CN117659138A

CN117659138A - Influenza virus NP protein mutant and application thereof

Info

Publication number: CN117659138A
Application number: CN202311796040.7A
Authority: CN
Inventors: 陶攀; 陈岑; 国爱丽; 周红波; 李梦玲; 郭鹏举
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2023-12-25
Filing date: 2023-12-25
Publication date: 2024-03-08

Abstract

The invention discloses an influenza virus NP protein mutant and application thereof, and the NP mutant protein is obtained by mutating the arginine at the 74 th, 75 th, 174 th, 175 th and 221 th positions of NP protein from wild type influenza virus A/human/Puerto Rico/8/34 (H1N 1) strain into glycine, and the other amino acid residues are unchanged. The amino acid sequence of the mutant is shown as SEQ ID No. 1. Compared with the wild NP protein, the NP mutant protein provided by the invention does not combine with bacterial RNA, solves the potential safety hazard of the wild NP protein, has high immunogenicity, and has a high application prospect as a candidate antigen of a universal influenza vaccine.

Description

Influenza virus NP protein mutant and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an influenza virus NP protein mutant and application thereof.

Background

Influenza is a highly pathogenic, infectious acute respiratory disease caused by influenza virus type a of the orthomyxoviridae family, and infects many animals such as humans, birds, pigs, etc. Because the virus has wide host range and high mutation speed, seasonal influenza occurs to different degrees, millions of severe cases and hundreds of thousands of deaths occur annually worldwide, which constitutes a great hazard to human health and social economy. Vaccination is the primary means of influenza prevention. Influenza virus nucleoprotein NPs are highly conserved among different subtypes, can induce a host to generate cellular immune response so as to clear infected cells, protect immune animals against infection of multiple subtype influenza viruses, and are one of the popular targets for developing universal influenza vaccines.

The NP protein plays an important role in the life cycle of the virus, and is combined with viral RNA (vRNA) to form a vRNP complex, and the vRNP complex is involved in various biological functions such as replication and transcription of the viral RNA. We have found that NP proteins expressed and purified using E.coli expression systems bind to bacterial RNA in large amounts non-specifically when the NP proteins are expressed. The NP protein containing the bacterial RNA can induce inflammatory reaction, has stronger toxic effect on mice, and has certain potential safety hazard. Therefore, removal of the activity of NP proteins that bind non-specifically to bacterial RNAs, while retaining their immunogenicity, is of great interest for the development of NP protein-based universal influenza vaccines.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: provides an influenza virus NP mutant protein with high immunogenicity and high safety, a preparation method and application thereof.

The technical scheme of the invention is as follows: an influenza virus NP protein mutant, the amino acid sequence of which is shown as SEQ ID No. 1.

A nucleotide sequence of the gene encoding the mutant is shown as SEQ ID No. 2.

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

An expression vector containing the above-mentioned gene.

Genetically engineered bacteria containing the expression vector.

The mutant or the gene or the mRNA or the expression vector or the genetically engineered bacterium are applied to the preparation of influenza virus vaccines.

An influenza virus recombinant protein vaccine contains the mutant.

Further, the vaccine also contains an adjuvant CpG1826, and the nucleotide sequence of the CpG1826 is shown as SEQ ID No. 12.

An influenza virus DNA vaccine comprising said gene.

An influenza virus mRNA vaccine comprising said mRNA.

The NP mutant protein of the present invention is obtained by mutating the arginine at the 74 th, 75 th, 174 th, 175 th and 221 th positions of NP protein from wild type influenza A/human/Puerto Rico/8/34 (H1N 1) strain to glycine, and the other amino acid residues are unchanged. The research is suitable for NP proteins of other strains, and the arginine at the corresponding site is mutated into glycine to obtain the safe NP protein antigen.

Compared with the prior art, the invention has the following beneficial effects:

compared with the wild NP protein, the NP mutant protein provided by the invention does not combine with bacterial RNA, solves the potential safety hazard of the wild NP protein, has high immunogenicity, and has a high application prospect as a candidate antigen of a universal influenza vaccine.

Drawings

Fig. 1: 4 PCR amplified fragments of NP mutant gene.

Fig. 2: electrophoresis results of the enzyme digestion identification of NP mutant protein expression plasmids.

Fig. 3: the NP mutant protein was purified by molecular sieve chromatography and SDS-PAGE and western blot was performed to verify the protein of interest.

Fig. 4: gel shift Assays detected that purified NP mutant proteins did not bind to bacterial RNA.

Fig. 5: ELISA mice that detected the immune NP mutant proteins produced specific total IgG, igG1, and IgG2a antibodies.

