A method for protein purification based on gem particles and uses thereof

Abstract

The present application relates to the field of bioengineering, and relates to a protein purification method based on GEM particles and application.The present application provides a fusion protein, which comprises a linker, an anchor hook protein and Gn-D III protein of fever with thrombocytopenia syndrome virus.The protein purification method provided by the present application inserts a protease cleavage site into the linker, and uses the linker to connect the anchor hook protein PA3 and the target protein Gn-D III protein of fever with thrombocytopenia syndrome virus: the fusion protein Gn-D III-Linker-PA3 is expressed by using an insect-baculovirus expression system, the prepared fusion protein Gn-D III-Linker-PA3 is displayed on the surface of GEM particles, and the protease is used for cutting, so that the purified target protein is obtained.The purification method has high impurity protein removal rate, short time consumption, low cost and simple operation.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to a protein purification method and its application based on GEM particles. Background Technology

[0002] Protein isolation and purification are widely used in biochemical research and are an important operational technique. In recent years, with the rapid development of biopharmaceuticals, in vitro diagnostics, and some emerging technology fields, the demand for high-purity, high-activity proteins has been increasing. Currently, using affinity tags to assist protein purification is the preferred method for academic research and industrial production. With appropriate tag design, sufficiently high purity and yield of the final protein product can be provided while preserving its inherent structure and function, greatly saving time and cost in the protein production process. However, due to the complexity of proteins themselves and the limitations of existing technologies, traditional affinity chromatography methods for purifying eukaryotically expressed proteins are often costly, time-consuming, and difficult to avoid non-specific contaminants. Large-scale recovery and purification of target proteins still faces significant challenges.

[0003] Gram-positive bacterial enhancer matrix (GEM) particles are obtained by pretreating *Lactococcus lactis* (L. lactis) with trichloroacetic acid to remove all nucleic acids and proteins, resulting in a complete cell wall peptidoglycan backbone. Protein anchor (PA3), acting as a carrier protein, specifically binds to GEM particles to display exogenous proteins on their surface. The GEM-PA surface display system, as a safe, effective, high-load protein antigen, and low-cost multifunctional platform, is widely used in vaccine adjuvants, vaccine vectors, and protein purification. Current applications in protein purification include: non-chromatographic tagging purification of canine alpha interferon using the self-cleavage function of integrins; and purification of porcine reproductive and respiratory syndrome virus (PRRSV) and foot-and-mouth disease virus (FMDV) by utilizing the specific binding between proteins to first form a fusion protein-target protein complex, followed by low-speed centrifugation. Summary of the Invention

[0004] In view of this, the present invention provides a protein purification method and application based on GEM particles. The protein purification method provided by the present invention mainly involves inserting a protease cleavage site (HRV 3C site) into a linker, using the linker to tandemly connect the anchor protein PA3 and the target protein to be purified, such as the third domain of the Gn protein of Severe fever with thrombocytopenia syndrome virus (SFTSV). The Gn-DⅢ-Linker-PA3 is expressed using an insect-baculovirus expression system, and the fusion protein Gn-DⅢ-Linker-PA3 prepared using a Bac-to-Bac expression system is displayed on the surface of GEM particles. The particles are then cleaved using a protease (HRV 3C) to obtain the purified target protein SFTSV Gn-DⅢ. This purification method has a high rate of removal of impurities, is time-efficient, low-cost, and simple to operate.

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

[0006] This invention provides a fusion protein comprising: a linker, an anchoring protein, and the Gn-DⅢ protein of fever with thrombocytopenia syndrome virus.

[0007] In some embodiments of the present invention, the anchoring protein in the above-mentioned fusion protein includes PA3.

[0008] In some embodiments of the present invention, the Linker in the fusion protein described above has a protease cleavage site.

[0009] In some embodiments of the present invention, the protease cleavage site in the fusion protein includes: HRV 3Csite.

[0010] In some embodiments of the present invention, the Gn-DⅢ protein in the above-described fusion protein has:

[0011] (1) An amino acid sequence as shown in SEQ ID NO:1; or

[0012] (2) A sequence based on the amino acid sequence shown in (1) by substitution, deletion, addition and / or replacement of one or more amino acids; or

[0013] (3) A sequence that is more than 90% homologous to the amino acid sequence shown in (1); and / or

[0014] The Linker has:

[0015] (4) An amino acid sequence as shown in SEQ ID NO:2; or

[0016] (5) A sequence based on the amino acid sequence shown in (4) by substitution, deletion, addition and / or replacement of one or more amino acids; or

[0017] (6) A sequence that is more than 90% homologous to the amino acid sequence shown in (4); and / or

[0018] The anchoring protein has:

[0019] (7) An amino acid sequence as shown in SEQ ID NO:3; or

[0020] (8) A sequence based on the amino acid sequence shown in (7) by substitution, deletion, addition and / or replacement of one or more amino acids; or

[0021] (9) A sequence that is more than 90% homologous to the amino acid sequence shown in (7).

