Construction and identification method of influenza B DNA vaccine

Abstract

The invention discloses a construction and identification method of an influenza B DNA vaccine, which comprises the following steps: step 1, downloading sequence information of an influenza B recommended strain B / Austria / 1359417 / 2021 in vaccine strain recommended components in 2022-2023, carrying out codon optimization on an HA gene of the strain, and then carrying out gene synthesis, and naming the gene as BHA; 2, highly conservative sequences of HA protein, M2e protein and NA protein are selected for series combination, gene synthesis is performed after codon optimization, and the gene is named as G3; 3, respectively extracting plasmid DNAs of the synthetic fragments for later use; 4, amplifying a target fragment by using a PCR technology so as to connect a carrier; and 5, carrying out a target gene and vector connection experiment. According to the present invention, the HA protein sequence of the DNA vaccine can be rapidly replaced by using the restriction enzyme cutting site, the preparation method is simple, the immunogenicity is good, the cross protection is good, the preparation cost is low, the preparation requirement of the large-scale influenza vaccine can be met, and the important scientific significance and the important social value are provided.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a method for constructing and identifying a type B influenza DNA vaccine. Background Art

[0002] Influenza is a serious acute respiratory infectious disease caused by influenza virus infection. Globally, influenza virus infection can cause 3-5 million severe cases and 290,000-650,000 deaths each year. According to the antigenicity of influenza virus nucleoprotein (NP) and matrix protein (M), it can be divided into four types: A, B, C, and D. Among them, influenza B virus is divided into two lineages, Victoria and Yamagata, which mainly infect humans and a small number of mammals, such as seals and pigs. Influenza B virus often co-circulates with influenza A virus worldwide, accounting for a high proportion of local outbreaks or seasonal epidemics of influenza, posing a serious threat to human public health.

[0003] The most effective way to prevent and control influenza B is vaccination. Most of the current commercial inactivated influenza vaccines are trivalent or quadrivalent vaccines prepared based on candidate strains predicted by the World Health Organization (WHO). Whether the prediction is successful will directly affect the protection rate of the vaccine of the season. It takes a long time from the determination of candidate strains to the commercialization of vaccines. Failure to predict and the inability to quickly prepare effective influenza vaccines will lead to a flu pandemic in the season.

[0004] Therefore, developing an efficient universal influenza vaccine with broad-spectrum activity is of great significance for preventing influenza epidemics. Among them, DNA vaccines have certain advantages. DNA vaccines clone one or more antigen encoding genes on a eukaryotic expression vector, and then directly immunize the constructed recombinant plasmid into the body, thereby activating the body's immune system. Compared with traditional vaccines, it has the advantages of low cost, high safety, convenient storage and transportation, can express antigens for a long time, is not interfered by maternal antibodies, has the same immunogenicity as natural antigens, and can stimulate the body to produce humoral immunity, cellular immunity and mucosal immunity at the same time. Therefore, based on the problems existing in the influenza B vaccine in the prior art, it is very necessary to design a new universal influenza B vaccine. Summary of the invention

[0005] In view of this, the present invention provides a method for constructing and identifying an influenza B DNA vaccine.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for constructing and identifying an influenza B DNA vaccine comprises the following steps:

[0008] Step 1: Download the sequence information of the influenza B recommended strain B / Austria / 1359417 / 2021 in the recommended vaccine strain components for 2022-2023, perform gene synthesis after codon optimization of the HA gene of the strain, and name the gene BHA;

[0009] Step 2: Select highly conserved sequences of HA protein, M2e protein and NA protein for tandem combination, perform gene synthesis after codon optimization, and name the gene G3;

[0010] Step 3: extract the plasmid DNA of the synthetic fragments separately for later use;

[0011] Step 4: Use PCR technology to amplify the target fragment to facilitate the connection to the vector;

[0012] Step 5: Conduct a target gene and vector connection experiment.