Fig. 6: ELISA mice that detected the immune NP mutant proteins produced specific mucosal IgG and IgA antibodies.

Fig. 7: body weight change and survival rate of NP mutant protein immunized mice after challenge.

Fig. 8: the lung tissue pathological changes of the NP mutant protein immunized mice after toxin attack are amplified locally in the graph I.

Detailed Description

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.

Key material sources and description

pET28a vector: stored by the laboratory.

Coli BL21 (DE 3) competent: purchased from Shanghai Biotechnology limited only.

CpG1826: synthesized by the division of bioengineering (Shanghai).

BALB/c mice: purchased from the university of agricultural laboratory animal center in China.

Recombinant plasmid of PR8 virus NP gene: stored by the laboratory.

Example 1: construction of expression plasmids and purified proteins for NP mutant proteins

1.1PCR amplification of the Gene of interest

Firstly, 4 pairs of primers of NP mutant genes are designed, wherein the primers comprise protective bases, enzyme cutting sites and nucleotide sequences combined with templates, and the designed primers are sent to a biological engineering (Shanghai) stock company for synthesis.

The nucleotide at the desired mutation site was mutated using a 3-step PCR method. The recombinant plasmid of PR8 virus NP gene is used as a template, P1 Fw and P1 Rw, P2 Fw and P2 Rw, P3 Fw and P3 Rw and P4 Fw and P4 Rw are respectively used as upstream and downstream primers for PCR amplification, the lengths of 4 amplified products (P1, P2, P3 and P4) are 243bp, 315bp, 156bp and 855bp respectively, the amplified results are shown as A in figure 1, the target bands are obvious and meet expectations, and 4 amplified fragments are recovered by a gel recovery kit. And (3) taking the recovered P1 and P2, P3 and P4 PCR products as templates, taking P1 Fw and P2 Rw, P3 Fw and P4 Rw as primers, performing a second overlap PCR, obtaining fragments of P1-2 and P3-4 with lengths of 546bp and 990bp respectively by PCR, and recovering the P1-2 and P3-4 PCR products by using a gel recovery kit as shown in the amplification result B in FIG. 1. And performing a third overlap PCR by taking the products of P1-2 and P3-4 recovered by the gel as templates and P1 Fw and P4 Rw as primers, wherein the length of the amplified product P1-4 is 1524bp. The NP gene fragments P1-4 thus obtained, which specifically encode the point mutation of R74G, R75G, R174G, R175G, R G, were amplified as shown in FIG. 1C. The PCR product was subjected to 1.5% agarose gel electrophoresis, and the fragment was recovered by a gel recovery kit and stored for use after measuring the DNA concentration.

Amplification of NP P1 Gene fragment: the upstream primer P1 Fw:5' -AGCCATATGGCTAGCGCGTCCCAAGGCACCAAA-3' (SEQ ID No.4; underlined is the restriction site NdeI); downstream primer P1 Rw:5'-ATTCCCACCTTCGTCAAAAGC-3' (SEQ ID No. 5).

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

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

Amplification of NP P4 Gene fragment: the upstream primer P4 Fw:5'-TATGAAGGTATGTGCAACATTCTC-3' (SEQ ID No. 10); downstream primer P4 Rw:5' -GTGGTGGTGCTCGAGATTGTCGTACTCCTCTGCATT-3' (SEQ ID No.11; underlined is the restriction site XhoI).

The PCR amplification system is as follows: 1. Mu.L of template DNA, 10. Mu.L of high-fidelity enzyme, 1. Mu.L of each of the upstream and downstream primers, and 8. Mu.L of ultrapure water. After mixing uniformly, setting a PCR amplification program, carrying out denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 50-60 ℃ for 15s, extension at 72 ℃ for 15-45s, and extension at 72 ℃ for 5min, wherein the denaturation is carried out for 30 cycles until the extension process, and preserving at 16 ℃ for standby.

1.2 construction of expression plasmids

The gel recovery product and pET28a empty vector plasmid were subjected to double digestion with NdeI and XhoI. And (5) cutting and recycling through agarose gel electrophoresis, and storing for standby after measuring the concentration by a spectrophotometer. The enzyme digestion reaction system is as follows:

the enzyme digestion reaction system is placed in a water bath kettle at 37 ℃ for incubation for 30-60min, enzyme digestion products are identified by agarose gel electrophoresis, and then the enzyme digestion products are subjected to gel digestion recovery by adopting an OMEGA gel recovery kit, and are stored for standby after concentration measurement.