[0022] In some embodiments of the present invention, the sequence of SEQ ID NO:1 is:

[0023] In some embodiments of the present invention, the sequence of SEQ ID NO:2 is:

[0024] In some embodiments of the present invention, the sequence of SEQ ID NO:3 is as follows:

[0025] In some embodiments of the present invention, the fusion protein has the following characteristics:

[0026] (10) An amino acid sequence as shown in SEQ ID NO:4; or

[0027] (11) A sequence based on the amino acid sequence shown in (10) by substitution, deletion, addition and / or replacement of one or more amino acids; or

[0028] (12) A sequence that is more than 90% homologous to the amino acid sequence shown in (10).

[0029] In some embodiments of the present invention, the sequence of SEQ ID NO:4 is as follows:

[0030] The present invention also provides a nucleic acid molecule encoding the above-mentioned fusion protein.

[0031] In some embodiments of the present invention, the above-mentioned nucleic acid molecules have:

[0032] (13) A nucleotide sequence as shown in SEQ ID NO:5; or

[0033] (14) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (13), but is different from the nucleotide sequence shown in (13) due to the degeneracy of the genetic code; or

[0034] (15) A nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (13) or (14), and which has the same or similar function as the nucleotide sequence shown in (13) or (14).

[0035] In some embodiments of the present invention, the sequence of SEQ ID NO:5 in the above-mentioned nucleic acid molecule is as follows:

[0036] In some embodiments of the present invention, the sequence of the nucleic acid molecule encoding the Gn-DⅢ protein, such as the sequence of SEQ ID NO:6, is as follows:

[0037] In some embodiments of the present invention, the sequence of the nucleic acid molecule encoding the Linker is shown in SEQ ID NO:7:

[0038] In some embodiments of the present invention, the sequence of the nucleic acid molecule encoding the anchoring hook protein is shown in SEQ ID NO:8:

[0039] The present invention also provides recombinant plasmids comprising: the above-described nucleic acid molecules and acceptable gene elements.

[0040] In some embodiments of the present invention, the gene element in the recombinant plasmid includes a promoter; the promoter includes P10 and / or PH.

[0041] In some embodiments of the present invention, the number of nucleic acid molecules in the recombinant plasmid is greater than or equal to 1.

[0042] The present invention also provides a host, transformation and / or transfection of the above recombinant plasmid.

[0043] In some embodiments of the present invention, the host includes Sf9 cells.

[0044] The present invention also provides a method for preparing the fusion protein, wherein the above-mentioned host is obtained, cultured, centrifuged, and the supernatant is collected to obtain the fusion protein.

[0045] The present invention also provides bacterial-like particles, which are obtained by combining the above-described fusion protein or the fusion protein obtained by the above-described preparation method with GEM particles.

[0046] The present invention also provides a method for preparing the above-mentioned bacterial-like particles, wherein the fusion protein is mixed with the GEM particles, centrifuged, and the precipitate is collected to obtain the bacterial-like particles.

[0047] In some embodiments of the present invention, in the above preparation method, the fusion protein is added in the form of a solution; the volume ratio of the solution to the GEM particles is 12:1 mL / U.

[0048] The present invention also provides a method for preparing the protein, wherein the above-mentioned bacterial-like particles and / or bacterial-like particles obtained by the above preparation method are centrifuged, the precipitate is collected and purified to obtain the protein.

[0049] In some embodiments of the present invention, the purification in the above preparation method includes a step of binding with HRV 3C protease.

[0050] In some embodiments of the present invention, the purification in the above preparation method further includes the steps of centrifugation, column loading, flow-through, and concentration.

[0051] The present invention also provides the use of the above-mentioned fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned recombinant plasmid, the above-mentioned host, the fusion protein obtained by the above-mentioned preparation method, the above-mentioned bacterial-like particles, the bacterial-like particles obtained by the above-mentioned preparation method, and / or the protein obtained by the above-mentioned preparation method in the preparation of products for the treatment and / or prevention of fever with thrombocytopenia syndrome.

