Novel self-encapsulated MVP-INT nano protein particle and application thereof

Abstract

The invention relates to the technical field of molecular biology, and discloses a novel self-encapsulated MVP-INT nano protein particle and application thereof. The invention relates to a novel self-encapsulated MVP-INT nano protein particle, which is formed by connecting an MVP gene, a self-cleavage inclusion gene and an INT gene in series. According to the novel self-encapsulated MVP-INT nano-protein particle provided by the invention, MVP nano-particles and INT protein are designed together through self-cutting intein, so that protein nano-particles capable of self-encapsulating drugs are prepared, the tedious process of encapsulating the drugs by using the nano-particles is reduced, the use problem and challenge of MVP as a nano-carrier are optimized, and the application prospect is wide. The drug encapsulation efficiency of the nano-carrier is improved, and market value is provided for research and development of universal nano-particle carriers.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and more specifically, to a novel self-encapsulated MVP-INT nanoprotein particle and its applications. Background Technology

[0002] Significant progress has been made in nanoparticle drug delivery systems (NDDS) in recent years, encompassing various types including liposomes, polymer nanoparticles, solid lipid nanoparticles (SLNs), nanocapsules, inorganic nanoparticles, and self-assembled nanoparticles. These nanoparticle drug delivery systems are widely used in fields such as cancer treatment, vaccine delivery, and gene therapy by improving drug solubility, bioavailability, and targeted delivery capabilities. Among them, protein nanoparticles based on polymer nanoparticles have received increasing attention in recent years due to their excellent biocompatibility, biodegradability, and low toxicity. Protein nanoparticles can not only efficiently encapsulate drugs but also achieve targeted delivery and intelligent responsive release through surface functionalization, demonstrating enormous application potential. Recent studies have shown that protein nanoparticles have been successfully applied in multiple fields such as anticancer drugs, vaccine delivery, and gene therapy, becoming an important direction for development in the pharmaceutical industry.

[0003] Self-assembled nanoparticles (SANPs) have become a research hotspot in the field of nanomedicine delivery in recent years. SANPs spontaneously form nanoscale structures through intermolecular interactions (such as hydrophobic interactions and hydrogen bonding), enabling effective drug loading and improving drug bioavailability, targeting, and controlled release properties. However, the core competitive advantage of SANPs lies in the simplicity and low cost of their preparation process. Traditional nanocarriers typically require complex physicochemical methods for synthesis, while SANPs rely on the natural spontaneous aggregation of molecules, allowing for efficient drug loading and controlled drug release through molecular design adjustments. Over the past decade, researchers have optimized the design of SANPs in various ways to adapt to different therapeutic needs. While SANPs have attracted significant attention in drug delivery systems due to their efficient drug loading capacity, their biocompatibility remains a significant concern. Firstly, SANPs may trigger immune responses, such as recognition by the complement system or phagocytosis by macrophages, leading to rapid nanoparticle clearance or inducing local inflammation. Secondly, the degradation products of some self-assembled nanoparticles may be cytotoxic or cause organ damage, especially in polymer-based carriers, where the degradation products may be toxic to organs such as the liver and kidneys. Furthermore, nanoparticles may accumulate in vivo, particularly in immune organs such as the liver and spleen; prolonged accumulation may lead to long-term activation of the immune system or organ dysfunction. In addition, while surface modifications (such as PEGylation) can improve biocompatibility, excessive modification may affect the targeting and drug release performance of nanoparticles. Therefore, despite the potential of self-assembled nanoparticles in drug delivery, biocompatibility remains a major obstacle limiting their clinical application. Summary of the Invention

[0004] The purpose of this invention is to provide a novel self-encapsulated MVP-INT nanoprotein particle and its application.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] One of the technical solutions of this invention:

[0007] A novel self-encapsulated MVP-INT nanoprotein particle is composed of an MVP gene, a self-cleaving intron gene, and an INT gene linked together.