Then, connecting the digested PCR target fragment and plasmid fragment by using T4 ligase at 16 ℃ overnight, adding the connection product into competent cells of escherichia coli DH5 alpha, and placing the cells on ice for 30min; after the ice bath is finished, placing the competence with the connection product in a water bath kettle at 42 ℃ for heat shock for 90 seconds, and immediately placing the mixture on ice for standing for 2 minutes after the heat shock is finished; adding 500 μl of LB culture medium, and incubating in a shaker at 37deg.C for 45min; after the incubation was completed, the supernatant was discarded at 450. Mu.L, the remaining liquid was resuspended in cells, and plated onto kanamycin-resistant plates for overnight incubation at 37 ℃. The connection system is as follows:

the single colony on the flat plate is randomly picked up to 10mL of LB liquid medium containing 50 mug/mL kanamycin in the next day, plasmids are extracted for enzyme digestion identification after 12h of culture, the identification result is shown in figure 2, two plasmids are provided with two strips after enzyme digestion, one is the carrier after enzyme digestion, the other is the NP mutant gene fragment after enzyme digestion, and the connection of the expression plasmids is proved to be successful. And (3) sending the two plasmids to a company for sequencing, and carrying out blast sequence analysis on the sequencing result and a plasmid map to determine that the sequence is correct.

1.3 expression and purification of proteins

The plasmid with correct sequencing is transformed into the expression competence of escherichia coli BL21 (DE 3), after ice bath for 30min, the plasmid is thermally shocked for 45s in a water bath kettle at 42 ℃, after the thermal shock is finished, a vertical horse is placed in ice bath for 2min, 500 mu L of LB culture medium is added into the competence, the mixture is put into a shaking table at 37 ℃ for 45min, centrifugation is carried out at 5000rpm for 3min after the incubation is finished, 500 mu L of supernatant is discarded, and after the thallus is resuspended by the residual supernatant, the supernatant is coated on an LB solid culture dish containing 50 mu g/mL kanamycin, and the culture is carried out in a constant temperature incubator at 37 ℃ for overnight.

Positive clones were picked up and cultured overnight in 10mL LB liquid medium containing kanamycin, transferred to 1L LB medium at a ratio of 1:100 the next day, placed in a shaking table at 37℃for shaking for about 3h to OD value of about 0.8, cooled to 28℃20min in advance, and added with IPTG at a final concentration of 1mM for induction of expression for 4h. E.coli cells were collected by centrifugation at 7000rpm for 15 min and the pellet was washed 3 times with PBS. After the last centrifugation, the supernatant was discarded and the cell pellet was resuspended in 100mL Binding buffer (50mM Tris,300mM NaCl,10mM imidazole, pH 8.0 with concentrated hydrochloric acid), lysed by a high pressure cell disrupter at 4℃and cell debris removed by centrifugation at 35000g,20min,4℃the collected supernatant was filtered through a 0.22 μm filter and loaded onto His-tagged protein purification cartridge by peristaltic pump, non-specifically bound heteroproteins were removed with about 30mL Wash buffer (50mM Tris,300mM NaCl,20mM imidazole, pH 8.0 with concentrated hydrochloric acid), and finally the protein of interest bound to the His cartridge was eluted with an partition buffer (50mM Tris,300mM NaCl,400mM imidazole, pH 8.0 with concentrated hydrochloric acid).

About 5mL of the collected protein eluate was passed through a chromatographic column by an AKTA system, the protein with the peak height (peak 1) in the gel filtration elution curve was collected (FIG. 3A), then concentrated using a 10kDa protein ultrafiltration tube, and split-packed into 1.5mL sterile EP tubes, 50. Mu.L each, and stored in a refrigerator at-80℃after split-packing for use.

10. Mu.L of NP mutant protein purified by molecular sieve chromatography was taken and put into a PCR tube containing 15. Mu.L of PBS and 5. Mu.L of 5X SD S sample buffer, and after mixing, the mixture was boiled at 100℃for 10min, and SDS-PAGE was performed, after 80V voltage was applied to the concentrated gel, 120V voltage was applied to the separated gel. After electrophoresis, the first block of albumin glue was removed from the glass plate and stained with coomassie brilliant blue for 2 hours, followed by decolorization. The other piece of albumin glue is used for Western blot verification, after a film is transferred at a constant voltage of 65V for 50min, a PVDF film is washed by TBST buffer for 3 times and 5min each time, then 10% skimmed milk powder is used for sealing for 2h at room temperature, and TBST is washed for 3 times and 5min each time; slowly shaking with primary antibody of mouse anti-his-tag (company: abclon, cat# AE 003) at room temperature for 2 hr, washing with 1 XTBE for 5min each time after the primary antibody incubation is completed, and washing 5 times; incubation with HRP-labeled goat anti-mouse IgG (company: abbkine, cat# A21010) for 45min at room temperature, washing with 1 XTBE 5 times for 5min each; ECL luminescence color developing solution (company: biosharp, cat# bl520 a) was mixed in equal volumes with solution A and solution B, the membrane was taken out, the liquid on the membrane was sucked off with filter paper, the mixture was uniformly immersed, and the purified NP mutant protein was imaged by exposure to a chemiluminescent instrument, and the band position was expected (B-C in FIG. 3).

