Ultra-large self-assembly virus-like particle mutant and application

Abstract

The invention belongs to the field of bionanomaterials, virology and protein design, and particularly relates to an ultra-large self-assembly virus-like particle mutant and application. The applicant mutates lysine at the 299th site of SV40 capsid protein VP1 into cysteine and glutamic acid at the 342nd site into cysteine in a site-specific mutagenesis mode, after the obtained mutant M1 is self-assembled to form the VLP, the particle size of the VLP is remarkably increased, the VLP is more suitable for being used as a delivery carrier of a medicine or a medicament, and the invention explores that the SV40 VLP serving as the delivery carrier has higher potential application value.

Description

Technical Field

[0001] The present invention belongs to the fields of biological nanomaterials, virology and protein design, and particularly relates to a super-large self-assembled virus-like particle mutant and its application. Background Art

[0002] Virus-like particles (VLPs), typical representatives of clathrin, are composed of viral capsid proteins to form regular hollow spherical or tubular structures, with sizes ranging from a dozen nanometers to several hundred nanometers. They have structural symmetry similar to that of natural virus capsids, do not contain genomes, are non-infectious, and are a kind of natural biological nanomaterials. Compared with other delivery carriers such as liposomes and polymer carriers, VLPs have the following characteristics: good monodispersity; large drug loading capacity; can be expressed in a variety of cells; assembled from single or multiple proteins into a hollow symmetric structure; the inner cavity, outer surface or the interaction interface between subunits can be modified and transformed to enable precise and selective single or multi-functionalization at different sites, etc. Therefore, they have advantages such as efficient loading, controllable release, specific targeting, and good biocompatibility.

[0003] SV40 belongs to the Polyomaviridae family, a circular dsDNA virus with a genome of 5.2 kb. The SV40 virus capsid has a T = 7d icosahedral structure, assembled from 72 pentamers of the VP1 protein. The structural proteins VP2 and VP3 are wrapped in the inner cavity of the virus capsid, connecting the VP1 capsid and the DNA genome. The research on the SV40 protein structure focuses on the major capsid protein VP1. Among the 72 pentamers, 12 pentamers are located on the 12 five-fold rotation axes of the icosahedron, forming 12 vertex pentamers, called "strict pentamers"; the remaining 60 pentamers are located on the faces of the icosahedron and are symmetric with respect to the six-fold rotation axis, called "local pentamers". The interfacial interaction between pentamers is closely related to the assembly and structural stability maintenance of the SV40 capsid protein.

[0004] SV40 has the ability to form virus - like particles (VLPs) with a single composition, high symmetry, controllable assembly, and moderate size. These advantages make it one of the ideal systems for studying VLP drug carriers. However, the VP1 pentamer can self - assemble into four conformations in vitro solution, including a T = 1 icosahedron with an outer diameter of 24 nm, an intermediate state or octahedron with a diameter of 32 nm, a T = 7 icosahedron with a diameter of 45 nm, and a tubular structure. Moreover, factors such as pH value, salt ion strength, and temperature in the solution have a significant impact on the morphology of VLPs. This diversity of VLP morphology is a unique feature of the self - assembly mode of SV40 different from other VLPs, and also reflects the instability of its self - assembly. In previous studies, it was found that co - assembly of quantum dots (QDs) with VLPs can promote the transformation of VLPs from polymorphism to T = 1 homogeneity, and the particle size of self - assembled VLPs (when the inner cavity is not loaded with cargo) is mainly distributed around 20 - 35 nm. How to improve the particle size of self - assembled VLPs and enhance the loading capacity through the design of the self - assembly interface will explore greater potential application value of SV40 VLPs as delivery carriers. Summary of the Invention

[0005] The object of the present invention is to provide an artificially synthesized mutant M1, and the amino acid sequence of the mutant M1 is shown in SEQ ID NO.2.

[0006] Another object of the present invention is to provide the application of the mutant M1 in the preparation of a delivery carrier.