[0008] Furthermore, the amino acid sequence of the MVP gene is shown in SEQ ID No. 1;

[0009] SEQ ID No.1

[0010] MATEEAIIRIPPYHYIHVLDQNSNVSRVEVGPKTYIRQDNERVLFAPVRMVT

[0011] VPPRHYCIVANPVSRDAQSSVLFDVTGQVRLRHADQEIRLAQDPFPLYPGEL

[0012] LEKDITPLQVVLPNTALHLKALLDFEDKNGDKVMAGDEWLFEGPGTYIPQK

[0013] EVEVVEIIQATVIKQNQALRLRARKECFDRDGKERVTGEEWLVRSVGAYLP

[0014] AVFEEVLDLVDAVILTEKTALHLRARQNFKDLRGVAHRTGEEWLVTVQDT

[0015] EAHVPDVYEEVLGVVPITTLGPRHYCVILDPMGPDGKNQLGQKRVVKGEK

[0016] SFFLQPGERLERGIQDVYVLSEQQGLLLKALQPLEEGEGEEKVAHQAGDRW

[0017] LIRGPLEYVPSAKVEVVEERQAIPLDQNEGIYVQDVKTGKVRAVIGSTYML

[0018] TQDEVLWEKELPSGVEELLNLGHDPLADRGQKGTAKVLQPSAARNKTRVV

[0019] SYRVPHNAAVQVYDYRAKRARVVFGPELVSLDPEEQFTVLSLSAGRPKRPH

[0020] ARRALCLLLGPDFFTDVITIETADHARLQLQLAYNWHFELKNRNDPEETAK

[0021] LFSVPDFVGDACKAIASRVRGAVASVTFDDFHKNSARIIRMAVFGFEMSED

[0022] AGPDGALLPRARDRAVFPQNGLVVSSVDVQSVEPVDQRTRDALQRSVQLA

[0023] IEITTNSQEAAAKHEAQRLEQEARGRLERQKILDQSEAEKARKELLELEAMS

[0024] MAVESTGNAKAEAESRAEAARIEGEGSVLQAKLKAQALAIETEAELERVKK

[0025] VREMELIYSRAQLELEVSKAQQLADVEAKKFKEMTEALGPGTIRDLAVAGPEMQVKLLQSLGLKSTLITDGSSPINLFNTAFGLLGLGSDGQPPVQK.

[0026] Furthermore, the amino acid sequence of the self-cleaving intron gene is shown in SEQ ID No. 2;

[0027] SEQ ID No.2

[0028] MSIRDEALLVGTKVTTKAGDKNIENITLEDEVLQFDMNTKDFSYTNPTKTQ

[0029] KVIRDEIYHFEGAGFDQKVSPNHRMIYEQGGEIKECLAKDFEPSEDKYFIIVE

[0030] GSHMQIKRIKSTDVKITHTKLDEPTEFHALSVPGKSFVVTDEHGNRSVTGASMHVEGKLGG.

[0031] Furthermore, the amino acid sequence of the INT gene is shown in SEQ ID No. 3;

[0032] SEQ ID No. 3

[0033] CTQHWQDAVPWTELLSLQTEDGFWKLTPELGLILNLNTNGLHSFLKQKGIQ

[0034] SLGVKGRECLLDLIATMLVLQFIRTRLEKEGIVFKSLMKMDDPSISRNIPWA

[0035] FEAIKQASEWVRRTEGQYPSICPRLELGNDWDSATKQLLGLQPISTVSPLHRVLHYSQG.

[0036] The second technical solution of this invention:

[0037] The preparation method of the above-mentioned novel self-encapsulated MVP-INT nanoprotein particles includes the following steps:

[0038] (1) Preparation of a novel self-encapsulated MVP-INT nanoprotein nanoparticle eukaryotic expression system:

[0039] The GAP promoter (PGAP) downstream of the yeast vector pGAPZA (Invitrogen) was subcloned into the EcoRI and KpnI sites of MVP to generate the yeast expression vector yMVP-pGAPZA. Subsequently, yMVP-pGAPZA and BspHI were linearized and transformed into the P. pastoris protease-deficient strain SMD1168. Electroporation was performed using a single pulse at 1.5 kV, 200 Ω, and 25 μF. The transformation medium was placed on YPDS agar containing 100 μg / mL of the antibiotic Zeocin. Zeocin-resistant transformants were screened by re-banding on fresh YPD plates containing Zeocin to select individual colonies. Eleven positive colonies were inoculated into 3 mL of agar containing 100 μg / mL Zeocin. Cells were cultured overnight in YPD medium at 30°C and 200 rpm. Cells were collected, lysed, and subjected to SDS-PAGE and Coomassie Brilliant Blue staining to confirm MVP expression. Colonies with the highest MVP production were selected for subsequent experiments. To assess the time course of MVP expression, selected colonies were inoculated into 3 mL of YPD medium containing 100 μg / mL Zeocin and cultured overnight at 30°C and 200 rpm. Then, 500 mL of YPD medium was inoculated until the OD600 reached 0.03. The culture was stored at 30°C and 200 rpm for 30 h. Cells were harvested in pre-weighed 50 mL tubes, centrifuged at 3000 rpm for 5 min at 4°C, washed with 10 mL of deionized water, and the wet cell microspheres were weighed and stored at -80°C.

[0040] (2) Preparation of a novel self-encapsulated MVP-INT nanoprotein nanoparticle prokaryotic expression system:

[0041] Take 1 mL of overnight BL21 bacterial culture and place it in a 1.5 mL microtube. Centrifuge at 1500 × g (4000 rpm) at 4°C for 5 min, and discard the supernatant. Add 100 μL of Solution A pre-chilled on ice to the microtube, and gently tap the microtube to suspend the precipitate; do not shake vigorously. Centrifuge at 1500 × g (4000 rpm) at 4°C for 5 min, and discard the supernatant. Add 100 μL of Solution A pre-chilled on ice to each microtube. B. Gently tap the microtube to suspend the precipitate; avoid vigorous shaking. The competent cells are now ready and stored at -80°C for later use. Thaw the -80°C competent cells on ice for 10 minutes. Transfer 100 μL of competent cells to a new transformation tube. Add 0.1 ng to 10 ng (3 μL to 10 μL) of transformation DNA to the competent cells, mix gently, and incubate on ice for 30 minutes. Incubate in a 42°C water bath for 45 seconds, then immediately place on ice for 1 to 2 minutes. Add 890 μL of pre-warmed LB medium at 37°C. Incubate at 37°C with shaking for 1 hour. Spread a suitable amount onto a plate and invert the plate in a 37°C incubator overnight. Identify and culture the bacterial colonies for the next step of the experiment; Activation culture: On the second day, pick a single colony and place it in liquid culture medium, incubate at 37℃ and 210 rpm for 4 hours; Scale-up culture: Add the activated bacterial culture to 300 mL of liquid culture medium at a volume ratio of 1:100 for scale-up culture; incubate at 20℃ and 210 rpm for 4 hours; Second scale-up culture: Mix the scaled-up bacterial culture with 300 mL of liquid culture medium and then separate them at a 1:1 ratio; incubate at 28℃ and 210 rpm for 1 hour; Add IPTG at a volume ratio of IPTG:bacterial culture = 1:1000, and incubate at 20℃ and 210 rpm for 6 hours with shaking to complete the induction; Take 1 mL of the culture before and after induction expression. Store at 4℃ for electrophoresis and as a control after induction; centrifuge at 8000-10000 rpm for 10 min, discard the supernatant, transfer the precipitate to another tube, and weigh the precipitate; resuspend the precipitate in TRIS solution at pH 8 (pH > protein isoelectric point 0.5); centrifuge at 10000 rpm for 10 min, discard the supernatant, transfer the precipitate to another tube, and weigh the precipitate; resuspend the precipitate in 3 mL / 10 mL TRIS solution at pH 8, and disrupt at 200 W for 5 min; centrifuge at 10000 rpm for 10 min after disruption, collect the supernatant, resuspend the precipitate in TRIS solution at a ratio of 1:100, and store at 4℃;

[0042] (3) Purification and characterization of novel self-encapsulated MVP-INT nanoprotein particles:

[0043] The MVP protein was purified, and the purification success was observed by SDS-PAGE protein electrophoresis. Then, the MVP nanoparticles were separated from the INT protein using self-cleaving inlets and incubated on ice for 0.5 h to obtain the novel self-encapsulated MVP-INT nanoparticles.

