Anti-HPV (human papillomavirus) infection recombinant III-type human collagen peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and provides an anti-HPV (human papillomavirus) infection recombinant III type human collagen peptide as well as a preparation method and application thereof, the amino acid sequence of the anti-HPV infection recombinant III type human collagen peptide is formed by three repetitions of a coding sequence UniProtKB: P02461 of III type human collagen from the 991st site to the 1032nd site, and is named as ATHPV3, and the amino acid sequence is shown as SEQ ID NO.1. The anti-HPV infection recombinant III type human collagen peptide can be used for preparing anti-HPV infection recombinant III type human collagen peptide. The recombinant III-type human collagen peptide with the molecular weight of about 13kD is obtained through expression and purification, the recombinant III-type human collagen peptide can be specifically combined with HPV 16 / 18 L1 protein, multiple pairs of hydrogen-bond interactions exist, and high-concentration and medium-concentration recombination can inhibit pseudovirus infection of 293T cells. The recombinant III-type human collagen peptide which is relatively good in purity, can be combined with HPV 16 / 18 L1 and can effectively inhibit HPV 16 / 18 pseudovirus infection of 293T cells is successfully obtained, and an effective candidate medicine is provided for preventing and treating HPV infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a recombinant type III human collagen peptide that is resistant to HPV infection and a preparation method and application thereof, and more specifically relates to a recombinant type III human collagen peptide that can bind to HPV L1 protein and its use in the preparation of a drug for preventing and / or treating HPV infection. Background Art

[0002] Human papillomavirus (HPV) is a circular DNA virus that is susceptible to the squamous epithelium of the epidermis and mucosa. The HPV infection rate of healthy women in different regions of my country is 15%-28%. Persistent HPV infection is the main cause of cervical cancer, of which 70%-84% of cervical cancer is related to HPV 16 and 18 infection. In addition to vaccination prevention, there is currently no specific drug. Existing treatments mainly rely on physical therapy and adjuvant therapy such as interferon, which is difficult to solve the problems of subclinical and persistent infection, resulting in symptomatic treatment and easy recurrence. For patients with high-risk HPV infection who cannot clear themselves, it is of great significance to block the persistent infection of the virus, reduce the viral load, and thus prevent cervical lesions. Human type III collagen is widely present in human tissues and plays an important structural and functional role. It is mainly distributed in the skin, blood vessels, muscles, intestinal mucosa, and the outer membrane of organs. Exogenous supplementation of active human type III collagen can moisturize and hydrate the vagina, restore the normal tissue function and elasticity of the vaginal mucosa, and help maintain the normal function of the cervix. Summary of the invention

[0003] The present invention provides a recombinant type III human collagen peptide for resisting HPV infection and a preparation method and application thereof, and more specifically relates to a recombinant type III human collagen peptide capable of binding to HPVL1 protein and use thereof in preparing a drug for preventing and / or treating HPV infection.

[0004] The present invention is achieved by the following technical scheme: a recombinant type III human collagen peptide for resisting HPV infection, whose amino acid sequence is three repetitions of the 991st to 1032nd coding sequence of type III human collagen UniProtKB: P02461, named ATHPV3, and the specific amino acid sequence is shown in SEQ ID NO.1.

[0005] The present invention also provides a gene encoding the recombinant type III human collagen peptide for resisting HPV infection as described above, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.2.

[0006] The present invention also provides a method for preparing the recombinant type III human collagen peptide for resisting HPV infection, comprising the following steps: (1) Culture: The coding sequence of type III human collagen at positions 991-1032 UniProtKB: P02461 was repeated three times and named ATHPV3; the 3' end of the sequence was connected with a histidine tag and a translation stop codon TGA, and constructed into a pCAGGS vector through restriction endonuclease sites EcoRI and XhoI, and transfected into HEK 293F cells for eukaryotic expression, and the supernatant of 293F cells after culture was collected; (2) Elution: Purify the target protein using a HisTrap HP 5 mL affinity chromatography column and elute in an elution buffer containing pH 8.0, 20 mM Tris, 150 mM NaCl, and 300 mM imidazole; (3) Purification: Purification was performed using a HiLoad 16 / 600 Superdex 75 prep gel filtration column at a flow rate of 1 ml / min in a buffer containing pH 8.0, 20 mM Tris and 150 mM NaCl. The purified protein was identified by SDS-PAGE and the molecular weight was 13 KD, which was the target collagen peptide.