[0007] In order to achieve the above - mentioned objects, the present invention takes the following technical measures:

[0008] The applicant obtained the mutant M1 by site - directed mutagenesis, which mutated the lysine at position 299 and the glutamate at position 342 of the SV40 capsid protein VP1 to cysteine. After the VLP self - assembled by the obtained mutant M1, the particle size of the VLP increased significantly, making it more suitable as a delivery carrier for drugs or medicaments. The sequence of the mutant M1 is shown in SEQ ID NO.2. The protection scope of the present invention includes:

[0009] A fusion protein obtained by fusing the mutant protein shown in SEQ ID NO.2 with a protein tag.

[0010] The coding gene of the mutant shown in SEQ ID NO.2 or the above - mentioned fusion protein.

[0011] An expression cassette, a recombinant vector, a recombinant microorganism, or an in vitro recombinant cell having the above - mentioned coding gene.

[0012] The above - mentioned mutant M1, fusion protein, the above - mentioned coding gene, and the application of the expression cassette, recombinant vector, recombinant microorganism, or in vitro recombinant cell having the above - mentioned coding gene in the preparation of self - assembled virus - like particles.

[0013] The above-mentioned mutant M1, fusion protein, the above-mentioned coding gene, and the expression cassette, recombinant vector, recombinant microorganism or isolated recombinant cell having the above-mentioned coding gene are used in the preparation of a delivery vector.

[0014] In the above-mentioned application, preferably, the self-assembled virus-like particles are SV40 self-assembled virus-like particles.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] In the present invention, by means of site-directed mutagenesis, lysine at position 299 of the SV40 capsid protein VP1 is mutated to cysteine, and glutamate at position 342 is mutated to cysteine. The present invention is named mutant M1. The results show that the particle size of the VLP self-assembled by this mutant is significantly increased. Description of the Drawings

[0017] Figure 1 It is a comparative transmission electron microscopy result diagram of VLPs self-assembled by mutant M1, M2 and wild-type SV40 capsid protein VP1.

[0018] Figure 2 It is a comparative particle size statistics diagram of VLPs self-assembled by mutant M1 and wild-type SV40 capsid protein VP1. Detailed Embodiments

[0019] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art; the reagents or materials, unless otherwise specified, are all from commercial channels. To make the technical solutions and advantages of the present invention clearer and more understandable, the following specific embodiments are used to further elaborate on the present invention in detail, but it does not constitute a limitation to the present invention. In the embodiments of the present invention, the M1 mutant protein is expressed and purified by means of prokaryotic expression. By using other conventional microbial expression means in the art, or directly synthesizing the M1 mutant protein, the self-assembly of virus-like particles can also be achieved.

[0020] The culture media involved in the following examples are as follows:

[0021] LB liquid medium: yeast powder 5 g·L -1 、peptone 10 g·L -1 、NaCl 10 g·L -1 。

[0022] LB solid medium: yeast powder 5 g·L -1 、peptone 10 g·L -1 、NaCl 10 g·L -1 、agar powder 13.5 g·L -1 。

[0023] Example 1:

[0024] Obtaining of HisVP1-M1 mutant protein:

[0025] (1) Construction of recombinant plasmid HisVP1-M1 mutation:

[0026] 1) Using site-directed mutagenesis technology, cysteine was introduced at position 299 of the VP1 protein of SV40.

[0027] Taking the pET32a-hisVP1 wild-type recombinant plasmid (F.Li, H.Chen, L.Ma, K.Zhou, Z.-P.Zhang, C.Meng, X.-E.Zhang, Q.Wang, Small 2014, 10, 536.) as a template, the forward and reverse primers K299C-sense and K299C-antisense were designed. The PCR reaction conditions were: 95°C for 2 min, 30×(95°C for 30 sec, 55°C for 1 min, 72°C for 7 min), 72°C for 30 sec, 16°C for 5 min.

[0028] Among them, the sequences of K299C-sense and K299C-antisense are:

[0029] K299C-sense: 5'-ACCCTTAGAAAGCGGTCTGTGTGCAACCCCTACCCAATTTCCTTT-3'

[0030] K299C-antisense: 5'-AAAGGAAATTGGGTAGGGGTTGCACACAGACCGCTTTCTAAGGGT-3'

[0031] The obtained PCR product was recovered by gel extraction, digested with Dpn I endonuclease for 4 h, and then incubated at 65°C for 10 min. The obtained PCR product was transformed into Escherichia coli DH5α by the CaCl 2 method. It was screened on a medium containing ampicillin resistance at 37°C. The transformants were obtained, and the plasmids of the transformants were picked for colony PCR verification. The primers used were the commercially available universal primers T7 and T7-ter of pET32a. If the PCR verification result of the transformant showed an electrophoresis band at 1277 bp, it indicated that the mutant expression vector was successfully constructed, and the above transformant was a positive transformant, namely pET32a-hisVP1-K299C.

