Preparation method and application of papilloma virus antigen composite peptide

Abstract

The invention relates to the technical field of papilloma virus antigen composite peptides, in particular to a preparation method and application of a papilloma virus antigen composite peptide. The composite peptide is selectively combined and activates specific immune cells, so that the immunity of a host to HPV infection is enhanced. The preparation method comprises the following steps: screening antigenic determinants with high immunogenicity from various HPV types, and combining the antigenic determinants with helper T cell epitopes to form a polypeptide chain. The polypeptide chain is produced through chemical synthesis or a DNA recombination technology, and then the high-purity composite peptide is obtained through a purification step. The composite peptide can be used as a vaccine component or a therapeutic agent, is applied to prevention of HPV related diseases such as cervical cancer, anal cancer and other skin and mucous membrane lesions, and can also be used as a key component in a diagnostic reagent. In addition, the composite peptide is also suitable for developing personalized cancer immunotherapy, and provides a more effective treatment scheme for patients affected by HPV.

Description

Technical Field

[0001] The present invention relates to the technical field of papillomavirus antigen composite peptides, and more specifically, to a preparation method and application of a papillomavirus antigen composite peptide. Background Art

[0002] Human papillomavirus (HPV) is a spherical DNA virus that widely exists in nature, with humans as the only host. It is very resistant to drying and can be stored for a long time. Currently, only more than 150 known HPV subtypes are classified into low-risk and high-risk types according to the severity of the diseases they cause. The clinical symptoms of the diseases include various skin warts, such as common warts, flat warts, plantar warts, etc. In addition, high-risk HPV can cause various tumors, such as cervical cancer, anal cancer, penile cancer, etc. The transmission routes mainly include contact transmission, sexual transmission, and mother-to-child transmission, among which sexual contact is the most common infection route. The general population is susceptible to HPV, but people with factors such as premature sexual intercourse, hormonal imbalance, low immunity, multiple sexual partners, and smoking are more likely to be infected.

[0003] The existing treatment methods for HPV infection mainly need to formulate personalized treatment plans according to the infection type, disease severity, and specific conditions of the patients, such as: drug treatment, physical treatment, surgical treatment, immunotherapy, and traditional Chinese medicine adjuvant treatment, etc. Among them, immunotherapy activates the patient's own immune system to make it more effectively identify and eliminate HPV virus. However, there is still a lack of effective immunotherapy means for preventing or treating HPV infection in this field. Summary of the Invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a preparation method and application of a papillomavirus antigen composite peptide. The present invention combines a highly immunogenic HPV antigenic determinant with a helper T cell epitope to form a fusion peptide. This fusion peptide can be used as a vaccine component or a therapeutic agent for preventing HPV-related diseases such as cervical cancer, anal cancer, and other skin and mucosal lesions, and can also be used as a key component in diagnostic reagents. It is also suitable for developing personalized cancer immunotherapy to provide more effective treatment plans for patients affected by HPV.

[0005] Specifically, the present invention first provides a highly immunogenic HPV antigenic determinant, and the HPV antigenic determinant is one or more selected from SEQ ID NO.1-3.

[0006] Preferably, the HPV antigenic determinant is selected from SEQ ID NO.1.

[0007] Preferably, the HPV antigenic determinant is selected from SEQ ID NO.2.

[0008] Preferably, the HPV antigenic determinant is selected from SEQ ID NO.3.

[0009] Furthermore, on the other hand, the present invention provides a Th antigen epitope, which is one or more selected from SEQ ID NOs. 4 - 6.

[0010] Preferably, the HPV antigenic determinant is selected from SEQ ID NO.4.

[0011] Preferably, the HPV antigenic determinant is selected from SEQ ID NO.5.

[0012] Preferably, the HPV antigenic determinant is selected from SEQ ID NO.6.

[0013] Furthermore, on the other hand, the present invention provides an HPV fusion antigenic determinant, which is characterized in that the HPV fusion antigenic determinant combines the above-mentioned highly immunogenic HPV antigenic determinant with the above-mentioned helper T cell epitope to form a fusion peptide.

[0014] Preferably, in the HPV fusion antigenic determinant, the HPV antigenic determinant and the helper T cell epitope are connected by a peptide linker.

[0015] Preferably, the peptide linker is selected from (GS) n , (GGSS) n , (GGGSSS) n or one or more of them, where n is a natural number, preferably 0, 1, 2.

