A kind of influenza virus NP protein mutant and its application

Abstract

The present invention discloses an influenza virus NP protein mutant and its application. The NP mutant protein of the present invention is obtained by mutating the arginine at positions 74, 75, 174, 175, and 221 of the NP protein from the wild-type influenza virus A / human / Puerto Rico / 8 / 34 (H1N1) strain to glycine, while other amino acid residues remain unchanged. The amino acid sequence of the mutant is shown in SEQ ID No. 1. Compared with the wild-type NP protein, the NP mutant protein provided by the present invention does not bind to bacterial RNA, solves the safety hazard of the wild-type NP protein, and also has high immunogenicity, and has high application prospects as a candidate antigen for a universal influenza vaccine.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an influenza virus NP protein mutant and an application thereof. Background Art

[0002] Influenza is a highly pathogenic, contagious acute respiratory disease caused by influenza virus type A of the Orthomyxoviridae family, which infects humans, poultry, pigs and other animals. Due to the wide host range and rapid mutation rate of the virus, seasonal influenza occurs to varying degrees, with millions of severe cases and hundreds of thousands of deaths worldwide each year, posing a huge threat to human health and social economy. Vaccination is the main means of preventing influenza. The influenza virus nucleoprotein NP is highly conserved among different subtypes and can induce a cellular immune response in the host to eliminate infected cells and protect immune animals against infection by multiple subtypes of influenza viruses. It is one of the hot targets for the development of universal influenza vaccines.

[0003] NP protein plays an important role in the viral life cycle. It binds to viral RNA (vRNA) to form a vRNP complex and participates in multiple biological functions such as viral RNA replication and transcription. When expressing NP protein, we found that NP protein expressed and purified using the E. coli expression system would non-specifically bind to bacterial RNA in large quantities. NP protein containing bacterial RNA can induce inflammatory responses and has a strong toxic effect on mice, posing certain safety risks. Therefore, it is of great significance to remove the activity of NP protein in non-specific binding to bacterial RNA while retaining its immunogenicity for the development of universal influenza vaccines based on NP protein. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an influenza virus NP mutant protein with high immunogenicity and high safety, and a preparation method and application thereof.

[0005] The technical solution of the present invention is: an influenza virus NP protein mutant, the amino acid sequence of the mutant is shown in SEQ ID No.1.

[0006] A gene encoding the mutant described above, the nucleotide sequence of the gene is shown as SEQ ID No.2.

[0007] An mRNA encoding the mutant, the nucleotide sequence of the mRNA is shown as SEQ ID No.3.

[0008] An expression vector containing the gene described above.

[0009] A genetically engineered bacterium containing the above-mentioned expression vector.

[0010] The use of the mutant or gene or mRNA or expression vector or genetically engineered bacteria described above in the preparation of influenza virus vaccines.

[0011] A recombinant influenza virus protein vaccine contains the mutant.

[0012] Furthermore, the vaccine also contains adjuvant CpG1826, and the nucleotide sequence of CpG1826 is shown in SEQ ID No.12.

[0013] An influenza virus DNA vaccine contains the gene.

[0014] An influenza virus mRNA vaccine contains the mRNA.

[0015] The NP mutant protein of the present invention is obtained by mutating the arginine at positions 74, 75, 174, 175, and 221 of the NP protein from the wild-type influenza virus A / human / Puerto Rico / 8 / 34 (H1N1) strain to glycine, while the other amino acid residues remain unchanged. This study is applicable to the NP proteins of other strains, and the arginine at the corresponding sites is mutated to glycine to obtain a safe NP protein antigen.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] Compared with the wild-type NP protein, the NP mutant protein provided by the present invention does not bind to bacterial RNA, thereby solving the potential safety hazard of the wild-type NP protein. It also has high immunogenicity and has a high application prospect as a candidate antigen for a universal influenza vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Four PCR amplified fragments of NP mutant gene.

[0019] Figure 2 :The electrophoresis results of restriction enzyme digestion identification of NP mutant protein expression plasmid.