[0032] 2) Using site-directed mutagenesis technology, cysteine was introduced at position 342 of the VP1 protein of SV40.

[0033] Using the constructed pET32a-hisVP1-K299C plasmid as a template, the forward and reverse primers E342C-sense and E342C-antisense were designed. The PCR reaction conditions were: 95°C for 2 min, 30×(95°C for 30 sec, 55°C for 1 min, 72°C for 7 min), 72°C for 30 sec, and 16°C for 5 min.

[0034] Among them, the sequences of E342C-sense and E342C-antisense are as follows:

[0035] E342C-sense: 5'-GGTTTATGAGGACACAGAGTGCCTTCCTGGGGATCCAGACA-3'

[0036] E342C-antisense: 5'-TGTCTGGATCCCCAGGAAGGCACTCTGTGTCCTCATAAACC-3'

[0037] The obtained PCR product was recovered by gel extraction and digested with Dpn I endonuclease for 4 h, followed by incubation at 65°C for 10 min. The obtained PCR product was transformed into Escherichia coli DH5α by the CaCl 2 method. The transformants were screened on a medium containing ampicillin resistance at 37°C. The plasmids of the selected transformants were subjected to colony PCR verification, and the primers used were the commercially available universal primers T7 and T7-ter of pET32a. If the PCR verification result of the transformant shows an electrophoretic band at 1277 bp, it indicates that the M1 mutant expression vector was successfully constructed, and the above transformant is a positive transformant.

[0038] This recombinant point mutant plasmid was named pHisVP1-M1. This mutant plasmid contains double-site mutations of K299C and E342C.

[0039] (2) Construction of HisVP1-M1 mutant expression strain

[0040] The obtained recombinant plasmid pHisVP1-M1 was transformed into competent E. coli Rosetta(DE3) cells by the CaCl 2 method. The transformed product was spread on an LB solid medium containing ampicillin resistance and chloramphenicol resistance for screening, and incubated at 37°C in an inverted position for 12 - 15 h. The selected transformants were subjected to colony PCR screening, and the primers used were the commercially available universal primers T7 and T7-ter of pET32a. After sequencing verification, the HisVP1-M1 mutant expression strain was obtained.

[0041] (3) Expression of mutant HisVP1-M1 strain and purification of HisVP1-M1 protein

[0042] The mutant HisVP1-M1 strain constructed in Example 3 was inoculated into 5 mL of LB test tube medium, and ampicillin and chloramphenicol were added. It was cultured overnight at 37°C with constant temperature and 180 r / min. The next day, it was transferred into a 500 mL Erlenmeyer flask, the corresponding antibiotics were added, and it was cultured with shaking at 37°C and 180 r / min until the OD 600 value was between 0.4 and 0.6, and IPTG was added to a final concentration of 0.5 mM. After continued induction culture at 25°C for 16 h, the cells were collected. The cell pellet was washed once with Binding buffer and then resuspended in 50 mL of Binding buffer for ultrasonic disruption. It was centrifuged at 10,000 r / min for 30 min, and the supernatant was loaded onto a Ni 2+ -NTA affinity chromatography column, and then the column was washed with Wash buffer A and Wash buffer B in sequence. Finally, the target protein was eluted with Elution buffer containing 1 M imidazole. The purified protein obtained was HisVP1-M1, which contained the protein shown in SEQ ID NO.2.

[0043] The buffer used was the same as that in Example 3.

[0044] Example 2:

[0045] Obtaining the control HisVP1-M2 mutant protein:

[0046] (1) Construction of the recombinant plasmid HisVP1-M2 mutant in the control group:

[0047] 1) Using the site-directed mutagenesis technique, cysteine was introduced at position 93 of the VP1 protein of SV40.

