A recombinant gene of rabies virus G protein virus-like particle, virus-like particle and construction method and application thereof

Abstract

The present invention relates to a recombinant gene of a rabies virus G protein virus-like particle, a virus-like particle, and a construction method and application thereof. The nucleotide sequence of the recombinant gene is shown in SEQ ID NO: 1. The present invention is based on a lentiviral expression system, optimizes the design of rabies virus envelope protein, replaces its transmembrane structure with the transmembrane structure of the GP5 protein of blue ear virus, and the prepared virus-like particle not only has the biological characteristics of the natural rabies virus, but also only contains a single fragment G protein that produces neutralizing antibodies, is safe and effective, has high immunogenicity, and the prepared virus-like particle has a high secretion expression amount, and can be used as a candidate vaccine for a new virus rabies virus vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant gene of a rabies virus G protein virus-like particle (VLP), a virus-like particle, and a construction method and application thereof. Background Art

[0002] Rabies is an infectious zoonotic disease caused by the rabies virus (RABV), and it is still a serious public health problem in developing countries. The current rabies vaccine is mainly prepared by inactivating the vaccine strain that is continuously proliferated and cultured in Vero cells. The production safety requirements are strict and the cost is high. When used, it still requires multiple intensive vaccinations to induce the body to produce sufficient protective neutralizing antibodies as soon as possible. At the same time, after a high degree of exposure, the rabies virus may still enter the central nervous system before the human body produces sufficient neutralizing antibodies, leading to illness and death. Therefore, it is still necessary to develop new rabies vaccines with lower costs, fewer injections, and stronger immunogenicity. Summary of the invention

[0003] In view of the above problems existing in the prior art, a new type of rabies virus G protein virus-like particle (VLP) recombinant gene, virus-like particle and its construction method and application are provided, which have lower cost, fewer injection times and stronger immunogenicity.

[0004] The technical solution of the present invention is:

[0005] In a first aspect of the present invention, a recombinant gene of rabies virus G protein virus-like particles is provided, and the nucleotide sequence of the recombinant gene is shown in SEQ ID NO:1.

[0006] The recombinant gene of the present invention is obtained by optimizing the design of the rabies virus envelope protein, replacing its transmembrane structure with the transmembrane structure of the GP5 protein of the blue ear virus. The virus-like particles (VLPs) formed by the expression of the lentiviral vector are secreted and expressed in the cell supernatant, producing a spatial conformation and function similar to the natural G protein of the rabies virus, having the biological characteristics of the natural rabies virus, and only containing the G protein that produces neutralizing antibodies. It is safe and effective, has high immunogenicity, and the secretion expression amount of the prepared virus-like particles is high.

[0007] Furthermore, the amino acid sequence of the recombinant gene is shown in SEQ ID NO:2.

[0008] The second aspect of the present invention further provides a method for constructing rabies virus-like particles, comprising the following steps in sequence:

[0009] 1) constructing a recombinant plasmid PLV-RABV-G-TM, wherein the recombinant plasmid PLV-RABV-G-TM is constructed by inserting the above-mentioned recombinant gene into a pLV-eGFP vector;

[0010] 2) The above-mentioned recombinant plasmid PLV-RABV-G-TM, envelope plasmid VSV-G and psPAX2 were co-transfected into host cells, cultured and the supernatant was collected to obtain packaged virus;

[0011] 3) Infect host cells with the packaged virus, screen monoclonal cells expressing fluorescence, culture and collect supernatant to obtain virus-like particles.

[0012] Preferably, step 1) comprises the following steps:

[0013] 1.1 Insert the above recombinant gene into the expression plasmid pUC57 vector to form vector I;

[0014] 1.2 The lentiviral expression plasmid pLV-eGFP and vector I were double-digested with Nhe I and Mlu I restriction endonucleases, and the large and small fragments were recovered by agarose gel electrophoresis;

[0015] 1.3 After ligation and transformation, the recombinant plasmid PLV-RABV-G-TM was obtained.