[0013] Preferably, in step 4, the amplification primers are as shown in Table 1:

[0014] Table 1 Primer sequences

[0015]

[0016] Preferably, in step 4, the PCR system is as shown in Table 2:

[0017] Table 2 PCR system

[0018]

[0019] Preferably, in step 4, the PCR reaction procedure is as shown in Table 3:

[0020] Table 3 PCR reaction program

[0021]

[0022] Preferably, in step 4, the amplified product is subjected to nucleic acid electrophoresis using 1% agarose gel.

[0023] Preferably, in step 5, the specific method steps for conducting the target gene and vector connection experiment are:

[0024] Step 5.1: After single digestion of the pVAX1-IRES vector with restriction endonuclease NdeⅠ, HA was ligated to the linearized pVAX1-IRES vector using a seamless cloning kit;

[0025] Step 5.2: After single digestion of the pVAX1-BHA-IRES vector with restriction endonuclease XhoⅠ, G3 was ligated to the linearized pVAX1-BHA-IRES using a seamless cloning kit;

[0026] Step 5.3: Transform the ligation product into competent cells Trans-T1, expand the culture and extract the plasmid DNA, select restriction endonucleases NdeⅠ and XhoⅠ for double digestion identification, and use 1% agarose gel for nucleic acid electrophoresis;

[0027] Step 5.4: The plasmid DNA sequence was confirmed to be correct by sequencing, and the recombinant plasmid pVAX-BHAG3 was successfully constructed.

[0028] Preferably, in step 5.1, the connection system is as shown in Table 4:

[0029] Table 4 Connection system

[0030]

[0031] Preferably, in step 5.2, the connection system is as shown in Table 5:

[0032] Table 5 Connection system

[0033]

[0034]

[0035] Compared with the prior art, the present invention has achieved the following technical effects:

[0036] The HA protein sequence of the DNA vaccine of the present invention can be quickly replaced using enzyme cleavage sites, and the preparation method is simple, the immunogenicity is good, the cross protection is good, the preparation cost is low, and it can meet the preparation needs of large quantities of influenza vaccines, and has important scientific significance and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a design structure diagram of the target gene pVAX1-BHAG3 of the method for constructing and identifying a type B influenza DNA vaccine of the present invention;

[0038] Figure 2 This is a PCR product electrophoresis result diagram of the construction and identification method of influenza B DNA vaccine of the present invention; wherein, M: D2000 marker; 1: BHA 1750bp; 2: Negative; 3: G3 1050bp; 4: Negative;

[0039] Figure 3 The figure is a result of electrophoresis of enzyme digestion products of the method for constructing and identifying a type B influenza DNA vaccine of the present invention; wherein, M1: DL15000 marker; M2: DL5000 marker; 1: pVBHAG3; 2: pVBHAG3 double digestion product;

[0040] Figure 4 This is a WB identification diagram of the construction and identification method of an influenza B DNA vaccine of the present invention;

[0041] Figure 5 This is an IFA identification diagram of a method for constructing and identifying an influenza B DNA vaccine according to the present invention;

[0042] Figure 6 The present invention is an immunization program diagram for the construction and identification method of influenza B DNA vaccine;

[0043] Figure 7 This is a diagram showing the specific antibody detection results of a method for constructing and identifying a type B influenza DNA vaccine according to the present invention;

[0044] Figure 8 This is a diagram of the hemagglutination inhibition antibody detection results of the method for constructing and identifying a type B influenza DNA vaccine of the present invention. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The present invention discloses a method for constructing and identifying an influenza B DNA vaccine, comprising the following steps:

[0047] Step 1: Download the sequence information of the influenza B recommended strain B / Austria / 1359417 / 2021 in the recommended vaccine strain components for 2022-2023, perform gene synthesis after codon optimization of the HA gene of the strain, and name the gene BHA;

[0048] Step 2: Select highly conserved sequences of HA protein, M2e protein and NA protein for tandem combination, perform gene synthesis after codon optimization, and name the gene G3;