[0048] Using the pET32a-hisVP1 wild-type recombinant plasmid as a template, the forward and reverse primers V93C-sense and V93C-antisense were designed. The PCR reaction conditions were: 95°C for 2 min, 30×(95°C for 30 sec, 55°C for 1 min, 72°C for 7 min), 72°C for 30 sec, 16°C for 5 min.

[0049] Among them, the sequences of V93C-sense and V93C-antisense were:

[0050] V93C-sense: 5'-CAAAGAACAACTGCCTTGCTACAGTTGCGCTAGAATTCCTTTGCCT-3'

[0051] V93C - antisense: 5'-AGGCAAAGGAATTCTAGCGCAACTGTAGCAAGGCAGTTGTTCTTTG-3'

[0052] The obtained PCR product was recovered by gel extraction, digested with Dpn I endonuclease for 4 h, and then incubated at 65 °C for 10 min. The obtained PCR product was transformed into Escherichia coli DH5α by the CaCl 2 method, and screened on a medium containing ampicillin resistance at 37 °C. The transformants were obtained, and the plasmids of the transformants were picked for colony PCR verification. The primers used were the commercial universal primers T7 and T7-ter of pET32a. If the PCR verification result of the transformant showed an electrophoretic band at 1277 bp, it indicated that the mutant expression vector was successfully constructed, and the above transformant was a positive transformant, namely pET32a-hisVP1-V93C.

[0053] 2) Using site-directed mutagenesis technology, cysteine was introduced at position 321 of the VP1 protein of SV40.

[0054] Using the constructed pET32a-hisVP1-V93C plasmid as a template, the forward and reverse primers E342C-sense and E342C-antisense were designed. The PCR reaction conditions were: 95 °C for 2 min, 30×(95 °C for 30 sec, 55 °C for 1 min, 72 °C for 7 min), 72 °C for 30 sec, 16 °C for 5 min.

[0055] Among them, the sequences of G321C-sense and G321C-antisense were:

[0056] G321C-sense: 5'-AGGACACAGAGGGTGGATTGCCAGCCTATGATTGG-3'

[0057] G321C-antisense: 5'-CCAATCATAGGCTGGCAATCCACCCTCTGTGTCCT-3'.

[0058] The obtained PCR product was recovered by gel extraction, digested with Dpn I endonuclease for 4 h, and then incubated at 65 °C for 10 min. The obtained PCR product was transformed into Escherichia coli DH5α by the CaCl 2The gene was transformed into E. coli DH5α by the transformation method, and screened in a medium containing ampicillin resistance at 37°C to obtain transformants. The transformants were selected for plasmid colony PCR verification, and the primers used were pET32a commercial universal primers T7 and T7-ter. If the PCR verification result of the transformant is: an electrophoresis band appears at 1277bp, it means that the M2 mutant expression vector is successfully constructed, and the above transformant is a positive transformant.

[0059] This recombinant point mutation plasmid was named pHisVP1-M2. This mutant plasmid contained V93C and G321C double mutations and served as a double mutation control group.

[0060] (2) Construction of HisVP1-M2 mutant expression strain

[0061] The obtained recombinant plasmid pHisVP1-M2 was treated with CaCl 2 The transformed product was coated on LB solid medium containing ampicillin resistance and chloramphenicol resistance for screening, and inverted culture was performed at 37°C for 12-15h. Transformants were selected for colony PCR screening, and the primers used were pET32a commercial universal primers T7 and T7-ter. After sequencing verification, the HisVP1-M2 mutant expression strain was obtained.

[0062] (3) Expression of mutant HisVP1-M2 strain and purification of HisVP1-M2 protein

[0063] The mutant HisVP1-M2 strain constructed above was inoculated into 5 mL LB test tube medium, ampicillin and chloramphenicol were added, and cultured overnight at 37°C and 180 r / min. The next day, it was transferred into a 500 mL LB conical flask, the corresponding antibiotics were added, and cultured at 37°C and 180 r / min until OD 600 The value was between 0.4 and 0.6, and IPTG was added to a final concentration of 0.5 mM. After induction culture at 25°C for 16 h, the bacterial cells were collected, the bacterial precipitate was washed once with Binding buffer, and then resuspended in 50 mL Binding buffer for ultrasonic disruption, centrifuged at 10,000 r / min for 30 min, and the supernatant was loaded on Ni equilibrated with Binding buffer. 2+ -NTA affinity chromatography column, then wash the column with Wash buffer A and Wash buffer B in sequence, and finally elute the target protein with Elution buffer containing 1M imidazole. The purified protein obtained is HisVP1-M2, which contains the protein shown in SEQ ID NO.3.