[0016] Preferably, in the step 2), during the transfection process, the transfection is performed according to the DNA mass ratio of plasmid PLV-RABV-G-TM:VSV-G:psPAX2 = 4:1:3.

[0017] Preferably, the host cell is a human embryonic kidney cell HEK-293FT.

[0018] Preferably, during the transfection process, PEI transfection reagent is used.

[0019] In one embodiment, step 2) co-transfection comprises the following steps:

[0020] 2.1 Prepare the transfection system according to the following conditions (200 μl in total):

[0021] ① Solution A: PEI (5 μl) + basic DMEM = 100 μl;

[0022] ② Solution B: 2 μg plasmid DNA + basic DMEM = 100 μl, mix plasmids pLV-eGFP, pPAX2 and pCMV-VSV-G at a mass ratio of 4:3:1, and add DMEM to 100 μl;

[0023] 2.2 Mix solution A and solution B, add solution A to solution B, and mix gently; incubate at room temperature for 20 minutes. Add the transfection system evenly to the plate, shake gently to mix, and incubate in an incubator.

[0024] 2.3 Observe cells every day to determine the status of cell transfection and harvest the viral supernatant after 72 hours.

[0025] Preferably, in step 3), the cell confluence of the host cells is ensured to reach more than 50% within 20-24 hours before virus infection.

[0026] In one embodiment, the cells were plated at an appropriate density one day in advance to ensure that the host cells reached a cell confluence of more than 50% for 20-24 hours before virus infection. Fluorescence was observed after 48 hours, and supernatant was harvested after 72 hours. The supernatant was subjected to WB detection, etc. At the same time, fluorescent cells were subjected to clonal cell screening. Cell lines were established from the screened monoclonal cells. Cells were cultured with serum-free culture medium, supernatants were collected for 48-72 hours, and purified and observed under an electron microscope.

[0027] The third aspect of the present invention provides rabies virus-like particles prepared by the above construction method.

[0028] The fourth aspect of the present invention provides the use of the virus-like particles in rabies virus vaccines.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention is based on the lentivirus expression system, optimizes the design of the rabies virus envelope protein, replaces its transmembrane structure with the transmembrane structure of the GP5 protein of the blue ear virus, obtains the recombinant gene, and then inserts the recombinant gene into the expression vector and the lentivirus envelope vector to package the virus, and transfects 293FT cells to prepare recombinant virus particles. The virus-like particle (VLP) produces a spatial conformation and function similar to the natural G protein of the rabies virus, directly stimulating the body to produce an immune response.

[0031] In addition to having the biological characteristics of natural rabies virus, the virus-like particles of the present invention only contain G protein that produces neutralizing antibodies, are safe and effective, have high immunogenicity, and the prepared virus-like particles have a high secretion expression level.

[0032] When the rabies virus-like particles (VLPs) of the present invention produce an immune response, their immune principle is similar to that of inactivated virus vaccines, their components are single, their side effects are small, and their production process does not require additional steps such as cell lysis. The preparation method is simpler, the cycle for producing recombinant virus-like particles in cells is shorter, the cost is lower, the safety is higher, the number of injections is reduced, and the immunogenicity is stronger. They can be used as candidate vaccines for new virus rabies virus vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The membrane structure of RABV-G-TM is shown in red, with the displaced transmembrane region (460-478);

[0034] FIG. 2 is the test result of Example 2 of the present invention, wherein Figure 2A The result of restriction enzyme digestion identification of pLV-RABV-G; Figure 2B This is a microscopic observation of PLV-RABV-G-TM packaged virus; Figure 2C This is the microscope observation picture of the blank control sample;

[0035] FIG3 is the detection result of Example 3 of the present invention, Figure 3A This is a fluorescence microscopy image of 293FT cells infected with RABV-G-TM packaged viruses. Figure 3B The electron micrograph of the collected virus-like particles RABV-G-TM VLP;

[0036] Figure 4 The Western Blot identification result of the virus-like particle RABV-G-TM VLP of Example 3 of the present invention;