Example 2: identification of NP mutant proteins not binding to bacterial RNA

To confirm that the purified NP mutant proteins did not bind to bacterial RNA, we tested by gel retardation analysis experiments. Taking out the prepared 6.5% non-modified polyacrylamide gel, adding a proper amount of 0.5 XTBE electrophoresis buffer into an electrophoresis tank, washing a sample adding hole, and pre-electrophoresis at 80V voltage for 30-60 min; 10. Mu.L of purified protein, 5. Mu.L of DEPC water and 2. Mu.L of 10 XDNA loading buffer were added to the PCR tube, gently swirled and mixed, and reacted at 26℃for 30 minutes. After the pre-electrophoresis, the buffer solution for the pre-electrophoresis was poured out, a new 0.5 XTBE was added, and the wells were rinsed and spotted. The electrophoresis tank was then placed on ice and electrophoresed at 100V for 1-2h. After electrophoresis, the gel block was put in 40mL of EB staining solution (10. Mu.L of EB solution was dissolved in 40mL of pure water), and shaken on a decolorizing shaker for 30min. As a result, as shown in FIG. 4, no bacterial RNA was detected in the NPmut wells, and we also set 2 control wells, in which the wild-type NP protein wells contained a large amount of bacterial RNA, while the wild-type NP protein (NP-Tx) wells treated with the RNase reagent still contained a small amount of RNA material. Therefore, the NP mutant protein is used as a candidate antigen of the universal influenza vaccine, and the potential safety hazard caused by the combination of the wild NP protein and bacterial RNA can be effectively solved.

Example 3: evaluation of immunoprotection effects of NP mutant proteins

1.1 animal immunization experiments

To assess the immunogenicity of the NP mutant (NPmut) proteins, we randomly divided 77 female BALB/c mice (purchased from the university of agriculture laboratory animal center in china) 6-8 weeks old into 7 groups of 11 mice each immunized with 15 μg protein, 50 μl volume per mouse, and the mice were immunized by nasal drip on days 0, 14, 28. We also set up wild-type NP protein, NP protein treated with rnase containing reagent (NP-Tx) as control group, detailed experimental groupings and immunizing doses as follows:

No.	experimental grouping	Immunization dose
			1	PBS+CpG1826	3μg
2	NP	15μg
			3	NPmut	15μg
4	NPmut+CpG1826	15μg+3μg
			5	NP	15μg
6	NP-Tx	15μg
			7	NP-Tx+CpG1826	15μg+3μg

1.2 Indirect ELISA detection of antigen-specific humoral immune response

14 days after the third immunization, the mice were bled from their tail veins and serum was isolated and tested for antigen-specific IgG antibody titers using an indirect ELISA. Diluting the purified NP protein to 2 mug/mL with coating buffer, adding 100 mug/hole to ELISA plate, coating at 4 deg.C overnight; the next day was washed 5 times with PBST, 200. Mu.L of 3% BSA blocking solution was added to each well, and incubated at 37℃for 1h; after blocking was completed, the serum was diluted 2-fold to 12 th well starting with PBST washed 5 times, at a ratio of 1:200, and incubated at 37℃for 1h. After the incubation, the ELISA plate was washed 5 times with PBST, 100. Mu.L of HRP-labeled goat anti-mouse IgG diluent (1:8000 dilution) was added to each well and incubated at 37℃for 45min. The secondary anti-dilution solution is discarded, the ELISA plate is washed for 5 times by PBST, 100 mu L of TMB color development solution is added into each hole, after the color development is carried out at room temperature and in dark place until the color is not changed, 100 mu L of 2M sulfuric acid stop solution is added into each hole to stop the color development, and the OD value at the wavelength of 450nm is measured by an ELISA. The results are shown in figure 5 a, with the mouse antibody titers of the immunized npmut+cpg1826 group and the NP protein group being significantly higher than the mouse antibody titers of the NPmut and PBS group and the differences being insignificant compared to the NP group. In addition, NP-Tx induced NP-specific antibody titers were also significantly higher in the NP-Tx group in combination with CpG1826 adjuvant. PBS control group failed to induce NP-specific antibodies. Demonstrating that the NP mutant protein and CpG1826 molecular adjuvant-mixed group, the RNase A-treated NP protein (NP-Tx) and CpG1826 molecular adjuvant-mixed group and NP protein group all induced similar and high-level humoral immune response.

