Recombinant human collagen type III peptide against HPV infection and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and provides a recombinant type III human collagen peptide for resisting HPV infection and a preparation method and application thereof, an amino acid sequence of which is a coding sequence of positions 991-1032 of type III human collagen UniProtKB: P02461 repeated three times, named as ATHPV3, and an amino acid sequence of which is shown as SEQ ID NO. 1. After expression and purification, the recombinant type III human collagen peptide with a molecular weight of about 13 kD is obtained, can be specifically combined with HPV 16 / 18 L1 protein, has multiple hydrogen bond actions, and can inhibit pseudo-virus infection of 293T cells at high and medium concentrations. The application successfully obtains a recombinant type III human collagen peptide with good purity, which can be combined with HPV 16 / 18 L1 and can effectively inhibit HPV 16 / 18 pseudo-virus infection of 293T cells, and provides an effective candidate drug for preventing and treating HPV infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a recombinant type III human collagen peptide against HPV infection and a preparation method and application thereof, and more particularly relates to a recombinant type III human collagen peptide capable of binding to HPV L1 protein and use of the recombinant type III human collagen peptide in preparation of a medicine for preventing and / or treating HPV infection. BACKGROUND

[0002] Human papillomavirus (HPV) is a circular DNA virus susceptible to epidermis and mucosal squamous epithelium. The HPV infection rate of healthy women in different regions of China is 15%-28%, and HPV persistent infection is the main cause of cervical cancer, and 70%-84% of the incidence of cervical cancer is related to HPV 16 and 18 type infection. Except for the prevention means of vaccination, there is no specific medicine at present. The existing treatment mainly through physical therapy and interferon and other adjuvant therapy, which is difficult to solve the problem of subclinical and persistent infection, causes the problem of treating the symptoms but not the disease and easy recurrence. For the high-risk HPV infection patients who cannot self-clear, it is of great significance to block the persistent infection of the virus, reduce the viral load, and then prevent cervical lesions. Human type III collagen widely exists in human tissues and plays an important structural and functional role, which is mainly distributed in skin, blood vessels, muscles, intestinal mucosa, outer membrane of organs and the like. Exogenous supplement of active human type III collagen can play a role in moisturizing and moisturizing the vagina, restoring the normal tissue function of vaginal mucosa and the elasticity of vagina, and help to maintain the normal function of cervix. SUMMARY

[0003] The present application provides a recombinant type III human collagen peptide against HPV infection and a preparation method and application thereof, and more particularly relates to a recombinant type III human collagen peptide capable of binding to HPV L1 protein and use of the recombinant type III human collagen peptide in preparation of a medicine for preventing and / or treating HPV infection.

[0004] The present application is realized by the following technical solutions, a recombinant type III human collagen peptide against HPV infection, the amino acid sequence of which is three repetitions of the coding sequence of the 991th-1032th position of type III human collagen UniProtKB: P02461, named as ATHPV3, and the specific amino acid sequence is shown in SEQ ID NO. 1.

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

[0006] The present application also provides a method for preparing the recombinant type III human collagen peptide against HPV infection, comprising the following steps:

[0007] (1) Cultivation: the sequence encoding 991-1032 of human type III collagen UniProtKB: P02461 is repeated three times and named ATHPV3; a histidine tag and a translation termination codon TGA are added to the 3' end of the sequence, which is constructed into a pCAGGS vector through restriction enzyme sites EcoRI and XhoI, transfected into HEK 293F cells for eukaryotic expression, and the supernatant of the 293F cells after cultivation is collected;

[0008] (2) Elution: the HisTrap HP 5 mL affinity column is used to purify the target protein, and the target protein is eluted in an elution buffer containing pH 8.0, 20 mM Tris, 150 mM NaCl and 300 mM imidazole;

[0009] (3) Purification: the HiLoad 16 / 600 Superdex 75 prep gel filtration column is used for purification in a buffer containing pH 8.0, 20 mM Tris and 150 mM NaCl at a flow rate of 1 ml / min; the purified protein is identified by SDS-PAGE, and the molecular weight is 13KD, which is the target collagen peptide.

[0010] The application further provides a use of the recombinant type III human collagen peptide for resisting HPV infection in the preparation of a medicine for preventing and / or treating HPV infection.

[0011] Further, the recombinant type III human collagen peptide ATHPV3 is combined with HPV 16 / 18 L1 protein, and the affinities are 51.6 nM and 58.2 nM, respectively.

[0012] Further, the recombinant type III human collagen peptide ATHPV3 forms 5 pairs of hydrogen bonds with HPV 16 L1 and 4 pairs of hydrogen bonds with HPV 18 L1.

[0013] The application designs a recombinant protein peptide ATHPV3 capable of being combined with HPV L1 protein on the basis of human type III collagen sequence, and then the recombinant protein peptide is transfected into a plasmid suitable for HEK 293F cell expression for producing the recombinant type III human collagen peptide. The SPR method is used to detect the affinity of the recombinant type III human collagen peptide with HPV 16 / 18 L1 protein; the molecular docking is used to prove the interaction force between the two proteins; and the luciferase reporter gene experiment is used to detect the neutralization activity of the recombinant type III human collagen peptide on HPV 16 / 18 pseudovirus.