[0020] Figure 3 :The NP mutant protein was purified by molecular sieve chromatography and the target protein was verified by SDS-PAGE and western blot.

[0021] Figure 4 :Gel shift assays detected that purified NP mutant protein does not bind to bacterial RNA.

[0022] Figure 5 : ELISA detected that mice immunized with NP mutant protein produced specific total IgG, IgG1 and IgG2a antibodies.

[0023] Figure 6 : ELISA detected that mice immunized with NP mutant protein produced specific mucosal IgG and IgA antibodies.

[0024] Figure 7 :Body weight changes and survival rate of mice immunized with NP mutant protein after challenge.

[0025] Figure 8 :Pathological changes in lung tissue of mice immunized with NP mutant protein after challenge, Ⅱ-Ⅳ are Figure Ⅰ Local magnification. DETAILED DESCRIPTION

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0027] Sources and descriptions of key materials

[0028] pET28a vector: maintained by this laboratory.

[0029] Escherichia coli BL21 (DE3) competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0030] CpG1826: synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0031] BALB / c mice were purchased from the Experimental Animal Center of Huazhong Agricultural University.

[0032] Recombinant plasmid of PR8 virus NP gene: preserved by this laboratory.

[0033] Example 1: Construction of expression plasmids for NP mutant proteins and purification of proteins

[0034] 1.1 PCR amplification of target gene

[0035] First, four pairs of primers for the NP mutant gene were designed. The primers included protective bases, restriction sites, and template-binding nucleotide sequences. The designed primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0036] The nucleotides at the expected mutation sites were mutated using a three-step PCR method. The recombinant plasmid of the PR8 virus NP gene was used as a template, and P1 Fw and P1 Rw, P2 Fw and P2 Rw, P3 Fw and P3 Rw, and P4 Fw and P4 Rw were used as upstream and downstream primers for PCR amplification. The lengths of the four amplified products (P1, P2, P3, and P4) were 243 bp, 315 bp, 156 bp, and 855 bp, respectively. The amplification results are shown in Figure 1As shown in A, the target band is obvious and in line with expectations. The 4 amplified fragments were recovered by gel recovery kit. The recovered P1 and P2, P3 and P4 PCR products were used as templates, and P1 Fw and P2 Rw, P3 Fw and P4Rw were used as primers for the second overlap PCR. The lengths of the P1-2 and P3-4 fragments obtained by PCR were 546 bp and 990 bp, respectively. The amplification results are shown in Figure 1 As shown in B, the P1-2 and P3-4 PCR products were recovered by gel recovery kit. The third overlap PCR was performed using the gel-recovered P1-2 and P3-4 products as templates and P1 Fw and P4 Rw as primers. The length of the amplified product P1-4 was 1524 bp. In this way, the NP gene fragment P1-4 encoding the R74G, R75G, R174G, R175G, and R221G point mutations was obtained. The amplification results are shown in Figure 1 As shown in C. The PCR product was electrophoresed by 1.5% agarose gel, and the fragment was recovered by gel recovery kit and the DNA concentration was measured and stored for future use.

[0037] Amplification of NP P1 gene fragment: Upstream primer P1 Fw: 5'-AGC CATATG GCTAGCGCGTCCCAAGGCACCAAA-3' (SEQ ID No. 4; the restriction enzyme site NdeI is underlined); downstream primer P1 Rw: 5'-ATTCCCACCTTCGTCAAAAGC-3' (SEQ ID No. 5).

[0038] Amplification of NP P2 gene fragment: upstream primer P2 Fw: 5'-CGAAGGTGGGAATAAATACCTG-3' (SEQ ID No. 6); downstream primer P2 Rw: 5'-TCCAGAACCGCCAGGGAGAGTTGA-3' (SEQ ID No. 7).

[0039] Amplification of NP P3 gene fragment: upstream primer P3 Fw: 5'-GGCGGTTCTGGAGCCGCAGGTGCTGCAGTCAAA-3' (SEQ ID No. 8); downstream primer P3 Rw: 5'-AATGTTGCACATACCTTCATAAGC-3' (SEQ ID No. 9).