[0044] The third technical solution of this invention:

[0045] The aforementioned novel self-encapsulated MVP-INT nanoprotein particles serve as efficient and safe drug delivery carriers for applications in anticancer drugs, vaccine delivery, and gene therapy.

[0046] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0047] This invention provides a novel self-encapsulated MVP-INT nanoprotein particle that solves the technical problems of poor stability, low encapsulation efficiency, and difficulty in achieving controlled release of existing nanoparticles.

[0048] This invention provides a novel self-encapsulated MVP-INT nanoprotein particles, which use MVP protein derived from organisms themselves, exhibiting good biocompatibility and low toxicity, thus reducing tissue compatibility issues during application.

[0049] This invention provides a novel method for preparing self-encapsulated MVP-INT nanoprotein nanoparticles, which strategically simplifies the expression and purification process and utilizes Escherichia coli as an expression vector to reduce the cost of the preparation process.

[0050] This invention provides a novel method for preparing self-encapsulated MVP-INT nanoprotein nanoparticles. It employs a specific self-assembly technique and significantly improves the assembly stability of nanoparticles and the encapsulation efficiency of drugs by regulating the interaction between proteins and target molecules. At the same time, it introduces purification tags, self-cleaving integrities, etc. to fuse and form monomers, polymers, or particles, thereby achieving controlled release and efficient delivery of drugs.

[0051] This invention provides a novel method for preparing self-encapsulated MVP-INT protein nanoparticles. By self-cleaving the inner peptides, MVP nanoparticles are designed to be combined with INT proteins to prepare protein nanoparticles that can self-encapsulate drugs. This reduces the cumbersome process of using nanoparticles to encapsulate drugs, optimizes the use of MVP as a nanocarrier and addresses the problems and challenges, improves the efficiency of drug encapsulation by nanocarriers, and provides market value for the development of universal nanoparticle carriers. Attached Figure Description

[0052] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0053] Figure 1 This is a flowchart illustrating the preparation process of the novel self-encapsulated MVP-INT nanoprotein particles of this invention.

[0054] Figure 2 This is the result of SDS-PAGE testing;

[0055] Figure 3 This is the result of a cytotoxicity test;

[0056] Figure 4 This is the result of a safety inspection.

[0057] Figure 5 This is the result of live imaging detection;

[0058] Figure 6 The results are from the compound immunogenicity test.

[0059] Figure 7 This is the result of an allergic reaction test. Detailed Implementation

[0060] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0061] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0062] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0063] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0064] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0065] Example

[0066] Preparation of a novel self-encapsulated MVP-INT nanoprotein particles

[0067] (1) Preparation of a novel self-encapsulated MVP-INT nanoprotein nanoparticle eukaryotic expression system:

[0068] The GAP promoter (PGAP) downstream of the yeast vector pGAPZA (Invitrogen) was subcloned into the EcoRI and KpnI sites of MVP to generate the yeast expression vector yMVP-pGAPZA. Subsequently, yMVP-pGAPZA and BspHI were linearized and transformed into the P. pastoris protease-deficient strain SMD1168. Electroporation was performed using a single pulse at 1.5 kV, 200 Ω, and 25 μF. The transformation medium was placed on YPDS agar containing 100 μg / mL of the antibiotic Zeocin. Zeocin-resistant transformants were screened by re-banding on fresh YPD plates containing Zeocin to select individual colonies. Eleven positive colonies were inoculated into 3 mL of agar containing 100 μg / mL Zeocin. Cells were cultured overnight in YPD medium at 30°C and 200 rpm. Cells were collected, lysed, and subjected to SDS-PAGE and Coomassie Brilliant Blue staining to confirm MVP expression. Colonies with the highest MVP production were selected for subsequent experiments. To assess the time course of MVP expression, selected colonies were inoculated into 3 mL of YPD medium containing 100 μg / mL Zeocin and cultured overnight at 30°C and 200 rpm. Then, 500 mL of YPD medium was inoculated until the OD600 reached 0.03. The culture was stored at 30°C and 200 rpm for 30 h. Cells were harvested in pre-weighed 50 mL tubes, centrifuged at 3000 rpm for 5 min at 4°C, washed with 10 mL of deionized water, and the wet cell microspheres were weighed and stored at -80°C.

