme-digested protein SDS-PAGE detection, lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa), lane 1: Refolded and purified protein, Lane 2: Purified protein after enzyme digestion.

Figures 5A to 5C show the results of expression and purification of the dominant epitope 3 protein of human cytomegalovirus obtained in the present invention, in which all arrows point to recombinant proteins.

Figure 5A shows the SDS-PAGE electrophoresis diagram of the whole bacterial protein of the positive strain in the small-scale expression SDS-PAGE test. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa), lane 1: host bacteria Whole bacterial protein, lane 2: IPTG induced whole bacterial protein;

Figure 5B shows the SDS-PAGE electrophoresis pattern of the disrupted supernatant and precipitated cells of a large number of expressed bacteria in SDS-PAGE detection. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa ), lane 1: induced expression of fragmented supernatant protein, lane 2: induced expression of fragmented insoluble protein;

Figure 5C shows the SDS-PAGE electrophoresis pattern of the eluted concentrated protein during SDS-PAGE detection of supernatant purification and enzyme-digested recombinant protein. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa). Lane 1: digested protein, lane 2: eluted concentrated protein.

Figures 6A to 6D show the results of expression and purification of the dominant epitope 4 protein of human cytomegalovirus obtained in the present invention, in which all arrows point to recombinant proteins.

Figure 6A shows the SDS-PAGE electrophoresis diagram of the whole bacterial protein of the positive strain in the small-scale expression SDS-PAGE test, the protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa), lane 1: the whole bacterial protein of the host strain , Lane 2: IPTG induces whole bacterial protein;

Figure 6B shows the SDS-PAGE electrophoresis pattern of the disrupted supernatant and precipitated cells of a large number of expressed bacteria in SDS-PAGE detection. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa ), lane 1: induced expression of fragmented supernatant protein, lane 2: induced expression of fragmented insoluble protein;

Figure 6C is an SDS-PAGE electrophoresis diagram of purified inclusion body protein in SDS-PAGE detection of purified protein. Lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa), lane 1: purified inclusion body. protein;

Figure 6D is the SDS-PAGE electrophoresis diagram of the eluted concentrated protein during renaturation purification and SDS-PAGE detection of enzyme-digested protein, lane M: protein molecular weight standard (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa), lane 1: Purified protein after enzyme digestion, Lane 2: Purified protein after renaturation.

It can be seen from the above protein expression verification results of dominant antigenic epitopes 1-4 that the present invention has successfully expressed P1, P2, P3 and P4 Escherichia coli recombinant proteins and completed large-scale expression for subsequent experiments for protein vaccine immunological evaluation.

Example 3

Immunological evaluation of different constructed E. coli recombinant proteins in mouse models

1. Vaccine safety assessment

1.1 Mouse weight detection

The weight of the mice was monitored during the immunization experiment. Compared with the negative control group, the weight of the mice in the experimental group did not decrease significantly. The results are shown in Figure 7. The date pointed by the arrow in the figure is the date of vaccine injection, and a total of three shots were immunized.

2. Vaccine cellular immune response detection

2.1 Vaccine induces cellular immune response

Mice were immunized with 15 μg of Escherichia coli system recombinant protein vaccines P1, P2, P3, and P4 respectively by intramuscular injection. A total of three immunizations were performed, with an interval of 14 days between each time. Lymphocytes were isolated from the spleens of mice 14 days after the last immunization to prepare single cells. Cell suspension was used to detect T lymphocyte immune response by flow cytometry. The results are shown in Figures 8 and 9.

Figure 8 shows the expression of IL2, IL4 and TNF-α in mouse spleen CD3+/CD4+ T cells after three injections of vaccine immunization (14 days/injection). Figure 9 shows the expression of IL2, IFN-γ and TNF-α in mouse spleen CD3+/CD8+ T cells after three injections of vaccine immunization (14 days/injection). Experimental results show that compared with the control group, P1, P2, P3, and P4 can activate T lymphocytes and induce cytokine secretion to varying degrees.

The above are only a few exemplary embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention is disclosed in preferred embodiments as above, this is not intended to limit the present invention. Any skilled person familiar with this field, without departing from the scope of the technical solution of the present invention, may make slight changes or modifications using the technical content disclosed above, which are equivalent to equivalent implementation examples and fall within the scope of the technical solution of the present invention.

Claims

A human cytomegalovirus epitope chimeric peptide, which contains or consists of two, three or four human cytomegalovirus epitope peptides, wherein the amino acid sequence of the epitope peptide is selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.
The human cytomegalovirus epitope chimeric peptide according to claim 1, which includes or consists of four human cytomegalovirus epitope peptides, wherein the amino acid sequences of the antigenic epitope peptides are SEQ ID NO .1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.
A pharmaceutical composition for preventing and/or treating human cytomegalovirus infection or its related diseases, which contains the human cytomegalovirus epitope chimeric peptide according to claim 1 or 2, and a pharmaceutically acceptable carrier , diluents, excipients and/or adjuvants.
The pharmaceutical composition according to claim 3, which is a vaccine.
The pharmaceutical composition according to claim 3, wherein the pharmaceutically acceptable carrier is a pET28a carrier or a pET28a-SUMO carrier;

Preferably, when the amino acid sequence of the epitope peptide is SEQ ID NO.1, SEQ ID NO.2 and/or SEQ ID NO.4, the pharmaceutically acceptable carrier is the pET28a vector; when the When the amino acid sequence of the epitope peptide is SEQ ID NO. 3, the pharmaceutically acceptable carrier is the pET28a-SUMO carrier.
The pharmaceutical composition according to claim 3, which is an intramuscular injection.
The human cytomegalovirus epitope chimeric peptide according to claim 1 or 2 or the pharmaceutical composition according to any one of claims 3 to 6 is used in the preparation of the prevention and/or treatment of human cytomegalovirus infection or its related diseases. uses in medicines.
The use according to claim 7, wherein the disease related to human cytomegalovirus infection is human cytomegaloviremia in pregnant women.
The use according to claim 7, wherein the disease associated with human cytomegalovirus infection is human cytomegalovirus viremia in organ transplant patients.