[0049] Step 3: extract the plasmid DNA of the synthetic fragments separately for later use;

[0050] Step 4: Use PCR technology to amplify the target fragment to facilitate the connection to the vector;

[0051] Amplification primers are shown in Table 1:

[0052] Table 1 Primer sequences

[0053]

[0054] The PCR system is shown in Table 2:

[0055] Table 2 PCR system

[0056]

[0057]

[0058] The PCR reaction program is shown in Table 3:

[0059] Table 3 PCR reaction program

[0060]

[0061] The amplified products were subjected to nucleic acid electrophoresis using 1% agarose gel. Figure 2 As shown, the PCR product was consistent with the expected size;

[0062] The PCR products were recovered for subsequent experiments;

[0063] Step 5: Conduct the target gene and vector connection experiment;

[0064] Step 5.1: After single digestion of the pVAX1-IRES vector with restriction endonuclease NdeⅠ, HA was ligated to the linearized pVAX1-IRES vector using a seamless cloning kit;

[0065] The connection system is shown in Table 4:

[0066] Table 4 Connection system

[0067]

[0068] Step 5.2: After single digestion of the pVAX1-BHA-IRES vector with restriction endonuclease XhoⅠ, G3 was ligated to the linearized pVAX1-BHA-IRES using a seamless cloning kit;

[0069] The connection system is shown in Table 5:

[0070] Table 5 Connection system

[0071]

[0072]

[0073] Step 5.3: Transform the ligation product into competent cells Trans-T1, expand the culture and extract the plasmid DNA. Select restriction endonucleases NdeⅠ and XhoⅠ for double digestion identification. Use 1% agarose gel for nucleic acid electrophoresis. The results are as follows: Figure 3 As shown, the ligation product is consistent with the expected size;

[0074] Step 5.4: The plasmid DNA sequence was confirmed to be correct by sequencing, and the recombinant plasmid pVAX-BHAG3 was successfully constructed. The plasmid map is shown in Figure 1 shown.

[0075] Example 1: Plasmid DNA transfection HEK293T cell experiment

[0076] (1) HEK293T cells were digested with trypsin to prepare a cell suspension and counted. 1×10 cells were plated in each well of a 6-well plate. 6 2 mL of DMEM medium containing 1% penicillin-streptomycin and 10% FBS was added to each well, and the cells were cultured at 37°C and 5% CO2. Transfection was performed when the cell density reached 70-80%.

[0077] (2) Gently pipette and mix 6 μg of recombinant plasmid and pVAX1 empty vector in 6 μL INVIDNA / RNA Transfection Reagent, and let stand at room temperature for 15 min.

[0078] (3) Replace the DMEM medium containing 2% FBS in the 6-well plate, drip the plasmid-transfection reagent mixture into the medium 3-4 times, mix thoroughly and culture at 37°C, 5% CO2 for 24 hours.

[0079] Example 2: Western Blot Identification

[0080] like Figure 4 As shown, HEK293T cell samples transfected with the recombinant plasmid pVAX1-BHAG3 reacted with Influenza Bvirus Hemagglutinin (HA) antibody, and a specific band appeared at about 70 kDa, while no band appeared in the cell samples that were not transfected (MOCK) or transfected with the pVAX1 empty vector, indicating that the recombinant plasmid pVAX1-BHAG3 was successfully expressed in HEK293T cells.

[0081] Example 3: IFA Identification

[0082] like Figure 5 As shown, green fluorescence can be observed in HEK293T cells transfected with pVAX1-BHAG3 under a fluorescence microscope, while no fluorescence was observed in cells transfected with the pVAX1 empty vector under a microscope, indicating that the recombinant plasmid pVAX1-BHAG3 was successfully expressed in HEK293T cells.