[0069] (2) Preparation of a novel self-encapsulated MVP-INT nanoprotein nanoparticle prokaryotic expression system:

[0070] Take 1 mL of overnight BL21 bacterial culture and place it in a 1.5 mL microtube. Centrifuge at 1500 × g (4000 rpm) at 4°C for 5 min, and discard the supernatant. Add 100 μL of Solution A pre-chilled on ice to the microtube, and gently tap the microtube to suspend the precipitate; do not shake vigorously. Centrifuge at 1500 × g (4000 rpm) at 4°C for 5 min, and discard the supernatant. Add 100 μL of Solution A pre-chilled on ice to each microtube. B. Gently tap the microtube to suspend the precipitate; avoid vigorous shaking. The competent cells are now ready and stored at -80°C for later use. Thaw the -80°C competent cells on ice for 10 minutes. Transfer 100 μL of competent cells to a new transformation tube. Add 0.1 ng to 10 ng (3 μL to 10 μL) of transformation DNA to the competent cells, mix gently, and incubate on ice for 30 minutes. Incubate in a 42°C water bath for 45 seconds, then immediately place on ice for 1 to 2 minutes. Add 890 μL of pre-warmed LB medium at 37°C. Incubate at 37°C with shaking for 1 hour. Spread a suitable amount onto a plate and invert the plate in a 37°C incubator overnight. Identify and culture the bacterial colonies for the next step of the experiment; Activation culture: On the second day, pick a single colony and place it in liquid culture medium, incubate at 37℃ and 210 rpm for 4 hours; Scale-up culture: Add the activated bacterial culture to 300 mL of liquid culture medium at a volume ratio of 1:100 for scale-up culture; incubate at 20℃ and 210 rpm for 4 hours; Second scale-up culture: Mix the scaled-up bacterial culture with 300 mL of liquid culture medium and then separate them at a 1:1 ratio; incubate at 28℃ and 210 rpm for 1 hour; Add IPTG at a volume ratio of IPTG:bacterial culture = 1:1000, and incubate at 20℃ and 210 rpm for 6 hours with shaking to complete the induction; Take 1 mL of the culture before and after induction expression. Store at 4℃ for electrophoresis and as a control after induction; centrifuge at 8000-10000 rpm for 10 min, discard the supernatant, transfer the precipitate to another tube, and weigh the precipitate; resuspend the precipitate in TRIS solution at pH 8 (pH > protein isoelectric point 0.5); centrifuge at 10000 rpm for 10 min, discard the supernatant, transfer the precipitate to another tube, and weigh the precipitate; resuspend the precipitate in 3 mL / 10 mL TRIS solution at pH 8, and disrupt at 200 W for 5 min; centrifuge at 10000 rpm for 10 min after disruption, collect the supernatant, resuspend the precipitate in TRIS solution at a ratio of 1:100, and store at 4℃;

[0071] (3) Purification and characterization of novel self-encapsulated MVP-INT nanoprotein particles:

[0072] The MVP protein was purified, and the purification success was observed by SDS-PAGE protein electrophoresis. Then, the MVP nanoparticles were separated from the INT protein using self-cleaving inlets and incubated on ice for 0.5 h to obtain the novel self-encapsulated MVP-INT nanoparticles.

[0073] The preparation flow chart of the novel self-encapsulated MVP-INT nanoprotein particles of this invention is shown below. Figure 1 As shown.

[0074] The novel self-encapsulated MVP-INT nanoprotein particles prepared in the examples were analyzed by SDS-PAGE. The SDS-PAGE results are as follows: Figure 2 As shown;

[0075] Depend on Figure 2 It can be seen that after the target protein was processed by Sephacryl S-1000 gel chromatography column and Butyl hydrophobic column, the SDS-PAGE results showed a single band with a molecular weight of about 200 kDa, which meets the requirements of nanomedicine.