For analysis of IgG subtypes, the antibody titers of IgG1 and IgG2a in serum were detected by indirect ELISA using NP protein as coating antigen. The secondary antibody was changed to HRP-labeled goat anti-mouse IgG1 and IgG2a (1:3000 dilution), and the rest was the same. The results indicate that the npmut+cpg1826 group, NP-tx+cpg1826 and NP proteomes induced significantly higher levels of NP-specific IgG1 and IgG2a antibodies than the NPmut, PBS and NP-Tx groups, resulting in a balanced Th1/Th2 type immune response (B, C in fig. 5).

1.3 Indirect ELISA detection of antigen-specific mucosal immune response

IgG, igA antibodies produced by the local mucosa proved to play an important role in preventing influenza infection, to assess whether NPm ut protein induced mucosal antibodies, we euthanized mice 10 days after the third immunization, lavaged the lungs of mice 3 times with ice-cold 1mL PBS, and centrifuged at 2500 g at 4 ℃ for 10min, the supernatants were collected and antigen-specific IgG and IgA mucosal antibody titers of bronchoalveolar perfusate (BALF) were detected by indirect ELIS a. The results are shown in figures 6 a and B, where NP, NP-Tx and CpG1826 mixed groups and NPmut and CpG mixed groups all induced similar and high levels of IgG and IgA antibodies compared to NPmut groups; no significant NP-specific IgG and IgA antibodies were detected in the mucosa of PBS immunized mice. In conclusion, the NP mutant protein was combined with CpG1826 molecular adjuvant to induce high levels of local mucosal antibodies.

1.4 toxicity challenge test to evaluate the protective Effect of NP mutant proteins on mice

At 14 days post 3 rd immunization, 5 mice per group were anesthetized and infected 5LD via nasal drops ₅₀ Homology A/human/Puerto Rico/8/34 (H1N 1) influenza virus. Mice in each group were observed daily and recorded for 14 days of death, body weight (weight loss greater than 25% was considered dead and euthanized immediately). The results showed that all mice in the group began to recover gradually after the rate of change of weight average at balance 7 was minimized after challenge, and that mice in the PBS group, NP-Tx group and NPmut group all died on day 9 after virus infection, whereas 40% of mice in the NPmut mixed with CpG1826, NP-Tx mixed with CpG1826 and NP group survived (FIGS. 7A and B). Thus, npmut+cpg1826 mixed mice were immunized, providing the same immunoprotection efficiency as the NP proteome, and subsequently could be a candidate target for influenza universal vaccines.

1.5 evaluation of the protective Effect of NP-mutant proteins on mice by pulmonary histopathological morphology

At 14 days after the 3 rd immunization, 3 mice were anesthetized and infected with 5LD via nasal drops ₅₀ A/human/Puerto Rico/8/34 (H1N 1) influenza virus. On day 6 after challenge, mouse lung tissue was taken and HE stained, and the results are shown in fig. 8. The lung of immunized PBS, NP-Tx and NPmut mice was significantly diseased compared to healthy mice lung tissue, and was seen to be extensively substantive, showing significant thickening of the alveolar wall, a large number of inflammatory cell infiltrates around the bronchi and pulmonary vessels, whereas the mouse lung tissue immunized with NP protein, NP-Tx and CpG1826 adjuvant mixture, and NPmut and CpG1826 adjuvant mixture showed similar pathological changes, with a lighter extent of disease, clear alveolar structure, mild to moderate thickening of the alveolar wall, with a small number of inflammatory cell infiltrates.

Claims

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

2. A gene encoding the mutant of claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 2.

3. An mRNA encoding the mutant of claim 1, wherein the nucleotide sequence of the mRNA is set forth in SEQ ID No. 3.

4. An expression vector comprising the gene of claim 2.

5. A genetically engineered bacterium comprising the expression vector of claim 4.

6. Use of the mutant of claim 1 or the gene of claim 2 or the mRNA of claim 3 or the expression vector of claim 4 or the genetically engineered bacterium of claim 5 in the preparation of an influenza virus vaccine.

7. An influenza virus recombinant protein vaccine comprising the mutant of claim 1.

8. The vaccine of claim 7, further comprising an adjuvant CpG1826, wherein the CpG1826 has a nucleotide sequence set forth in SEQ ID No. 12.

9. An influenza DNA vaccine comprising the gene of claim 2.

10. An influenza virus mRNA vaccine comprising the mRNA of claim 3.