[0040] Amplification of NP P4 gene fragment: upstream primer P4 Fw: 5'-TATGAAGGTATGTGCAACATTCTC-3' (SEQ ID No. 10); downstream primer P4 Rw: 5'-GTGGTGGTG CTCGAGATTGTCGTACTCCTCTGCATT-3′ (SEQ ID No. 11; the underline indicates the restriction enzyme site XhoI).

[0041] The PCR reaction amplification system is as follows: 1 μL template DNA, 10 μL high-fidelity enzyme, 1 μL upstream and downstream primers, 8 μL ultrapure water. After mixing evenly, set the PCR amplification program, 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 50-60°C annealing for 15 s, 72°C extension for 15-45 s, 72°C post-extension for 5 min, including 30 cycles of denaturation to extension, and store at 16°C for future use.

[0042] 1.2 Construction of expression plasmid

[0043] The gel recovery product and the pET28a empty vector plasmid were double digested with NdeI and XhoI. After gel electrophoresis, the gel was recovered and the concentration was measured by spectrophotometer and stored for later use. The enzyme digestion reaction system is as follows:

[0044]

[0045] The enzyme digestion reaction system was incubated in a 37°C water bath for 30-60 min. The digestion product was identified by agarose gel electrophoresis and then recovered by using an OMEGA gel recovery kit. After measuring the concentration, the product was stored for later use.

[0046] Then, the digested PCR target fragment and plasmid fragment were connected with T4 ligase at 16°C overnight, and the connection product was added to the competent E. coli DH5α cells and placed on ice for 30 minutes; after the ice bath, the competent cells with the connection product were placed in a 42°C water bath for 90 seconds, and immediately placed on ice for 2 minutes after the heat shock; 500μL of LB culture medium was added and placed in a 37°C shaker for incubation for 45 minutes; after the incubation, centrifuged at 5000rpm for 3 minutes, discarded 450μL of the supernatant, and the remaining liquid was resuspended in the bacteria, and spread on a plate containing kanamycin resistance, and cultured at 37°C overnight. The connection system is as follows:

[0047]

[0048] The next day, a single colony on the plate was randomly picked and placed in 10 mL of LB liquid culture medium containing 50 μg / mL kanamycin. After culturing for 12 hours, the plasmid was extracted and digested for enzyme identification. The identification results were as follows: Figure 2 As shown in the figure, after enzyme digestion, both plasmids have two target bands, one for the vector after enzyme digestion and the other for the NP mutant gene fragment after enzyme digestion, proving that the expression plasmids were successfully connected. The two plasmids were sent to the company for sequencing, and the sequencing results were blasted with the plasmid map to confirm that the sequence was correct.

[0049] 1.3 Protein expression and purification

[0050] The above-mentioned correctly sequenced plasmid was transformed into the competent E. coli BL21 (DE3) expression medium, and after ice bathing for 30 minutes, heat shock was placed in a 42°C water bath for 45 seconds. After the heat shock, it was immediately placed on ice for 2 minutes. 500 μL of LB medium was added to the competent medium, and it was incubated in a 37°C shaker for 45 minutes. After the incubation, centrifugation was performed at 5000 rpm for 3 minutes, and 500 μL of supernatant was discarded. The bacteria were resuspended with the remaining supernatant and spread on an LB solid culture dish containing 50 μg / mL kanamycin, and cultured in a 37°C constant temperature incubator overnight.