[0052] The present invention also provides products comprising: the above-described fusion protein, the above-described nucleic acid molecule, the above-described recombinant plasmid, the above-described host, the fusion protein obtained by the above-described fusion protein preparation method, the above-described bacterial-like particles, the bacterial-like particles obtained by the above-described preparation method, and / or the protein obtained by the above-described preparation method.

[0053] This invention provides a fusion protein comprising: a linker, an anchoring protein, and the Gn-DⅢ protein of fever with thrombocytopenia syndrome virus.

[0054] The protein purification method provided by this invention mainly involves inserting a protease cleavage site (HRV 3C site) into a linker. The linker then tandemly links the anchor protein PA3 with the target protein to be purified, such as the third domain of the Gn protein in Severe fever with thrombocytopenia syndrome virus (SFTSV). The Gn-DⅢ-Linker-PA3 fusion protein, prepared using an insect-baculovirus expression system, is expressed. The fusion protein Gn-DⅢ-Linker-PA3, prepared using a Bac-to-Bac expression system, is displayed on the surface of GEM particles and cleaved using a protease (HRV 3C) to obtain the purified target protein SFTSV Gn-DⅢ. This purification method offers high impurity protein removal, is time-efficient, low-cost, and simple to operate. Attached Figure Description

[0055] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0056] Figure 1 A schematic diagram of the fusion protein SFTSV Gn-DⅢ-Linker-PA3 in Example 1 is shown; the red sequence is the HRV 3C protease recognition site, and the red arrow is the HRV 3C protease cleavage site.

[0057] Figure 2 The PCR amplification results of the target gene in Example 1 are shown; where: left shows: PCR amplification results of SFTSV Gn-DⅢ(PH) gene; right shows: amplification results of SFTSV Gn-DⅢ-Linker-PA3(P10) gene;

[0058] Figure 3 The identification of the recombinant plasmid in Example 1 is shown; where: left shows the double enzyme digestion identification results of pFBD-Gn-DⅢ-Linker-PA3; right shows the double enzyme digestion identification results of pFBD-Gn-DⅢ-Linker-PA3-Gn-DⅢ-Linker-PA3.

[0059] Figure 4 The image shows the PCR identification results of the recombinant rod-like particles in Example 1; where: lane 1 is the identification result of the rBacmid-Gn-DⅢ-Linker-PA3PH end; lane 2 is the identification result of the rBacmid-Gn-DⅢ-Linker-PA3P10 end;

[0060] Figure 5This shows the morphological changes of Sf9 cells after infection with recombinant baculovirus in Example 1; where: left shows: normal Sf9 cells; right shows: Sf9 cells infected with recombinant baculovirus rBV-Gn-DⅢ-Linker-PA3;

[0061] Figure 6 The identification of recombinant baculovirus rBV-Gn-DⅢ-Linker-PA3 in Example 1 is shown below; where: A shows: genomic PCR identification; B shows: indirect immunofluorescence identification (Ba shows: normal Sf9 cells; Bb shows: Sf9 cells infected with recombinant baculovirus rBV-Gn-DⅢ-Linker-PA3); C shows: Western Blot identification (lane 1: cell suspension of rBV-Gn-DⅢ-Linker-PA3; lane 2: culture supernatant of rBV-Gn-DⅢ-Linker-PA3; lane 3: supernatant after cell lysis of rBV-Gn-DⅢ-Linker-PA3; lane 4: precipitate after cell lysis of rBV-Gn-DⅢ-Linker-PA3);

[0062] Figure 7 Example 2 shows the identification of the optimal binding amount of the fusion protein Gn-DⅢ-Linker-PA3 with GEM; wherein lanes 1-6 are respectively: 0.5U GEM bound to 0, 2, 4, 6, 8 and 10 mL of rBV-Gn-DⅢ-Linker-PA3 suspension cell culture supernatant.

[0063] Figure 8 Example 2 shows the identification of the purity of the purified SFTSV Gn-DⅢ protein: Left shows: SDS-PAGE identification of the purified SFTSV Gn-DⅢ protein; Right shows: Thin-layer scanning results of the purified SFTSV Gn-DⅢ protein. Detailed Implementation

[0064] This invention discloses a protein purification method and its application based on GEM particles.