[0014] The results show that: after expression and purification, the recombinant type III human collagen peptide with a molecular weight of about 13 kD is obtained, compared with 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 point is located; the affinity results show that ATHPV3 can effectively bind with HPV 16 / 18 L1 protein, and has high specificity; ATHPV3 and HPV 16 / 18 L1 protein exist multiple hydrogen bond interactions; high and medium concentrations of ATHPV3 can inhibit the pseudovirus infection of 293T cells. For the organism, the collagen peptide prepared in the application can inhibit HPV invasion into target cells by binding with HPV L1, prevent virus replication, reduce virus quantity, thereby block the persistent infection of the virus, and achieve the prevention and treatment of diseases such as cervical cancer caused by HPV.

[0015] The ATHPV3 prepared in the application has significant anti-HPV activity, and provides an effective method for treating HPV infection diseases such as cervical cancer. The application successfully obtains a recombinant type III human collagen peptide ATHPV3 which has good purity, can bind with HPV 16 / 18 L1, and can effectively inhibit the infection of HPV 16 / 18 pseudovirus on 293T cells, thereby providing an effective candidate drug for preventing and treating HPV infection. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is the gel electrophoresis result of the purified ATHPV3; Figure 2 It is the SDS-PAGE identification result of the purified ATHPV3; in the figure, the left graph is the collagen peptide purification graph, and the right graph is the collagen peptide SDS-PAGE electrophoresis graph; Figure 3 It is the SPR method detection result of the affinity of the recombinant type III human collagen peptide and HPV 16 / 18 L1 protein; in the figure, A is the affinity detection graph of the negative control 2F2E5 and HPV 16 L1 protein; B is the affinity detection graph of the negative control 2F2E5 and HPV 18 L1 protein; C is the affinity detection graph of the collagen peptide and HPV 16 L1 protein; D is the affinity detection graph of the collagen peptide and HPV 18 L1 protein; Figure 4 It is the interaction graph of the collagen peptide and HPV 16 / 18 L1; in the figure, A is the interaction graph of the collagen peptide and HPV 16 L1; B is the interaction graph of the collagen peptide and HPV 18 L1; Figure 5 It is the luciferase reporter gene experiment detection result graph of the neutralization activity of the recombinant type III human collagen peptide on HPV 16 / 18 pseudovirus. DETAILED DESCRIPTION

[0017] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0018] Unless otherwise defined, 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 application belongs, and the materials referred to in this disclosure are incorporated by reference.

[0019] The equivalent techniques of the described specific embodiments, which can be realized by the ordinary skill in the art without creative work, are included in the present application.

[0020] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The instruments and equipment used in the following embodiments are all conventional laboratory instruments and equipment unless otherwise specified. The experimental materials used in the following embodiments are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0021] I. Construction of recombinant human type III collagen peptide: The coding sequence of 991-1032 of human type III collagen (UniProtKB: P02461) is repeated three times, named ATHPV3, and the specific amino acid sequence is shown in SEQ ID NO. 1. The specific process is as follows:

[0022] GPIGPPGPRGNRGERGSEGSPGHPGMPGPPGPPGAPGPCCGGGPIGPPGPRGNRGERGSEGSPGHPGMPGPPGPPGAPGPCCGGGPIGPPGPRGNRGERGSEGSPGHPGMPGPPGPPGAPGPCCGG.

[0023] Then, a histidine tag and a translation termination codon TGA are connected at the 3' end, and the recombinant protein is constructed into a pCAGGS vector through restriction enzyme sites EcoRI and Xhol, transfected into HEK 293F cells for eukaryotic expression, and the supernatant after 293F cell culture is collected.

[0024] Using the same method, eight repeats (named ATHPV8) and ten repeats (named ATHPV10) of the coding sequence of 991-1032 are prepared as comparative experimental proteins.

[0025] Elution: The HisTrap HP 5 mL affinity column (Cytiva) was used to purify the target protein, and the target protein was eluted in an elution buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl and 300 mM imidazole.

[0026] Purification: The HiLoad 16 / 600 Superdex 75 prep gel filtration column (Cytiva) was used to purify the target protein in a buffer containing 20 mM Tris (pH 8.0) and 150 mM NaCl at a flow rate of 1 ml / min. After the purified protein was identified by SDS-PAGE, the molecular weight of the protein was about 13 kD, as shown in Figure 1 、 Figure 2 The purified protein was stored at -80°C for later use.

[0027] II. SPR method for detecting the affinity of recombinant type III human collagen peptide to HPV 16 / 18 L1 protein

[0028] The HPV16 / 18 L1 protein was immobilized on the Chip CM5 (Cytiva) chip, and the Biacore T200 surface plasmon resonance instrument was used to detect the binding kinetics of the fusion protein to HPV16 / 18 L1. 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).