[0076] Effect verification:

[0077] I. In vitro biological evaluation of novel self-encapsulated MVP-INT nanoprotein particles

[0078] 1. Cytotoxicity of novel self-encapsulated MVP-INT nanoprotein particles

[0079] A breast cancer cell line (4T1) in the logarithmic growth phase was prepared into a single-cell suspension by trypsin digestion, and then... 4 Cells were seeded at a density of 1000 ppm in 96-well plates and cultured overnight to allow cell adhesion. After adding MVP recombinant protein carrier particles that release PTX and CPG for 24 hours, the cells were incubated in culture medium containing CCK-8. The UV absorbance values ​​(λ = 450 nm) of the blank group (A0), PBS group (A1), and novel self-encapsulated MVP-INT nanoprotein particle group (A2) were measured using a microplate reader. Cell viability was calculated using the following formula.

[0080] Cell viability (%) = (A2-A0) / (A1-A0) × 100%;

[0081] Cytotoxicity test results as follows Figure 3 As shown;

[0082] Depend on Figure 3 It was found that when the MVP-INT concentration reached 400 μg / mL, the cell viability still exceeded 80%, and when the concentration was below 200 μg / mL, there was no significant impact on cell viability, demonstrating that MVP-INT exhibits good biocompatibility.

[0083] 2. Safety of MVP recombinant protein vector

[0084] 50 μL of red blood cells were dispersed in 950 μL of physiological saline containing different concentrations of MVP recombinant protein carrier particles (10, 25, 50, 100 μg) and mixed at 450 rpm at 37°C. The mixture was then centrifuged at 800 rpm for 5 min to collect intact red blood cells. The absorbance of the supernatant was measured at 540 nm. Physiological saline (0.9% NaCl) served as a negative control (0% lysis), and distilled water served as a positive control (100% lysis). The hemolysis rate was calculated using the following formula:

[0085] Hemolysis rate (%) = (Dt - Dnt) / (Dpc - Dnc) × 100

[0086] Where Dt, Dnc, and Dpc are the absorbance of the tested sample, negative control, and positive control, respectively;

[0087] Safety inspection results as follows Figure 4 As shown;

[0088] Depend on Figure 4 It was found that after an incubation time of 4 hours, none of the MVP-INT nanoparticles at any concentration caused significant hemolysis, and the hemolysis rate did not exceed 5%. This further verifies the biocompatibility of MVP-INT nanoparticles; when interacting with red blood cells, they do not cause red blood cell rupture and can well maintain their original structure, demonstrating good blood compatibility.

[0089] II. In vivo biological evaluation of novel self-encapsulated MVP-INT nanoprotein particles

[0090] 1. Tumor model establishment and in vivo imaging

[0091] Log-phase human breast cancer cell line (4T1) was collected, digested with trypsin, and resuspended to prepare a single-cell suspension. 0.2 mL of this suspension was subcutaneously injected into the right upper fat pad of BALB / c nude mice (1×10⁻⁶ cells / mL). 5 (One cell / mouse) were fed for two weeks to allow the tumor to grow; when the tumor reached a diameter of about 0.6-0.8 cm, near-infrared fluorescence in vivo imaging was performed on the tumor-bearing mice; the mice were stratified and randomly divided into PTX and CPG injection groups (n=10), novel self-encapsulated MVP-INT nanoprotein particle group (n=10), and blank control group PBS (n=10) according to their body weight. In vivo imaging was performed using the IVIS small animal optical in vivo imaging system at 0, 1, 2, 4, 6, 8, 24, and 48 h, and the imaging parameters were kept consistent.

[0092] Live imaging detection results as follows Figure 5 As shown;

[0093] Depend on Figure 5 It can be seen that, over time, the fluorescence signals at the tumor sites in mice decreased to varying degrees. However, the rate of decrease in fluorescence signal in the ICG / MVP-INT group was much smaller than that in the Free ICG group. This indicates that while ICG is rapidly metabolized by mice, MVP-INT can be effectively enriched at the tumor sites and its rate of metabolism by mice is greatly reduced.