[0051] Positive clones were picked and cultured overnight in 10 mL LB liquid medium containing kanamycin. The next day, the clones were transferred to 1 L LB medium at a ratio of 1:100 and placed in a 37°C shaker for about 3 hours until the OD value was about 0.8. The temperature of the shaker was lowered to 28°C 20 minutes in advance, and IPTG with a final concentration of 1 mM was added to induce expression for 4 hours. The E. coli cells were collected by centrifugation at 7000 rpm for 15 minutes, and the precipitate was washed 3 times with PBS. After the last centrifugation, the supernatant was discarded and the cell pellet was resuspended with 100 mL Binding buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, adjusted to pH 8.0 with concentrated hydrochloric acid). The cells were lysed at 4°C with a high-pressure cell disruptor and the cell debris was removed by centrifugation at 35,000 g, 20 min, 4°C. The collected supernatant was filtered through a 0.22 μm filter and loaded onto a his-tagged protein purification pre-loaded column by a peristaltic pump. About 30 mL Wash buffer (50 mM Tris, 300 mM NaCl, 20 mM imidazole, adjusted to pH 8.0 with concentrated hydrochloric acid) was used to remove non-specifically bound impurities. Finally, the target protein bound to the His column was eluted with Elution buffer (50 mM Tris, 300 mM NaCl, 400 mM imidazole, adjusted to pH 8.0 with concentrated hydrochloric acid).

[0052] The collected protein eluate of about 5 mL was passed through the chromatographic column by the AKTA system, and the protein with the highest peak (peak 1) in the gel filtration elution curve was collected ( Figure 3 A), and then concentrated using a 10KDa protein ultrafiltration tube, and dispensed into 1.5mL sterile EP tubes, 50μL per tube, and stored in a -80℃ refrigerator for later use.

[0053] Take 10 μL of NP mutant protein purified by molecular sieve chromatography and add it to a PCR tube containing 15 μL PBS and 5 μL 5×SD Ssample buffer. Mix well and boil at 100°C for 10 min. Perform SDS-PAGE electrophoresis. First run the stacking gel at 80 V, then run the separation gel at 120 V. After the electrophoresis, remove the first protein gel from the glass plate and stain with Coomassie brilliant blue for 2 h, then decolorize. Another protein gel was used for Western blot verification. After 50 minutes of transfer at a constant voltage of 65V, the PVDF membrane was washed 3 times with TBST buffer for 5 minutes each time, then blocked with 10% skim milk powder at room temperature for 2 hours, and washed 3 times with TBST for 5 minutes each time; the primary antibody of mouse anti-his-tag (company: abclonal, catalog number: AE003) was slowly shaken at room temperature for 2 hours, and washed with 1×TBST after the primary antibody incubation, each time for 5 minutes, and washed 5 times; the secondary antibody labeled with HRP goat anti-mouse IgG (company: Abbkine, catalog number: A21010) was incubated at room temperature for 45 minutes, and washed 5 times with 1×TBST for 5 minutes each time; the A of ECL luminescent colorimetric solution (company: biosharp, catalog number: bl520a) was added. Mix equal volumes of solution A and solution B, remove the membrane, absorb the liquid on the membrane with filter paper, and evenly immerse it in the mixed solution. Use a chemiluminescence instrument to expose and image. The purified NP mutant protein is about 55 kDa, and the band position is consistent with the expectation ( Figure 3 Chinese BC).

[0054] Example 2: Identification of NP mutant proteins that do not bind bacterial RNA

[0055] In order to confirm that the purified NP mutant protein will not bind to bacterial RNA, we conducted a gel retardation analysis experiment. Take out the prepared 6.5% non-denaturing polyacrylamide gel, add an appropriate amount of 0.5×TBE electrophoresis buffer to the electrophoresis tank, rinse the sample wells, and pre-electrophoresis at 80V for 30min-60min; add 10μL of purified protein, 5μL DEPC water and 2μL 10×DNA loading buffer to the PCR tube, gently blow and mix, and react at 26℃ for 30min. After the pre-electrophoresis, pour out the pre-electrophoresis buffer, add new 0.5×TBE, rinse the sample wells, and spot the sample. Then put the electrophoresis tank on ice and run electrophoresis at 100V for 1-2h. After the electrophoresis, place the gel block in 40mL of EB staining solution (10μL EB solution dissolved in 40mL pure water) and shake on a decolorizing shaker for 30min. The results are as follows Figure 4As shown in the figure, no bacterial RNA was detected in the NPmut gel wells. At the same time, we also set up 2 control wells, in which the wild-type NP protein gel wells contained a large amount of bacterial RNA, while the wild-type NP protein (NP-Tx) gel wells treated with RNase reagents still contained a small amount of RNA substances. Therefore, NP mutant protein, as a candidate antigen for universal influenza vaccine, can effectively solve the safety risks caused by wild-type NP protein binding to bacterial RNA.