[0016] Preferably, the sequence of the HPV fusion antigenic determinant is as shown in SEQ ID NOs. 7 - 9.

[0017] Preferably, the sequence of the HPV fusion antigenic determinant is as shown in SEQ ID NO.7.

[0018] Preferably, the sequence of the HPV fusion antigenic determinant is as shown in SEQ ID NO.8.

[0019] Preferably, the sequence of the HPV fusion antigenic determinant is as shown in SEQ ID NO.9.

[0020] On the other hand, the present invention provides the use of the above-mentioned HPV fusion antigenic determinant in the preparation of drugs for preventing or treating diseases related to HPV infection.

[0021] Preferably, the diseases related to HPV infection include cervical cancer, anal cancer, and other skin and mucosal lesions, etc.

[0022] On the other hand, the present invention provides the use of the above-mentioned HPV fusion antigen determinant in the preparation of reagents for diagnosing diseases related to HPV infection.

[0023] The advantages of the present invention are as follows: The present invention combines the highly immunogenic HPB antigen determinant with the helper T cell epitope to form a fusion peptide. This fusion peptide can be used as a vaccine component or therapeutic agent for preventing HPV-related diseases such as cervical cancer, anal cancer, and other skin and mucosal lesions, and can also be used as a key component in diagnostic reagents. It is also suitable for developing personalized cancer immunotherapy to provide more effective treatment options for patients affected by HPV. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 . Activity analysis of the HPV fusion antigen determinant. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following is a further detailed description of the present invention in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0026] The present invention combines the highly immunogenic HPB antigen determinant with the helper T cell epitope to form a fusion peptide.

[0027] The HPV antigen determinant is one or more selected from SEQ ID NO.1-3.

[0028] Preferably, the HPV antigen determinant is selected from SEQ ID NO.1.

[0029] Preferably, the HPV antigen determinant is selected from SEQ ID NO.2.

[0030] Preferably, the HPV antigen determinant is selected from SEQ ID NO.3.

[0031] The Th antigen epitope provided by the present invention is one or more selected from SEQ ID NO.4-6.

[0032] Preferably, the HPV antigen determinant is selected from SEQ ID NO.4.

[0033] Preferably, the HPV antigen determinant is selected from SEQ ID NO.5.

[0034] Preferably, the HPV antigen determinant is selected from SEQ ID NO.6.

[0035] The present invention provides an HPV fusion antigen determinant, which is formed by combining the above-mentioned highly immunogenic HPV antigen determinant with the above-mentioned helper T cell epitope to form a fusion peptide.

[0036] Preferably, in the HPV fusion antigen determinant, the HPV antigen determinant and the helper T cell epitope are connected by a peptide linker.

[0037] Preferably, the peptide linker is selected from (GS) n , (GGSS) n , (GGGSSS) n or one or more of them, where n is a natural number, preferably 0, 1, 2.

[0038] Preferably, the sequence of the HPV fusion antigen determinant is as shown in SEQ ID NO.7-9.

[0039] Preferably, the sequence of the HPV fusion antigen determinant is as shown in SEQ ID NO.7.

[0040] Preferably, the sequence of the HPV fusion antigen determinant is as shown in SEQ ID NO.8.

[0041] Preferably, the sequence of the HPV fusion antigen determinant is as shown in SEQ ID NO.9.

[0042] The present invention provides the use of the above-mentioned HPV fusion antigen determinant in the preparation of drugs for preventing or treating diseases related to HPV infection.

[0043] Preferably, the diseases related to HPV infection include cervical cancer, anal cancer and other skin and mucosal lesions.

[0044] The present invention provides the use of the above-mentioned HPV fusion antigen determinant in the preparation of reagents for diagnosing diseases related to HPV infection.

[0045] The following examples are only used to illustrate the present invention and do not limit the scope of the present invention. Based on the specific examples in the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0046] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art; in the embodiments of the present invention, if not specifically specified, the technical means used are conventional means well-known to those skilled in the art.