[0007] The present invention also provides use of the recombinant type III human collagen peptide that resists HPV infection in preparing a drug for preventing and / or treating HPV infection.

[0008] Furthermore, the recombinant type III human collagen peptide ATHPV3 binds to HPV 16 / 18 L1 protein with affinities of 51.6 nM and 58.2 nM, respectively.

[0009] Furthermore, the recombinant type III human collagen peptide ATHPV3 forms 5 pairs of hydrogen bonds with HPV16 L1 and 4 pairs of hydrogen bonds with HPV18 L1.

[0010] The present invention is based on the sequence of human type III collagen, and is truncated and modified to design a recombinant protein peptide ATHPV3 that can bind to HPVL1 protein, which is then transfected into a plasmid suitable for HEK 293F cell expression to produce recombinant type III human collagen peptide. The SPR method is used to detect the affinity of recombinant type III human collagen peptide and HPV 16 / 18 L1 protein; molecular docking proves the interaction between the two proteins; and the luciferase reporter gene experiment is used to detect the neutralizing activity of recombinant type III human collagen peptide against HPV 16 / 18 pseudovirus.

[0011] The results showed that after expression and purification, a recombinant type III human collagen peptide with a molecular weight of about 13 kD was obtained. Compared with the eight repeats (about 30 kD) and ten repeats (about 40 kD) of the basic sequence, due to its smaller molecular weight, it can better enter the protein domain where the target is located; the affinity results showed that ATHPV3 can effectively bind to HPV 16 / 18 L1 protein with high specificity; ATHPV3 and HPV 16 / 18 L1 protein have multiple pairs of hydrogen bonds; high and medium concentrations of ATHPV3 can inhibit pseudovirus infection of 293T cells. For the body, the collagen peptide prepared by the present invention can inhibit HPV from invading target cells by binding to HPV L1, preventing viral replication and reducing the amount of virus, thereby blocking persistent viral infection and preventing and treating diseases such as cervical cancer caused by HPV.

[0012] The ATHPV3 prepared by the present invention has significant anti-HPV activity, and provides an effective method for treating HPV-infected diseases such as cervical cancer. The present invention successfully obtains a recombinant type III human collagen peptide ATHPV3 with good purity, which can bind to HPV 16 / 18 L1 and effectively inhibit HPV 16 / 18 pseudovirus from infecting 293T cells, and provides an effective candidate drug for preventing and treating HPV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the gel electrophoresis result of purified ATHPV3; Figure 2 The results of SDS-PAGE identification of purified ATHPV3; in the figure: the left picture is the collagen peptide purification picture; the right picture is the SDS-PAGE electrophoresis picture of collagen peptide; Figure 3 The results of the affinity test between recombinant type III human collagen peptide and HPV 16 / 18 L1 protein detected by SPR method; in the figure: A is the affinity test graph between negative control 2F2E5 and HPV16 L1 protein; B is the affinity test graph between negative control 2F2E5 and HPV18 L1 protein; C is the affinity test graph between collagen peptide and HPV16 L1 protein; D is the affinity test graph between collagen peptide and HPV18 L1 protein; Figure 4 : The diagram of the interaction between collagen peptide and HPV16 / 18 L1; In the figure: A is the diagram of the interaction between collagen peptide and HPV16 L1; B is the diagram of the interaction between collagen peptide and HPV18 L1; Figure 5 This is a graph showing the results of a luciferase reporter gene experiment detecting the neutralizing activity of recombinant type III human collagen peptide against HPV 16 / 18 pseudovirus. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0015] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0016] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0017] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0018] 1. Construction of recombinant type III human collagen peptide: The coding sequence of type III human collagen at positions 991 to 1032 (UniProtKB: P02461) was repeated three times and named ATHPV3. The specific amino acid sequence is shown in SEQ ID NO.1. The details are as follows: GPIGPPGPRGNRGERGSEGSPGHPGMPGPPGPPGAPGPCCGGGPIGPPGPRGNRGERGSEGSPGHPGMPGPPGPPGAPGPCCGGGPIGPPGPRGNRGERGSEGSPGHPGMPGPPGPPGAPGPCCGG.