[0064] The buffer used is the same as that in Example 3.

[0065] Example 3:

[0066] Obtaining of HisVP1-WT wild-type protein:

[0067] The expression strain E. coli Rosetta(DE3)(pET32a-hisVP1) of wild-type HisVP1 was inoculated into 5 mL of LB test tube medium, added with ampicillin and chloramphenicol, and cultured overnight at 37 °C with constant temperature and 180 r / min. The next day, it was transferred into a 500 mL LB Erlenmeyer flask, added with the corresponding antibiotics, and cultured with shaking at 37 °C and 180 r / min until the OD 600 value was between 0.4 - 0.6, and IPTG was added to a final concentration of 0.5 mM. After continued induction culture at 25 °C for 16 h, the bacterial cells were collected. The bacterial cell precipitate was washed once with Binding buffer and then resuspended in 50 mL of Binding buffer for ultrasonic disruption. After centrifugation at 10,000 r / min for 30 min, the supernatant was loaded onto a Ni 2+ -NTA affinity chromatography column, and then the column was washed successively with Wash buffer A and Wash buffer B. Finally, the target protein was eluted with Elution buffer containing 1 M imidazole. The purified protein obtained was HisVP1-WT, which contained the protein shown in SEQ ID NO.1.

[0068] Among them, the formula of the buffer used is:

[0069] Binding buffer: 25 mM Tris-HCl pH 7.8, 500 mM sodium chloride, 5 mM imidazole, 5% glycerol;

[0070] Wash buffer A: 25 mM Tris-HCl pH 7.8, 500 mM sodium chloride, 40 mM imidazole, 5% glycerol;

[0071] Wash buffer B: 25 mM Tris-HCl pH 7.8, 500 mM sodium chloride, 80 mM imidazole, 5% glycerol;

[0072] Elution buffer: 25 mM Tris-HCl pH 7.8, 500 mM sodium chloride, 500 mM imidazole, 5% glycerol.

[0073] Example 4:

[0074] Disassembly and Concentration Determination of Three Proteins Prepared in Examples 1-3

[0075] The mutant HisVP1-M1 protein, mutant HisVP1-M2 protein, and HisVP1-WT wild-type protein prepared in Examples 1-3 were respectively dialyzed into Disassembly buffer using a dialysis bag with a molecular weight cut-off of 14 kDa. The dialysis fluid was changed every 8 h, for a total of 3-5 times, and finally the dialysis dilution ratio reached 1:10,000.

[0076] The concentration of the wild-type HisVP1-WT protein was measured by SDS-PAGE and a densitometer scanner. Take 10 μL of the wild-type HisVP1-WT protein to be measured, mix it with the same volume of protein loading buffer, and boil it in a water bath for 10 min. After centrifuging at 10,000 rpm for 1 min, the standard BSA protein with a working concentration of 0.2 mg / mL and the wild-type HisVP1-WT protein to be measured were respectively loaded into the SDS-PAGE gel wells, with volumes of 2 μL, 4 μL, 5 μL, 6 μL, and 8 μL. After decolorizing the SDS-PAGE, use a densitometer scanner for scanning and analyze with Image Lab software. By means of absolute quantification, construct a standard curve, and R 2 > 0.95 is an effective curve. Calculate the concentration of the protein to be measured for the bands of the target protein within the range of the standard curve. The measured concentrations of the mutant HisVP1-M1 protein, mutant HisVP1-M2 protein, and HisVP1-WT wild-type protein were 0.2 mg / mL, 0.12 mg / mL, and 0.03 mg / mL, respectively; the masses of the mutant HisVP1-M1 protein, mutant HisVP1-M2 protein, and HisVP1-WT wild-type protein purified from every 500 mL of liquid LB fermentation broth were calculated to be 1.52 mg, 1.38 mg, and 0.36 mg, respectively.

[0077] Among them, the buffer formulation used was:

[0078] Disassembly buffer: 10 mM Tris-HCl pH 8.8, 200 mM NaCl, 2 mM EDTA, 2 mM DTT, 5% glycerol.