[0056] Example 3: Evaluation of the immune protection effect of NP mutant protein

[0057] 1.1 Animal Immunization Experiment

[0058] To evaluate the immunogenicity of NP mutant (NPmut) protein, we randomly divided 77 6-8 week old female BALB / c mice (purchased from the Experimental Animal Center of Huazhong Agricultural University) into 7 groups, with 11 mice in each group. Each mouse was immunized with 15 μg protein, and the volume of each mouse was 50 μL. The mice were immunized by intranasal drops on days 0, 14, and 28. We also set up wild-type NP protein and NP protein treated with RNase reagent (NP-Tx) as control groups. The detailed experimental groups and immunization doses are as follows:

[0059] No. Experimental Grouping Immunization dose 1 PBS+CpG1826 3μg 2 NP 15 μg 3 NPmut 15 μg 4 NPmut+CpG1826 15μg+3μg 5 NP 15 μg 6 NP-Tx 15 μg 7 NP-Tx+CpG1826 15μg+3μg

[0060] 1.2 Indirect ELISA to detect antigen-specific humoral immune response

[0061] 14 days after the third immunization, blood was collected from the tail vein of mice and serum was separated. The titer of antigen-specific IgG antibodies in the serum was detected by indirect ELISA. The purified NP protein was diluted to 2 μg / mL with coating buffer, 100 μl per well, added to the ELISA plate, and coated overnight at 4°C; the next day, it was washed 5 times with PBST, 200 μL of 3% BSA blocking solution was added to each well, and incubated at 37°C for 1 hour; after blocking, it was washed 5 times with PBST, and the serum was diluted 2-fold at a ratio of 1:200 to the 12th well, and incubated at 37°C for 1 hour. After incubation, the plate was washed 5 times with PBST, 100 μL of HRP-labeled goat anti-mouse IgG dilution (1:8000 dilution) was added to each well and incubated at 37°C for 45 minutes. The secondary antibody diluent was discarded, and the ELISA plate was washed again with PBST for 5 times. 100 μL of TMB colorimetric solution was added to each well. After color development at room temperature and in the dark until no color change occurred, 100 μL of 2M sulfuric acid stop solution was added to each well to stop color development. The OD value at a wavelength of 450 nm was measured using an ELISA reader. The results are shown in Figure 2. Figure 5As shown in A, the antibody titers of mice in the immunized NPmut+CpG1826 group and the NP protein group were significantly higher than those in the NPmut and PBS groups, and the difference was not significant compared with the NP group. In addition, the NP-specific antibody titer induced by the NP-Tx and CpG1826 adjuvant mixed group was also significantly higher than that in the NP-Tx group. The PBS control group could not induce NP-specific antibodies. This indicates that the NP mutant protein mixed group with the CpG1826 molecular adjuvant, the NP protein (NP-Tx) treated with RNase A and the CpG1826 molecular adjuvant mixed group, and the NP protein group all induced similar and high levels of humoral immune responses.

[0062] In order to analyze the IgG subtype, NP protein was used as the coating antigen to detect the antibody titers of IgG1 and IgG2a in the serum by indirect ELISA. The secondary antibody replaced IgG with HRP-labeled goat anti-mouse IgG1 and IgG2a (1:3000 dilution), and the rest of the steps were the same as above. The results showed that compared with the NPmut, PBS and NP-Tx groups, the NPmut+CpG1826 group, NP-Tx+CpG1826 and NP protein groups could induce significantly higher levels of NP-specific IgG1 and IgG2a antibodies, producing a balanced Th1 / Th2 immune response ( Figure 5 (B, C).