[0019] Then, a histidine tag and a translation termination codon TGA were attached to the 3' end, and the gene was constructed into a pCAGGS vector through restriction endonuclease sites EcoRI and XhoI, and transfected into HEK 293F cells for eukaryotic expression, and the supernatant after 293F cell culture was collected.

[0020] The same method was used to prepare eight repeats (named ATHPV8) and ten repeats (named ATHPV10) of the coding sequence at positions 991-1032 as comparative experimental proteins.

[0021] Elution: The target protein was purified using a HisTrap HP 5 mL affinity chromatography column (Cytiva) in an elution buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl, and 300 mM imidazole.

[0022] Purification: Purification was performed using a HiLoad 16 / 600 Superdex 75 prep gel filtration column (Cytiva) in a buffer containing 20 mM Tris (pH 8.0) and 150 mM NaCl at a flow rate of 1 ml / min. The purified protein was identified by SDS-PAGE and its molecular weight was about 13 kD. Figure 1 , Figure 2 As shown, store at -80℃ until use.

[0023] 2. SPR method to detect the affinity between recombinant type III human collagen peptide and HPV 16 / 18 L1 protein HPV16 / 18 L1 protein was immobilized on Chip CM5 (Cytiva), and the binding kinetics of fusion protein and HPV16 / 18 L1 were detected by Biacore T200 surface plasmon resonance. The buffer system was PBST (10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.005% Tween 20).

[0024] Diluted ATHPV3, ATHPV8, and ATHPV10 (39, 78, 156, 312.5, 625, and 1250 nM) were flowed through the chip at 30 μL / min for 2 min, dissociated for 4 min, and HPV16 / 18 L1 binding was detected. After each test, 10 mM glycine (pH 3.0) was used for regeneration for 2 min.

[0025] The binding data were fitted to the Langmuir binding equation using Biacore™ T200 evaluation software. Figure 3 As shown, the results showed that ATHPV3 could bind to HPV 16 / 18 L1 with high affinity, with affinities of 51.6 nM and 58.2 nM, respectively, while ATHPV8 and ATHPV10 could not bind to HPV 16 / 18 L1 protein. It is speculated that this may be because ATHPV8 and ATHPV10 have larger molecular weights, more post-translational modifications, and more complex structural conformations, which make them unable to reach the binding site with HPV 16 / 18 L1.

[0026] 3. Molecular Docking Based on the amino acid sequence, AlphaFold2 was used to predict the three-dimensional structure of ATHPV3. The models were classified according to the pLDDT average value of 93.562, and the optimized structure with the highest ranking was selected. According to the pLDDT of the protein, UCSFChimera 1.16 was used for rendering. ZDock 3.0.2 software was used for molecular docking of HPV16 / 18 L1 and ATHPV3. The results are shown in Figure 4 As shown, the results show that the recombinant type III human collagen peptide ATHPV3 forms 5 pairs of hydrogen bonds with HPV16 L1 and 4 pairs of hydrogen bonds with HPV18 L1, allowing the recombinant type III human collagen peptide ATHPV3 to bind tightly to the two proteins. It can be seen from the figure that ATHPV3 can enter the groove area of ​​HPV16 / 18 L1 for binding. We speculate that ATHPV8 and ATHPV10 with larger molecular weight cannot be inserted into this area due to their more complex conformations, resulting in the inability to bind to HPV16 / 18 L1 to function.