[0079] Example 5:

[0080] Self-assembly of the Three Proteins Prepared in Examples 1-3

[0081] The three depolymerized mutant HisVP1-M1 proteins, mutant HisVP1-M2 proteins, and HisVP1-WT wild-type proteins after concentration determination in Example 4 were respectively adjusted or concentrated to 0.2 mg / mL and dialyzed to Assembly buffer using a dialysis bag with a molecular weight cut-off of 14 kDa. The dialysis fluid was changed every 8 h for a total of 3 - 5 times, and finally the dialysis dilution ratio reached 1:10,000. The buffer formulation used was the same as in Example 4.

[0082] The self-assembled VLPs formed were named VLP-M1, VLP-M2, and VLP-WT respectively.

[0083] Example 6:

[0084] Transmission electron microscopy characterization of the three proteins after self-assembly:

[0085] Before preparing the transmission electron microscopy (TEM) sample, the copper grid was placed in a plasma surface cleaner for hydrophilic treatment for 20 s. Take the VLP self-assembled protein (10 μL) and add it to the treated copper grid, let it stand for 5 min, and then use filter paper to absorb the excess protein liquid. Then, stain it negatively with 2% phosphotungstic acid for 5 min, and use filter paper to absorb the excess negative staining solution. After the copper grid was dried overnight, it was observed under a 100 KV transmission electron microscope, and the VLP particle size was statistically analyzed.

[0086] The wild-type VLP-WT, mutant VLP-M1, and mutant VLP-M2 were respectively observed by VLP transmission electron microscopy and the particle size was statistically analyzed. The transmission electron microscopy results are as Figure 1 shown. It can be seen from the electron microscopy results that the VLP particles formed by the mutant VLP-M1 are spherical in shape, which is consistent with the wild-type VLP-WT, but the particle size of most VLP particles has increased significantly. The VLP of SV40 is self-assembled from pentamers, and it can be seen in the electron microscope that the number of pentamers in the mutant VLP-M1 with a significantly increased particle size is significantly increased, which also proves that the mutant VLP-M1 has formed super-large self-assembled VLPs. In contrast, the self-assembly ability of the control mutant VLP-M2 is significantly reduced. Although it is also a double-site mutation, the mutated sites are closely related to the interaction of pentamer self-assembly, so it has lost the self-assembly ability. The discovery of the mutant VLP-M1 means that by rational site design and mutation, increasing the volume of the VLP lumen can increase the cargo loading capacity inside it, further enhancing the potential of SV40 self-assembled VLPs as nano-delivery carriers in the biomedical field.

[0087] The particle size statistical results are as Figure 2As shown. From the statistical measurement results of the particle size distribution, it can be seen that the minimum particle size of the VLP-M1 self-assembled particles is 26.933 nm, the maximum particle size is 72.748 nm, and the average particle size is 41.068 ± 7.571 nm. The minimum particle size of the wild-type VLP-WT self-assembled particles is 12.909 nm, the maximum particle size is 53.405 nm, and the average particle size is 27.732 ± 6.374 nm. The average particle size of the mutant VLP-M1 is 13.336 nm larger than that of the wild-type VLP-WT. From the statistical analysis results, it can be seen that through double-site mutation, the particle size significantly increases, forming ultra-large self-assembled VLPs.

Claims

1. An artificially synthesized mutant M1, the amino acid sequence of the mutant M1 is shown in SEQ ID NO.

2.

2. A fusion protein obtained by fusing the mutant protein according to claim 1 with a protein tag.

3. A gene encoding the mutant or the fusion protein according to claim 1.

4. An expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the coding gene according to claim 3.

5. Use of the mutant according to claim 1, the fusion protein according to claim 2, the coding gene according to claim 3, the expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the coding gene according to claim 3 in preparing self-assembling virus-like particles.

6. The use according to claim 5, wherein the self-assembled virus-like particles are SV40 self-assembled virus-like particles.

7. Use of the mutant according to claim 1, the fusion protein according to claim 2, the coding gene according to claim 3, an expression cassette, a recombinant vector, a recombinant microorganism or an in vitro recombinant cell having the coding gene according to claim 3 in preparing a delivery vector.

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