[0065] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0066] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0067] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0068] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0069] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0070] The plasmids involved in this invention are as follows: the plasmid containing the SFTSV Gn-DⅢ gene was provided by Sangon Biotech (Shanghai) Co., Ltd.; the plasmid pFBD-E-Linker-PA3(HRV 3C site) containing the Linker (containing the HRV 3C site) and the anchor hook protein gene PA3 was designed and synthesized by our laboratory.

[0071] In Examples 1 and 2 of this invention, the raw materials and reagents used can all be purchased from the market.

[0072] The present invention will be further illustrated below with reference to the embodiments:

[0073] Example 1: Construction and Identification of Recombinant Baculovirus

[0074] 1. Primer design and synthesis

[0075] Specific amplification primers were designed (Table 1). Referring to the Gn glycoprotein third domain gene of the SFTSV strain HBMC16_human_2015 (GenBank: KY440776.1), upstream primer Gn-DⅢ-PH-F (with a Not I restriction site added) and downstream primer Gn-DⅢ-PH-R (with a 24 bp HRV 3C site gene containing an Apa I restriction site added to the 3' end) were designed. Using the recombinant plasmid pFBD-Gn-DⅢ-Linker-PA3 constructed at the PH end as a template, upstream primer Gn-DⅢ-P10-F (with a NheI restriction site added) and downstream primer Gn-DⅢ-P10-R (with a Kpn I restriction site added) were designed.

[0076] Primers PH F and M13R for identification at the pH end and primers M13F and P10R for identification at the P10 end were designed based on the insect cell-baculovirus expression vector pFastBac Dual.

[0077] Table 1 Primer Information

[0078]

[0079]

[0080] Note: Underlined areas are enzyme cleavage sites.

[0081] 2. Construction and identification of recombinant plasmid pFBD-Gn-DⅢ-Linker-PA3-Gn-DⅢ-Linker-PA3

[0082] (1) Amplification of the target gene SFTSV Gn-DⅢ

[0083] Using the SFTSV Gn-DⅢ gene as a template, and Gn-DⅢ-PH-F and Gn-DⅢ-PH-R as primers, the SFTSV Gn-DⅢ gene was amplified under the reaction conditions shown in Table 2. The target fragment was separated by 1% agarose gel electrophoresis, and the amplified product was the same size as the target fragment. Figure 2 (Left) The SFTSV Gn-DⅢ gene was successfully amplified.

[0084] Table 2 PCR reaction conditions

[0085]

[0086]

[0087] (2) Identification of recombinant plasmid pFBD-Gn-DⅢ-Linker-PA3

[0088] The recovered SFTSV Gn-DⅢ gene and the plasmid pFBD-E-Linker-PA3 (HRV3C site) containing the Linker-PA3 gene were digested with Not I and Apa I. The digested products were ligated and transformed into DH5α competent cells, plated on ampicillin-resistant solid LB agar plates, and incubated upside down at 37°C for 12 h. Afterward, single clones were picked and cultured in ampicillin-resistant liquid LB agar plates with shaking for 12 h. The plasmid was extracted and identified by double digestion with Not I and Hind III. Figure 3 (Left) The enzyme digestion product is the same size as the Gn-DⅢ-Linker-PA3 gene. The correctly identified plasmid was sequenced and analyzed. The results showed that its sequence was 100% homologous to the synthesized SFTSV Gn-DⅢ gene sequence. The recombinant plasmid containing the SFTSV Gn-DⅢ gene was successfully constructed and named pFBD-Gn-DⅢ-Linker-PA3.

[0089] (3) Amplification of the target gene Gn-DⅢ-Linker-PA3(P10)

[0090] Using recombinant plasmid pFBD-Gn-DⅢ-Linker-PA3 as a template, and Gn-DⅢ-P10-F and Gn-DⅢ-P10-R as primers, the Gn-DⅢ-Linker-PA3(P10) gene was amplified. Figure 2 (Right), the reaction conditions are shown in Table 2. Agarose gel electrophoresis analysis showed that the amplified product was consistent with the size of the target gene fragment, and the Gn-DⅢ-Linker-PA3(P10) gene was successfully amplified.