[0047] Example 1

[0048] Screening of highly immunogenic HPV antigen determinants:

[0049] Online prediction was performed on the E6 protein of HPV18 virus and the T cell locus HLA-DRB1*01:02 commonly carried by Chinese people using the SYFPEITHI software to predict HPV antigenic epitopes and Th antigenic epitopes, and the top three epitope sequences were selected for standby according to the scores. The results are shown in Tables 1 and 2 below:

[0050] Table 1. Highly immunogenic HPV antigenic determinants

[0051]

[0052]

[0053] Table 2. Highly active helper T cell epitopes

[0054]

[0055] Example 2

[0056] Analysis of the activity of highly immunogenic HPV antigenic determinants:

[0057] The MTT assay was used to evaluate the response level of antigen-specific T cells in PBMC induced by the HPV antigenic determinants screened in Example 1. The detailed steps can be referred to the MTT detection kit (purchased from, BioVision, catalog number K299-1000). The results are shown in Table 3 below:

[0058] Table 3. Immune stimulation index of different concentrations of HPV antigenic determinants

[0059]

[0060]

[0061] Stimulation index (SI) = OD value of each group of samples / OD value of the blank group.

[0062] The results showed that: compared with the blank control, the HPV antigenic determinants screened in the present invention all had superior immune stimulation activity, especially the stimulation activity of SEQ ID NO.1 was the strongest.

[0063] Example 3

[0064] 3.1 Construction of HPV fusion antigenic determinants:

[0065] The HPV antigenic determinants screened in Example 1 were respectively fused with the helper T cell epitope shown in SEQ ID NO.4 to construct HPV fusion antigenic determinants. The specific fusion method can be selected to construct by chemical synthesis or recombinant DNA technology. Specifically, the present invention uses a de novo synthesis method to prepare the HPV fusion antigenic determinants, and their sequences are shown in SEQ ID NOs.7-9.

[0066] 3.2 Activity analysis of HPV fusion antigenic determinants:

[0067] A mouse cervical cancer model infected with HPV16 was constructed. Age-matched mice were selected, and each was subcutaneously inoculated with 0.2 mL of 1×10 6 / mL HPV16-positive HeLa cells on the right cervical back. The successfully modeled mice were randomly grouped. The treatment group was intraperitoneally injected with the above-mentioned HPV fusion antigenic determinants in sequence, and the blank group was injected with an equal volume of normal saline. The injection dose was 50 μg / kg, and the drug was administered once a week for 2 consecutive weeks. The tumor volume was observed and recorded during the experiment.

[0068] The results are as Figure 1 shown. Compared with the blank group, the HPV fusion antigenic determinants shown in SEQ ID NOs.7-9 of the present invention can effectively reduce the tumor volume in mice, inhibit the in vivo proliferation of HPV virus, and thus effectively prevent and treat diseases infected with HPV.

[0069] It is necessary to point out here that the above embodiments are only for further elaboration and explanation of the technical solutions of the present invention, and are not further limitations on the technical solutions of the present invention. The method of the present invention is only a preferred implementation scheme and is not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A highly immunogenic HPV antigenic determinant, characterized in that: The HPV antigenic determinant is one or more selected from SEQ ID NO.1-3.

2. Th antigen epitope, characterized in that The Th antigen epitope is one or more selected from SEQ ID NO.4-6.

3. HPV fusion antigen determinant, characterized in that The HPV fusion antigen determinant is a fusion peptide formed by combining the HPV antigen determinant with high immunogenicity described in claim 1 with the helper T cell epitope described in claim 2.

4. The HPV fusion antigen determinant according to claim 3, characterized in that: The HPV antigen determinant and the helper T cell epitope in the HPV fusion antigen determinant are connected via a peptide linker.

5. The HPV fusion antigen determinant according to claim 3, characterized in that: The peptide linker is selected from (GS) n 、(GGSS) n 、(GGGSSS) n One or more of , where n is a natural number.

6. The HPV fusion antigen determinant according to claim 3, characterized in that: The sequence of the HPV fusion antigen determinant cluster is shown in SEQ ID NO.7-9.

7. Use of the HPV fusion antigen determinant according to any one of claims 3 to 6 in the preparation of medicines for preventing or treating diseases related to HPV infection.

8. The use according to claim 7, characterized in that The HPV infection-related diseases include at least one of cervical cancer, anal cancer and other skin and mucosal lesions.

9. The use according to claim 7, characterized in that The medicament may be selected from therapeutic or preventive vaccines.

10. Use of the HPV fusion antigen determinant according to any one of claims 3 to 6 in the preparation of reagents for diagnosing diseases related to HPV infection.

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