[0083] Example 4: Immunization Grouping

[0084] like Figure 6As shown, the mice were divided into an immunization group and a control group. The immunization group was immunized with pVAX1-BHAG3, the control group was immunized with pVAX1, and the blank group was a Mock group. Each group had 15 mice, and all were injected intramuscularly at the tibialis anterior muscle of the hind leg of the mice. The immunization dose was 100 μg, and the immunization was three times in total, with an interval of three weeks. The mouse serum was collected before immunization, 14 days after the first immunization, 7 days after the second immunization, and 7 days after the third immunization. After being placed at room temperature for 2 hours, the upper serum was aspirated and stored for later use after centrifugation at 3000 rpm for 10 minutes.

[0085] Example 5: Specific antibody detection

[0086] The influenza B Victoria lineage HA protein was used as an antigen to coat the ELISA plate overnight, and the collected mouse sera were tested for specific antibodies using an ELISA reader. Figure 7 As shown, the level of specific antibodies in the immunization group increased with the increase in the number of immunizations.

[0087] Example 6: Hemagglutination inhibition antibody detection

[0088] Four units of antigen were prepared using influenza B Victoria lineage virus, and the collected mouse serum was tested for hemagglutination inhibition antibodies. The results were as follows: Figure 8 As shown, the immunized group can produce hemagglutination inhibition antibodies against the homologous virus, and the hemagglutination inhibition antibodies increase with the increase of immunization times, reaching a maximum of 1:160.

[0089] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for constructing and identifying an influenza B DNA vaccine, characterized in that: The following steps are involved: Step 1: Download the sequence information of the influenza B recommended strain B / Austria / 1359417 / 2021 in the recommended vaccine strain components for 2022-2023, perform gene synthesis after codon optimization of the HA gene of the strain, and name the gene BHA; Step 2: Select highly conserved sequences of HA protein, M2e protein and NA protein for tandem combination, perform gene synthesis after codon optimization, and name the gene G3; Step 3: extract the plasmid DNA of the synthetic fragments separately for later use; Step 4: Use PCR technology to amplify the target fragment to facilitate the connection to the vector; Step 5: Conduct a target gene and vector connection experiment.

2. The method for constructing and identifying an influenza B DNA vaccine according to claim 1, characterized in that: In step 4, the amplification primers are shown in Table 1: Table 1 Primer sequences 3. The method for constructing and identifying an influenza B DNA vaccine according to claim 1, characterized in that: In step 4, the PCR system is as shown in Table 2: Table 2 PCR system 4. The method for constructing and identifying an influenza B DNA vaccine according to claim 1, characterized in that: In step 4, the PCR reaction procedure is shown in Table 3: Table 3 PCR reaction program 5. The method for constructing and identifying an influenza B DNA vaccine according to claim 1, characterized in that: In step 4, the amplified product is subjected to nucleic acid electrophoresis using 1% agarose gel.

6. The method for constructing and identifying an influenza B DNA vaccine according to claim 1, characterized in that: In step 5, the specific method steps for conducting the target gene and vector connection experiment are: Step 5.1: After single digestion of the pVAX1-IRES vector with restriction endonuclease NdeⅠ, HA was ligated to the linearized pVAX1-IRES vector using a seamless cloning kit; Step 5.2: After single digestion of the pVAX1-BHA-IRES vector with restriction endonuclease XhoⅠ, G3 was ligated to the linearized pVAX1-BHA-IRES using a seamless cloning kit; Step 5.3: Transform the ligation product into competent cells Trans-T1, expand the culture and extract the plasmid DNA, select restriction endonucleases NdeⅠ and XhoⅠ for double digestion identification, and use 1% agarose gel for nucleic acid electrophoresis; Step 5.4: The plasmid DNA sequence was confirmed to be correct by sequencing, and the recombinant plasmid pVAX-BHAG3 was successfully constructed.

7. The method for constructing and identifying an influenza B DNA vaccine according to claim 6, characterized in that: In step 5.1, the connection system is shown in Table 4: Table 4 Connection system 8. The method for constructing and identifying an influenza B DNA vaccine according to claim 6, characterized in that: In step 5.2, the connection system is shown in Table 5: Table 5 Connection system

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