[0091] (4) Identification of recombinant plasmid pFBD-Gn-DⅢ-Linker-PA3-Gn-DⅢ-Linker-PA3

[0092] The recovered Gn-DⅢ-Linker-PA3(P10) gene and recombinant plasmid pFBD-Gn-DⅢ-Linker-PA3 were double-digested with NheI and KpnI. The digested products were ligated and transformed into DH5α competent cells. Single clones were picked, and the plasmids were extracted and identified by double digestion with NheI and KpnI. Figure 3 (Right), the enzyme digestion product is the same size as the Gn-DⅢ-Linker-PA3 (P10) gene, and a recombinant plasmid containing two Gn-DⅢ-Linker-PA3 genes was successfully constructed and named pFBD-Gn-DⅢ-Linker-PA3-Gn-DⅢ-Linker-PA3.

[0093] 3. Preparation and identification of recombinant rod-like particles rBacmid-Gn-DⅢ-Linker-PA3

[0094] The recombinant plasmid pFBD-Gn-DⅢ-Linker-PA3-Gn-DⅢ-Linker-PA3 was transformed into DH10Bac competent cells and plated on triple-antibiotic solid LB plates containing tetracycline (10 μg / mL), kanamycin sulfate (50 μg / mL), gentamicin (7 μg / mL), IPTG, and X-Gal. The plates were incubated upside down at 37°C for 40 h. White spots were picked and cultured at 37°C with shaking at 200 rpm for 16 h. Rod granules were extracted for PCR identification. Using rod granules as templates, the target gene under the PH promoter was identified using PHF and M13R primers; the target gene under the P10 promoter was identified using M13F and P10R upstream and downstream primers. The results showed that the amplified product was consistent with the size of the target fragment. Figure 4 This indicates that the recombinant rod-like particle was successfully prepared, and the correctly identified rod-like particle was named rBacmid-Gn-DⅢ-Linker-PA3.

[0095] Table 3 PCR reaction conditions

[0096]

[0097] 4. Rescue of recombinant baculovirus rBV-Gn-DⅢ-Linker-PA3

[0098] Sf9 cells were seeded into 6-well plates and cultured at 27°C for 12 hours. Once the cells reached over 80% confluence, the medium was replaced with Grace's medium free of both recombinant and dextrin-free media. The positive recombinant rod-like cell transfection with rBacmid-Gn III-III-Linker-PA3 (4 μg) and Cellfectin II Reagent (8 μL) were diluted separately with Grace's medium, gently mixed, and allowed to stand for 20 minutes before being evenly added to the 6-well plates. The plates were then incubated at 27°C for 5 hours, followed by a change to Grace's complete medium. The plates were then incubated at 27°C for 5 days, with cell status observed daily. Compared to normal adherent Sf9 cells, adherent Sf9 cells transfected with recombinant rod-like cell transfection with rBacmid-Gn III-III-Linker-PA3 exhibited significant cytopathic effects, including swelling, rounding, and extensive detachment. Figure 5 When 60% of the cells showed cytopathic effects on day 5, the supernatant was collected as the first-generation recombinant baculovirus, named rBV-Gn-DⅢ-Linker-PA3, and passed blindly for 3 more generations.

[0099] 5. Identification of recombinant baculovirus rBV-Gn-DⅢ-Linker-PA3

[0100] (1) Genomic PCR identification

[0101] Genomic DNA was extracted from cells containing P2 generation recombinant baculovirus and ordinary Sf9 cells, and PCR identification was performed using Gn-DⅢ-P10-F and Gn-DⅢ-P10-R primers, respectively. Figure 6 A), the reaction conditions are shown in Table 2 (Gn-DⅢ-Linker-PA3). The size of the amplified specific fragment is consistent with the size of the target gene, indicating that the Gn-DⅢ-Linker-PA3 gene was successfully integrated into the recombinant baculovirus.

[0102] (2) Indirect immunofluorescence identification

[0103] Sf9 cells in good growth condition were evenly seeded into six-well plates. When the cells reached 70% cell growth, 10 μL of recombinant baculovirus was inoculated into each well. The cells were cultured at 27°C for 48 h, then the culture medium was discarded. The cells were fixed with 70% ethanol for 30 min. A 100-fold diluted anti-SFTSVGn-DⅢ mouse serum (diluted with 1% BSA) was used as the primary antibody, and the cells were incubated at 37°C for 1 h. A 200-fold diluted FITC-labeled goat anti-mouse IgG was used as the secondary antibody, and the cells were incubated at 37°C for 1 h in the dark. The cells were washed three times with PBS after each step. The cells were observed under an inverted fluorescence microscope. The results showed (…). Figure 6 B) Compared with normal cell controls, adherent Sf9 cells infected with recombinant baculovirus showed obvious green fluorescence under a fluorescence microscope, indicating that the recombinant baculovirus can successfully express the protein Gn-DⅢ after infecting cells.