[0037] Figure 5 This is the evaluation result of the neutralizing antibody titer in the immunization application of Example 4 of the present invention. DETAILED DESCRIPTION

[0038] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0039] In the present invention, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0040] Example 1. Design and synthesis of the recombinant gene RABV-G-TM

[0041] Sequence design and expression optimization were performed based on the RABV-G gene sequence, and the optimized transmembrane region of the RABV-G gene sequence was replaced. Figure 1 In the present invention, in the design of the RABV-G-TM gene sequence, the transmembrane region gene of the RABV-G gene sequence is replaced by the transmembrane region of the blue ear virus GP5 protein (transmembrane structure: 160-478), and Nhe I and Mlu I restriction sites are added before the start codon and after the stop codon of the expression frame to obtain the nucleotide sequence shown in SEQ ID NO: 1.

[0042] The nucleotide sequence shown in SEQ ID NO:1 was commissioned to Anhui General Biotechnology Co., Ltd. to use a gene synthesizer to obtain the recombinant gene of RABV-G-TM of the present invention. The amino acid sequence of the recombinant gene is shown in SEQ ID NO:2.

[0043] The specific sequence is shown in the following table, wherein for SEQ ID NO: 1, the bold italic part is the restriction site; the underlined part is the transmembrane region of the replaced blue ear virus GP5 protein.

[0044]

[0045] Example 2: Construction of recombinant plasmid and packaging virus

[0046] 1. Insert the RABV-G-TM recombinant gene into the pLV-eGFP vector to form the vector PLV-RABV-G-TM. Specifically, the steps include:

[0047] 1) Insert the RABV-G-TM recombinant gene into the expression plasmid pUC57 vector to form vector I;

[0048] 2) The lentiviral expression plasmid pLV-eGFP and vector I were double-digested with Nhe I and Mlu I restriction endonucleases, and the large fragment and small fragment were recovered by agarose gel electrophoresis;

[0049] 3) After ligation and transformation, the recombinant plasmid PLV-RABV-G-TM was obtained.

[0050] The gene sequence of RABV-G-TM is 1594 bp. The restriction enzyme digestion results of PLV-RABV-G-TM are as follows: Figure 2A shown.

[0051] 2. The constructed PLV-RABV-G-TM and envelope plasmids VSV-G and psPAX2 were co-transfected into host cells 293FT, cultured and collected the supernatant to obtain packaged virus.

[0052] Specifically, using PEI transfection reagent, the co-transfection method includes the following steps:

[0053] 2.1 Prepare the transfection system according to the following conditions (200 μl in total):

[0054] ① Solution A: PEI (5 μl) + basic DMEM = 100 μl;

[0055] ② Solution B: 2 μg plasmid DNA + basic DMEM = 100 μl, according to the mass ratio, plasmids PLV-RABV-G-TM, pPAX2 and pCMV-VSV-G = 4:3:1, mix the PLV-RABV-G-TM plasmid and the envelope plasmids pCMV-VSV-G and psPAX2, add DMEM to 100 μl,

[0056] 2.2 Mix solution A and solution B, add solution A to solution B, and mix gently; incubate at room temperature for 20 minutes. Add the transfection system evenly to the plate, shake gently to mix, and incubate in an incubator.

[0057] 2.3 Observe the cells every day to determine the cell transfection status. After the cells are cultured for 48 hours, collect the cell culture supernatant to obtain the packaged virus and observe the images under a fluorescence microscope. Figure 2B As shown, Figure 2C The blank control sample is 293FT cells.

[0058] Example 3: Construction of rabies virus-like particles (RABV-G-TM VLP)

[0059] A RABV-G-TM VLP particle, using the packaging virus prepared in Example 2, and infecting 293FT cells with the packaging virus at an MOI of 3. Specifically comprising:

[0060] Plate the cells at an appropriate density one day in advance to ensure that the host cells have a cell confluence of more than 50% within 20-24 hours before virus infection.