[0063] 1.3 Indirect ELISA to detect antigen-specific mucosal immune response

[0064] IgG and IgA antibodies produced by local mucosa have been shown to play an important role in preventing influenza infection. To evaluate whether NPmut protein can induce mucosal antibodies, we euthanized mice 10 days after the third immunization, lavaged the lungs of mice three times with 1 mL of ice-cold PBS, and then centrifuged at 3500 g for 10 min at 4 °C. The supernatant was collected and the antigen-specific IgG and IgA mucosal antibody titers of bronchoalveolar lavage fluid (BALF) were detected by indirect ELISA. Figure 6 As shown in A and B, compared with the NPmut group, the NP group, the NP-Tx mixed with CpG1826 group, and the NPmut mixed with CpG group all induced similar and high levels of IgG and IgA antibodies; no obvious NP-specific IgG and IgA antibodies were detected in the mucosa of mice immunized with PBS. In summary, the mixture of NP mutant protein and CpG1826 molecular adjuvant can induce high levels of local mucosal antibodies.

[0065] 1.4 Challenge experiment to evaluate the protective effect of NP mutant protein on mice

[0066] 14 days after the third immunization, 5 mice from each group were anesthetized and infected with 5LD 50Homologous A / human / Puerto Rico / 8 / 34 (H1N1) influenza virus. The number of deaths and body weight of mice in each group were observed and recorded every day for 14 days (weight loss greater than 25% was considered death and euthanasia was performed immediately). The results showed that the average weight change rate of mice in all groups dropped to the minimum on the 7th day after infection and then began to gradually recover. The mice in the PBS group, NP-Tx group and NPmut group all died on the 9th day after virus infection, while 40% of the mice in the NPmut and CpG1826 mixed group, NP-Tx and CpG1826 mixed group and NP group survived ( Figure 7 (A and B). Therefore, the immune protection efficiency provided by the mixture of NPmut+CpG1826 after immunization of mice was the same as that of the NP protein group, and it can be used as a candidate target for universal influenza vaccine in the future.

[0067] 1.5 Evaluation of the protective effect of NP mutant protein on mice by lung tissue pathology

[0068] 14 days after the third immunization, three mice from each group were anesthetized and infected with 5LD 50 A / human / Puerto Rico / 8 / 34(H1N1) influenza virus. On the 6th day after the challenge, the lung tissues of the mice were taken and HE staining was performed. The results are as follows Figure 8 As shown. Compared with the lung tissues of healthy mice, the lungs of mice immunized with PBS, NP-Tx and NPmut showed obvious lesions, with large-scale consolidation, obvious thickening of the alveolar wall, and a large number of inflammatory cell infiltration around the bronchi and pulmonary blood vessels. The lung tissues of mice immunized with NP protein, NP-Tx and CpG1826 adjuvant mixture, and NPmut and CpG1826 adjuvant mixture showed similar pathological changes, with milder lesions, clear alveolar structure, mild to moderate thickening of the alveolar wall, and a small amount of inflammatory cell infiltration.

Claims

1. An influenza virus NP protein mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID No.

1. The NP mutant protein is obtained by mutating the arginine at positions 74, 75, 174, 175, and 221 of the NP protein from the wild-type influenza virus A / human / Puerto Rico / 8 / 34 (H1N1) strain to glycine, while keeping the other amino acid residues unchanged.

2. A gene encoding the mutant according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No.

2.

3. An mRNA encoding the mutant according to claim 1, characterized in that The nucleotide sequence of the mRNA is shown in SEQ ID No.

3.

4. An expression vector containing the gene according to claim 2.

5. A genetically engineered bacterium containing the expression vector according to claim 4.

6. Use of the mutant according to claim 1, the gene according to claim 2, the mRNA according to claim 3, the expression vector according to claim 4, or the genetically engineered bacteria according to claim 5 in the preparation of influenza virus vaccines.

7. A recombinant influenza virus protein vaccine, characterized in that: The vaccine contains the mutant according to claim 1, and further contains an adjuvant CpG1826, the nucleotide sequence of which is shown in SEQ ID No.

12.

8. An influenza virus DNA vaccine comprising the gene according to claim 2.

9. An influenza virus mRNA vaccine comprising the mRNA according to claim 3.

Images