[0029] The diluted ATHPV3, ATHPV8, ATHPV10 (39, 78, 156, 312.5, 625, 1250 nM) were flowed through the chip at a flow rate of 30 μL / min for 2 min, and the dissociation was 4 min. The binding of HPV16 / 18 L1 was detected. After each detection, 10 mM glycine (pH 3.0) was used for regeneration for 2 min.

[0030] The binding data was fitted to the Langmuir binding equation by Biacore™ T200 evaluation software, and the results are shown in Figure 3 The results show that ATHPV3 can bind to HPV 16 / 18 L1 with high affinity, with affinities of 51.6 nM and 58.2 nM, respectively, while ATHPV8 and ATHPV10 cannot bind to HPV 16 / 18 L1 protein, which is speculated to be due to the larger molecular weight of ATHPV8 and ATHPV10, more post-translational modifications, and more complex structural conformation, resulting in the inability to reach the binding site of HPV 16 / 18 L1.

[0031] III. Molecular docking

[0032] The three-dimensional structure of ATHPV3 was predicted using AlphaFold2 based on the amino acid sequence, and the optimized structure with the highest ranking was selected according to the pLDDT average value of 93.562. According to the pLDDT of the protein, rendering was performed using UCSF Chimera 1.16. HPV16 / 18 L1 and ATHPV3 molecular docking was performed using ZDock 3.0.2 software. The results are shown in Figure 4 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 tightly bind to both proteins. As can be seen from the figure, ATHPV3 can enter the HPV16 / 18 L1 groove region and bind, and we speculate that ATHPV8 and ATHPV10, which have a larger molecular weight, cannot insert into this region due to their more complex conformation, resulting in their inability to bind to HPV16 / 18 L1 to function.

[0033] Four, luciferase reporter gene experiment to detect the neutralizing activity of recombinant type III human collagen peptide on HPV 16 / 18 pseudovirus

[0034] 293FT cells were plated in a 96-well plate at a density of 1×10 5 / mL, and DMEM medium was diluted 1×10 −1 , 1×10 −2 , 1×10 −3 , 1×10 −4 , 1×10 −5 times. 100 μL of HPV 16 / 18 pseudovirus was added to the cells, and incubated at 37°C for 72 h. The presence of more than one lesion cell under an inverted fluorescence microscope was considered positive. The Reed-Muench method was used to calculate the median cell culture infectious dose (TCID50), which is the virus dilution when half of the cells are infected with the virus. The TCID50 endpoint dilution ratio was 1:1000.

[0035] 293FT cells were plated in a 6-well plate at a density of 1×10 5 / mL, and the structural gene expression plasmid and reporter plasmid were co-transfected into 293FT cells using Lipofectamine 2000. The transfected 293FT-HPV 16 / 18 L1 cells were plated in a 6-well plate at a density of 1×10 4The 293FT cells were seeded in 96-well plates at a density of 1 x 104 / mL, and the recombinant ATHPV3 was serially diluted in DMEM containing 10% fetal bovine serum, with a range of 1 μM ~ 0.05 nM. The diluted recombinant ATHPV3 and the pseudovirus (dilution ratio 1:1000) were sequentially added to the 96-well plates, and the ATHPV8 group was used as a negative control group, and incubated at 37°C for 1 h. 100 ul of the above mixture was added to the 293FT cell wells, and incubated at 37°C for 24 h. Then, green fluorescence (reporter gene production) was observed under a fluorescence microscope, and the results are shown in Figure 5 FIG. 6. The results show that ATHPV3 at a high concentration of 1 μM and a medium concentration of 0.5 μM can effectively inhibit the infection of 293FT cells by the pseudovirus, while ATHPV8 at a concentration of 1 μM has no significant inhibitory effect. The results of the pseudovirus-luciferase reporter gene experiment further show that ATHPV3 can neutralize the HPV 16 / 18 pseudovirus by binding to the HPV 16 / 18 L1 protein, and can effectively reduce the viral load.

[0036] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some or all of the technical features; and these modifications or substitutions do not cause the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A recombinant human collagen type III peptide against HPV infection, characterized in that: The amino acid sequence is shown as SEQ ID NO.

1.

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

2.

3. A method of preparing the recombinant human collagen type III peptide against HPV infection according to claim 1, characterized in that: The method comprises the following steps: (1) Cultivation: the 3' end of the sequence shown as SEQ ID NO. 1 is connected with a histidine tag and a translation termination codon TGA, which is constructed into a pCAGGS vector through restriction enzyme sites EcoRI and XhoI, transfected into HEK 293F cells for eukaryotic expression, and the supernatant of the 293F cells after culture is collected; (2) Elution: the HisTrap HP 5 mL affinity chromatography column is used to purify the target protein, and the target protein is eluted in an elution buffer containing pH 8.0, 20 mM Tris, 150 mM NaCl and 300 mM imidazole; (3) Purification: the HiLoad 16 / 600 Superdex 75 prep gel filtration chromatography column is used to purify the target protein in a buffer containing pH 8.0, 20 mM Tris and 150 mM NaCl at a flow rate of 1 ml / min; the purified protein is identified by SDS-PAGE, and the molecular weight size is 13KD, which is the target collagen peptide.

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

Images