[0094] 2. Immunogenicity of novel self-encapsulated MVP-INT nanoprotein particles

[0095] Five BALB / c mice were injected intramuscularly into the abdomen and inner thigh with novel self-encapsulated MVP-INT nanoprotein particles and physiological saline, with an immunization dose of 0.5 mL. The mice were immunized three times, once every 7 days. Blood was collected from the tail at 7, 14 and 21 days after immunization to measure the antibody titer in the serum.

[0096] The results of the compound immunogenicity test are as follows Figure 6 As shown;

[0097] Depend on Figure 6 It can be seen that comparing the OD450-630 of serum diluted 800 times after 21 days of MVP-INT and PBS triple immunization showed no significant difference (P value < 0.01), proving that MVP-INT does not stimulate or only slightly stimulates the production of low levels of antibodies in mice.

[0098] 3. Allergic reactions to novel self-encapsulated MVP-INT nanoprotein particles

[0099] Five adult BALB / c mice were used to sensitize each mouse by intraperitoneal injection of novel self-encapsulated MVP-INT nanoprotein particles and 0.5 mL (2 mg / kg) of PBS solution once a week for a total of three times. The behavior and signs of each mouse were observed every 5 days for a total of 25 days. The average weight of each group of animals was recorded before the first sensitization and challenge.

[0100] Allergy test results as follows Figure 7 As shown;

[0101] Depend on Figure 7 It can be seen that the weight of the mice fluctuated around 20g, their living conditions remained at a normal level, no adverse reactions were observed during the immunization period, their appetite and mental state were good, and no deaths occurred in either group during the observation period.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A novel self-encapsulated MVP-INT nanoprotein particle, characterized in that, The novel self-encapsulated MVP-INT nanoprotein particles are composed of the MVP gene, the self-cleaving intron gene, and the INT gene linked together.

2. The novel self-encapsulated MVP-INT nanoprotein particles according to claim 1, characterized in that, The amino acid sequence of the MVP gene is shown in SEQ ID No. 1; SEQ ID No.1 .

3. The novel self-encapsulated MVP-INT nanoprotein particles according to claim 1, characterized in that, The amino acid sequence of the self-cleaving intron gene is shown in SEQ ID No. 2; SEQ ID No.2 MSIRDEALLVGTKVTTKAGDKNIENITLEDEVLQFDMNTKDFSYTNPTKTQKVIRDEIYHFEGAGFDQKVSPNHRMIYEQGGEIKECLAKDFEPSEDKYFIIVEGSHMQIKRIKSTDVKITHTKLDEPTEFHALSVPGKSFVVTDEHGNRSVTGASMHVEGKLGG.

4. The novel self-encapsulated MVP-INT nanoprotein particles according to claim 1, characterized in that, The amino acid sequence of the INT gene is shown in SEQ ID No. 3; SEQ ID No. 3 CTQHWQDAVPWTELLSLQTEDGFWKLTPELGLILNLNTNGLHSFLKQKGIQSLGVKGRECLLDLIATMLVLQFIRTRLEKEGIVFKSLMKMDDPSISRNIPWAFEAIKQASEWVRRTEGQYPSICPRLELGNDWDSATKQLLGLQPISTVSPLHRVLHYSQG.

5. A method for preparing novel self-encapsulated MVP-INT nanoprotein particles as described in claims 1-4, characterized in that, Includes the following steps: (1) Preparation of a novel self-encapsulated MVP-INT nanoprotein nanoparticle eukaryotic expression system: The GAP promoter (PGAP) downstream of the yeast vector pGAPZA (Invitrogen) was subcloned into the EcoRI and KpnI sites of MVP to generate the yeast expression vector yMVP-pGAPZA. Subsequently, yMVP-pGAPZA and BspHI were linearized and transformed into the P. pastoris protease-deficient strain SMD1168. Electroporation was performed using a single pulse at 1.5 kV, 200 Ω, and 25 μF. The transformation medium was placed on YPDS agar containing 100 μg / mL of the antibiotic Zeocin. Zeocin-resistant transformants were screened by re-banding on fresh YPD plates containing Zeocin to select individual colonies. Eleven positive colonies were inoculated onto a plate containing 100 μg / mL of Zeocin. Cells were cultured overnight in 3 mL YPD medium containing Zeocin at 30°C and 200 rpm. Cells were collected, lysed, and subjected to SDS-PAGE and Coomassie Brilliant Blue staining to confirm MVP expression. Colonies with the highest MVP production were selected for subsequent experiments. To assess the time course of MVP expression, selected colonies were inoculated into 3 mL YPD medium containing 100 μg / mL Zeocin and cultured overnight at 30°C and 200 rpm. Then, 500 mL of YPD medium was inoculated until the OD600 reached 0.