[0027] 4. Luciferase reporter gene assay to detect the neutralizing activity of recombinant type III human collagen peptide against HPV 16 / 18 pseudovirus 293FT cells were cultured at 1×10 5 / mL were plated in a 96-well plate, and DMEM culture medium was used at a density of 1×10 −1 , 1×10 −2 , 1×10 −3 , 1×10 −4 , 1×10 −5 HPV 16 / 18 pseudovirus was diluted 100 μL and added to the cells. The cells were incubated at 37°C for 72 hours and observed under an inverted fluorescence microscope. The positive wells were those with more than one diseased cell. The Reed-Muench method was used to calculate the cell culture half infection dose (TCID50), i.e., the virus dilution when the virus infects half of the cells. The TCID50 endpoint dilution ratio was 1:1000.

[0028] 293FT cells were cultured at 1×10 5 / mL density was plated in a 6-well plate, and the structural gene expression plasmid and the reporter plasmid were co-transfected into 293FT cells using Lipofectamine2000. The transfected 293FT-HPV 16 / 18 L1 cells were plated at 1×10 4 / mL density was plated in a 96-well plate, and ATHPV3 was serially diluted in DMEM containing 10% fetal bovine serum, ranging from 1μM to 0.05nM. The diluted recombinant ATHPV3 and pseudovirus (dilution ratio 1:1000) were added to the 96-well plate in sequence, and the ATHPV8 group was used as the negative control group, and incubated at 37℃ for 1h. 100ul of the above mixture was added to the 293FT cell wells, incubated at 37℃ for 24h, and then the green fluorescence (reporter gene production) was observed under a fluorescence microscope. The results are as follows Figure 5 As shown, the results showed that ATHPV3 at high concentrations of 1μM and medium concentrations of 0.5μM could effectively inhibit pseudovirus infection of 293FT cells, while 1μM ATHPV8 had no significant inhibitory effect. The results of the pseudovirus-luciferase reporter gene experiment further showed that ATHPV3 further neutralized HPV 16 / 18 pseudovirus by binding to HPV 16 / 18 L1 protein, effectively reducing the viral load.

[0029] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant type III human collagen peptide for resisting HPV infection, characterized in that: The amino acid sequence is three repetitions of the coding sequence UniProtKB: P02461 of type III human collagen at positions 991 to 1032, named ATHPV3, and the specific amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the recombinant human type III collagen peptide for resisting HPV infection according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

3. The method for preparing the recombinant human type III collagen peptide for resisting HPV infection according to claim 1, characterized in that: The steps include: (1) Culture: The coding sequence of type III human collagen at positions 991-1032 UniProtKB: P02461 was repeated three times and named ATHPV3; the 3' end of the sequence was connected with a histidine tag and a translation stop codon TGA, and constructed into a pCAGGS vector through restriction endonuclease sites EcoRI and XhoI, and transfected into HEK 293F cells for eukaryotic expression, and the supernatant of 293F cells after culture was collected; (2) Elution: Purify the target protein using a HisTrap HP 5 mL affinity chromatography column and elute in an elution buffer containing pH 8.0, 20 mM Tris, 150 mM NaCl, and 300 mM imidazole; (3) Purification: In a buffer containing pH 8.0, 20 mM Tris and 150 mM NaCl, purification was performed using a HiLoad 16 / 600 Superdex 75 prep gel filtration column at a flow rate of 1 ml / min. The purified protein was identified by SDS-PAGE, and the molecular weight was 13 KD, which was the target collagen peptide.

4. Use of the recombinant human type III collagen peptide against HPV infection according to claim 1 in the preparation of a drug for preventing and / or treating HPV infection.

5. The use according to claim 4, characterized in that: The recombinant type III human collagen peptide ATHPV3 binds to HPV 16 / 18 L1 protein with affinities of 51.6 nM and 58.2 nM, respectively.

6. The use according to claim 5, characterized in that: The recombinant type III human collagen peptide ATHPV3 forms 5 pairs of hydrogen bonds with HPV16 L1 and 4 pairs of hydrogen bonds with HPV18 L1.

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