[0061] After 48 hours, the fluorescence was observed. Figure 3A As shown, the supernatant was harvested after 72 hours to collect VLP particles. The supernatant was subjected to WB detection, etc. At the same time, the fluorescent cells were screened for monoclonal cell lines, and the screened monoclonal cells were used to establish cell lines, which were cultured in serum-free SFM82 culture medium. The supernatant was harvested after 48-72 hours to collect virus-like particles RABV-G-TM VLP, and observed under an electron microscope after purification, as shown Figure 3B shown.

[0062] The rabies virus virus-like particles RABV-G-TM VLP were subjected to Western Blot detection. RABV-G-TM VLP virus-like particles reacted with RABV-G-053 antibodies to produce specific bands. The methods involved in the process can be methods well known in the art. Figure 4 Western Blot identification results of RABV-G-TM VLP in sample No. 1 showed that the size was approximately 58KDA.

[0063] Figure 4Sample No. 2 is the identification result of virus-like particles RABV-G VLP without replacing the transmembrane structure constructed under the same conditions. Calculated by imageJ software, the expression level of virus-like particles after replacing the transmembrane structure is 2 times higher than that without replacing the transmembrane structure.

[0064] Example 4: Application of rabies virus-like particles (RABV-G-TM VLP) in mouse immunization

[0065] The virus-like particles RABV-G-TM VLP provided in Example 3 were used to immunize mice with RABV-G-TM VLP as antigen, and blood was drawn from the mice for rabies virus neutralizing antibody detection.

[0066] Test results such as Figure 5 As shown, it can be seen that the neutralizing antibody titer produced is high, and the aluminum adjuvant group is better than the imported commercial inactivated vaccine.

[0067] The above description shows that the rabies virus-like particle RABV-G-TM VLP of the present invention has the biological characteristics of the inactivated rabies vaccine, has a single component, is safer and more effective, and can be used as a new rabies virus vaccine candidate.

[0068] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0069] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A recombinant gene of rabies virus G protein virus-like particles, characterized in that: The nucleotide sequence of the recombinant gene is shown in SEQ ID NO:

1.

2. A method for constructing rabies virus-like particles, characterized in that: The steps are as follows: 1) constructing a recombinant plasmid PLV-RABV-G-TM; the recombinant plasmid is constructed by inserting the recombinant gene described in claim 1 into a pLV-eGFP vector; 2) co-transfecting the above-mentioned recombinant expression plasmid PLV-RABV-G-TM, envelope plasmid VSV-G and packaging plasmid psPAX2 into host cells, culturing and collecting the supernatant to obtain packaged virus; 3) The packaged virus is used to infect host cells, and the supernatant is collected to obtain virus-like particles.

3. The construction method according to claim 2, characterized in that: Step 1) includes the following steps: 1.1 Insert the recombinant gene described in claim 1 into the expression plasmid pUC57 vector to form vector I; 1.2 The lentiviral expression plasmid pLV-eGFP and vector I were double-digested with Nhe I and Mlu I restriction endonucleases, and the large and small fragments were recovered by agarose gel electrophoresis; 1.3 After ligation and transformation, the recombinant plasmid PLV-RABV-G-TM was obtained.

4. The construction method according to claim 2, characterized in that: In the step 2), during the transfection process, the DNA mass ratio of plasmid PLV-RABV-G-TM:VSV-G:psPAX2 is 4:1:

3.

5. The construction method according to claim 2, characterized in that: In step 3), ensure that the cell confluence of the host cells reaches more than 50% within 20-24 hours before packaging the virus for infection.

6. The construction method according to claim 2, characterized in that: The host cell is a human embryonic kidney cell HEK-293FT.

7. The construction method according to claim 2, characterized in that: During transfection, PEI transfection reagent was used.

8. The rabies virus-like particles obtained by the construction method according to any one of claims 2 to 7.

9. Use of the virus-like particles according to claim 8 in the preparation of rabies virus vaccine.

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