03. The culture was stored at 30°C and 200 rpm for 30 h. Cells were harvested in pre-weighed 50 mL tubes, centrifuged at 3000 rpm for 5 min at 4°C, washed with 10 mL of deionized water, and the wet cell microspheres were weighed and stored at -80°C. (2) Preparation of a novel self-encapsulated MVP-INT nanoprotein nanoparticle prokaryotic expression system: Take 1 mL of overnight BL21 bacterial culture and place it in a 1.5 mL microtube. Centrifuge at 1500 × g (4000 rpm) at 4°C for 5 min, and discard the supernatant. Add 100 μL of Solution A pre-chilled on ice to the microtube, and gently tap the microtube to suspend the precipitate; do not shake vigorously. Centrifuge at 1500 × g (4000 rpm) at 4°C for 5 min, and discard the supernatant. Add 100 μL of Solution A pre-chilled on ice to each microtube. B. Gently tap the microtube to suspend the precipitate; avoid vigorous shaking. The competent cells are now ready. Store at -80°C for later use. Thaw the competent cells stored at -80°C on ice for 10 minutes. Transfer 100 μL of competent cells to a new transformation tube. Add 0.1 ng to 10 ng (3 μL to 10 μL) of transformation DNA to the competent cells, mix gently, and place on ice for 30 minutes. After incubating in a 42°C water bath for 45 seconds, immediately place on ice for 1 to 2 minutes. Add 890 μL of... LB medium pre-warmed to 37℃; cultured at 37℃ with shaking for 1 hour; spread an appropriate amount onto a plate and invert the plate in a 37℃ incubator overnight; confirm the colonies and proceed to the next experiment; activation culture: the next day, pick a single colony and place it in liquid medium, culture at 37℃ and 210 rpm for 4 hours; scale-up culture: add the activated bacterial culture to 300 mL of liquid medium at a volume ratio of 1:100 for scale-up culture; culture at 20℃ and 210 rpm for 4 hours; secondary scale-up culture: mix the scaled-up bacterial culture with 300 mL of liquid medium and then separate them at a 1:1 ratio; culture at 28℃ and 210 rpm for 1 hour; add IPTG at a volume ratio of IPTG:bacterial culture = 1:1000, culture at 20℃ and 210 rpm for 6 hours with shaking, induction complete; take 1 mL before and after induction expression and store at 4℃ for electrophoresis and as a control after induction; 8000~10000r Centrifuge for 10 min, discard the supernatant, transfer the precipitate to another tube, and weigh the precipitate. Resuspend the precipitate in TRIS solution at pH 8 (pH > protein isoelectric point 0.5). Centrifuge at 10000 rpm for 10 min, discard the supernatant, transfer the precipitate to another tube, and weigh the precipitate. Resuspend the precipitate in 3 mL / 10 mL TRIS solution at pH 8, and disrupt it at 200 W for 5 min. After disruption, centrifuge at 10000 rpm for 10 min, collect the supernatant, and resuspend the precipitate in TRIS solution at a ratio of 1:

100. Store at 4 °C. (3) Purification and characterization of novel self-encapsulated MVP-INT nanoprotein particles: The MVP protein was purified, and the purification success was observed by SDS-PAGE protein electrophoresis. Then, the MVP nanoparticles were separated from the INT protein using self-cleaving inlets and incubated on ice for 0.5 h to obtain the novel self-encapsulated MVP-INT nanoparticles.

6. The application of a novel self-encapsulated MVP-INT nanoprotein particle as described in claims 1 to 4 as a highly efficient and safe drug delivery carrier in anticancer drugs, vaccine delivery, and gene therapy.

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