Japanese encephalitis recombinant adenovirus shuttle plasmid, adenovirus plasmid, virus and preparation method and application thereof

Abstract

The invention discloses a recombinant adenovirus shuttle plasmid and a preparation method thereof, a recombinant adenovirus and an application thereof, and a Japanese encephalitis recombinant adenovirus plasmid, belonging to the field of biomedicine. The Japanese encephalitis recombinant adenovirus shuttle plasmid provided by the invention uses pShuttle as a carrier, carries a Japanese encephalitis virus wild-type E protein gene or a Japanese encephalitis virus E protein mutant gene, the 5' end of the Japanese encephalitis virus wild-type E protein gene or the Japanese encephalitis virus E protein mutant gene is connected to a signal peptide gene, the 5' end of the signal peptide gene is connected to a Kozak sequence, and the 5' end of the Kozak sequence is connected to a restriction site 1. The recombinant shuttle plasmid, recombinant plasmid, and recombinant adenovirus provided by the invention can be applied to the preparation of a Japanese encephalitis virus adenovirus vaccine, and have broad development and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to a Japanese encephalitis recombinant adenovirus shuttle plasmid, an adenovirus plasmid, a virus, and a preparation method and application thereof; more particularly, to a Japanese encephalitis recombinant adenovirus shuttle plasmid, a preparation method thereof, a Japanese encephalitis recombinant adenovirus, and an application thereof, and a Japanese encephalitis recombinant adenovirus plasmid. Background Art

[0002] Japanese encephalitis is an acute infectious disease caused by Japanese encephalitis virus (JEV), and its main vector is Culex tritaeniorhynchus. It is an important zoonosis in Asia and the Pacific, threatening the health of the general public and economic development. At present, the prevention and treatment methods of Japanese encephalitis mainly include vaccination, drug treatment, etc., but there are still many problems in prevention and control. For example: the determination of JEV infection is mostly carried out through PCR and ELISA, and the detection steps are cumbersome; the antibodies induced by JEV vaccination can aggravate the infection of other flaviviruses such as Zika virus (ZIKV) through antibody-dependent enhancement (ADE) effect in vivo and in vitro. In summary, it is imperative to find a new method for the prevention and detection of Japanese encephalitis virus. Summary of the invention

[0003] The problem to be solved by the present invention is how to improve the safety of the prevention or detection method of Japanese encephalitis virus.

[0004] In order to solve the above problems, the present invention provides a recombinant adenovirus shuttle plasmid and a preparation method thereof, a recombinant adenovirus and an application thereof, and a Japanese encephalitis recombinant adenovirus plasmid, wherein the Japanese encephalitis recombinant adenovirus shuttle plasmid carries a Japanese encephalitis virus wild-type E protein gene or a Japanese encephalitis virus E protein mutant gene, the amino acid sequence encoded by the Japanese encephalitis virus wild-type E protein gene is shown in SEQ ID No.1, and the amino acid sequence encoded by the Japanese encephalitis virus E protein mutant gene is shown in SEQ ID No.3.

[0005] E protein is the main structural protein of Japanese encephalitis virus. It is a hydrophobic glycoprotein and is closely related to the pathogenicity of Japanese encephalitis virus. It is highly biologically conservative and is a commonly used target protein for Japanese encephalitis virus detection and treatment. Most vaccines developed using the E protein gene can protect animals from lethal doses of Japanese encephalitis virus attack. Therefore, the E protein gene is an excellent vaccine target. Adenovirus vectors are a type of commonly used genome modification technology that uses adenovirus as a vector. Adenovirus is a double-stranded DNA virus with high gene transfer efficiency, which can express or integrate exogenous target genes into host cells. Adenovirus vectors are usually constructed based on the wild-type adenovirus genome backbone, which is achieved by destroying the replication and packaging ability of the adenovirus and inserting the sequence of the exogenous gene to be expressed. The location of the insertion of the exogenous gene is usually the region where the E1 / E3 of the original adenovirus is removed. Adenovirus vectors have the following characteristics: 1. They can infect various types of mammalian cells, including non-dividing cells, slow-proliferating cells, and normal cells; they can stably exist in human cells; 2. They can stably express foreign genes without affecting host gene expression and function; 3. They have lower immunogenicity and cytotoxicity than other gene transfer vectors; 4. They are easy to prepare on a large scale and extract with high purity. Therefore, adenovirus vectors have broad application prospects in gene therapy, gene vaccines, and gene research.

[0006] Preferably, the nucleotide sequence of the wild-type E protein gene of the Japanese encephalitis virus is shown as SEQ ID No.2, and the nucleotide sequence of the mutant E protein gene of the Japanese encephalitis virus is shown as SEQ ID No.4.