[0104] (3) Western Blot identification

[0105] Sf9 cells in suspension culture were infected with recombinant baculovirus and cultured at 27℃ and 120 rpm for 96 h. Cells and culture supernatant were collected by low-speed centrifugation. The cell pellet was resuspended in cell lysis buffer, lysed on ice for 30 min, and centrifuged at 4℃ and 12000 rpm for 15 min. The supernatant and pellet were collected, and the pellet was resuspended in an equal volume of PBS. Samples were prepared from the cell culture product, cell culture supernatant, cell lysis supernatant, and cell lysis pellet. Western blotting was performed using 1:300 dilution of anti-SFTSV Gn-DⅢ mouse serum as the primary antibody and 1:20000 dilution of HRP-labeled goat anti-mouse IgG as the secondary antibody. The results showed ( Figure 6 C) A specific protein band of approximately 45 kDa was detected in the supernatant of the recombinant baculovirus culture. In summary, this indicates that Sf9 cells infected with the recombinant baculovirus successfully expressed the Gn-DⅢ-Linker-PA3 fusion protein, which was secreted into the supernatant.

[0106] Example 2: Purification of SFTSV Gn-DⅢ

[0107] 1. Determination of the optimal binding amount of the fusion protein Gn-DⅢ-Linker-PA3

[0108] Cell culture products infected with P3 generation rBV-Gn-DⅢ-PA3 and cultured in suspension for 4 days were centrifuged at 3500 rpm for 15 min at 4°C. 0, 2, 4, 6, 8, and 10 mL of cell culture supernatant were then mixed with 0.5 U GEM particles and incubated at room temperature for 1 h with shaking. The precipitate was then centrifuged at 10000 rpm for 10 min at 4°C, resuspended in PBS, and washed 5 times. After the final centrifugation, the precipitate was resuspended in PBS to prepare protein samples. SDS-PAGE analysis was performed. Figure 7 The highest amount of SFTSV-Gn-DⅢ-PA3 protein was obtained after binding 6 mL of cell culture supernatant with 0.5 U of GEM particles. Therefore, the optimal binding volume of rBV-Gn-DⅢ-PA3 suspension cell culture supernatant with GEM is 12 mL / U.

[0109] 2. Purification of SFTSV Gn-DⅢ protein

[0110] Take 150 mL of supernatant from P4 generation rBV-Gn-DⅢ-PA3 culture suspension, bind it with 12.5 U GEM at room temperature for 1 h, wash 5 times with PBS, resuspend the precipitate in 1.2 mL of 1×HRV 3C buffer, add 6 μL of HRV 3C protease, bind at 4℃ for 16 h, centrifuge, and bind the supernatant with a nickel column for 6 h. Concentrate the flow-through to 900 μL using ultrafiltration tubes to obtain the purified product. SDS-PAGE analysis of the purified product (…) Figure 8 (Left). After purification, SFTSV Gn-DⅢ protein was scanned using thin-layer chromatography, and the purity was approximately 100%. Figure 8 right).

[0111] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Bacterial-like particles, characterized in that, It was obtained by binding the fusion protein to GEM particles; The fusion protein includes: a linker, an anchor protein, and the Gn-D III protein of fever with thrombocytopenia syndrome virus; the linker has a protease cleavage site; The sequence of the fusion protein is as shown in SEQ ID NO:4; The sequence of the Gn-D Ⅲ protein is as shown in SEQ ID NO:1, and... The linker sequence is as shown in SEQ ID NO:2, consisting of an amino acid sequence; and The sequence of the anchoring protein is as shown in SEQ ID NO:3, which is an amino acid sequence.

2. The method for preparing bacterial-like particles as described in claim 1, characterized in that, The fusion protein was mixed with the GEM particles, centrifuged, and the precipitate was collected to obtain the bacterial-like particles.

3. The preparation method according to claim 2, characterized in that, The fusion protein is added in solution form; the volume ratio of the solution to the GEM particles is 12:1 mL / U.

4. A method for preparing Gn-D III protein of fever with thrombocytopenia syndrome virus, characterized in that, Centrifuge the bacterial-like particles as described in claim 1 and / or the bacterial-like particles obtained by the preparation method as described in claim 2 or 3, collect the precipitate, and purify it to obtain the protein.

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