[0007] Preferably, the 5' ends of the Japanese encephalitis virus wild-type E protein gene and the Japanese encephalitis virus E protein mutant gene are connected to a signal peptide gene, the 5' end of the signal peptide gene is connected to a Kozak sequence, the 5' end of the Kozak sequence is connected to an enzyme cleavage site one, the protein sequence encoded by the signal peptide gene is shown in SEQ ID No.5, the nucleotide sequence of the signal peptide gene is shown in SEQ ID No.6, the nucleotide sequence of the Kozak sequence is shown in SEQ ID No.7, and the specific nucleotide sequence is: gccacc, the nucleotide sequence of the enzyme cleavage site one is shown in SEQ ID No.8, and the specific nucleotide sequence is: gaattc.

[0008] Preferably, the 3' ends of the Japanese encephalitis virus wild-type E protein gene and the Japanese encephalitis virus E protein mutant gene are connected with a second restriction site, and the nucleotide sequence of the second restriction site is shown in SEQ ID No.9, and the specific nucleotide sequence is: ctcgag.

[0009] Preferably, the vector of the Japanese encephalitis recombinant adenovirus shuttle plasmid is pShuttle.

[0010] Furthermore, the second aspect of the present invention provides a method for preparing the aforementioned Japanese encephalitis recombinant adenovirus shuttle plasmid, comprising the following steps:

[0011] S1: Double digestion of pShuttle shuttle plasmid, Japanese encephalitis virus wild-type E protein gene or Japanese encephalitis virus E protein mutant gene with restriction endonucleases EcoRI and Xhol;

[0012] S2: subjecting the digestion product of step S1 to 0.8% agarose gel electrophoresis, cutting out the bands of the target gene and pShuttle, respectively, and recovering pShuttle and the target gene Japanese encephalitis virus wild-type E protein gene or Japanese encephalitis virus E protein mutant gene using an agarose gel purification recovery kit;

[0013] S3: Use T4 DNA ligase to connect pShuttle to the Japanese encephalitis virus wild-type E protein gene or the Japanese encephalitis virus E protein mutant gene;

[0014] S4: Add the reaction system after the connection in step S3 to the Stbl3 competent cells, flick to mix, place on ice for 30 minutes, place in a 42°C water bath for 90 seconds, quickly transfer to ice and place on ice for 2 minutes, add LB culture medium without antibiotics to the cells, and shake and culture at 180 rpm / 37°C for 1 hour;

[0015] S5: Centrifuge and remove the supernatant, mix the remaining bacterial solution and inoculate it on LB agar medium containing 50 μg / ml kanamycin, place it upright at 37°C until the liquid is absorbed, and then invert it and culture it at 37°C overnight;

[0016] S6: Select white bacterial clones and add them to 10 ml of LB culture medium (containing 50 μg / ml kanamycin), and culture with shaking at 220 rpm / 37°C for 18 h;

[0017] S7: Extract the recombinant plasmid from the bacterial culture using a plasmid miniprep kit and determine the plasmid concentration.

[0018] Furthermore, the third aspect of the present invention provides a Japanese encephalitis recombinant adenovirus plasmid, which carries an exogenous gene, a Japanese encephalitis virus wild-type E protein gene or a Japanese encephalitis virus E protein mutant gene, and is obtained by transforming the Japanese encephalitis recombinant adenovirus shuttle plasmid described in any one of claims 1 to 5 and the human adenovirus type 5 genome backbone plasmid pAdEasy-1 into Escherichia coli BJ5183 for homologous recombination and then screening.

[0019] Furthermore, it is obtained by transfecting the aforementioned Japanese encephalitis recombinant adenovirus plasmid into host cells.

[0020] Furthermore, the fifth aspect of the present invention provides a use of the aforementioned Japanese encephalitis recombinant adenovirus in the preparation of a Japanese encephalitis recombinant adenovirus vaccine.

[0021] Adenovirus vector vaccine is a new type of vaccine with replication-defective adenovirus as vector. It has the advantages of high transduction efficiency and high safety. However, adenovirus vector vaccine also has the defect of few target sites. At present, there is no adenovirus vector vaccine with Japanese encephalitis virus E protein as target site. The present invention is the first to use Japanese encephalitis E protein as target site, Japanese encephalitis recombinant adenovirus as raw material, and AdEasy as recombination system to prepare Japanese encephalitis recombinant adenovirus vaccine.

[0022] Preferably, the use comprises the following steps: introducing the Japanese encephalitis recombinant adenovirus Ad5-JEV-E WT and / or Ad5-JEV-E mut After being inoculated into host cells, the cells are serially passaged, and when the transcription and expression of the recombinant adenovirus E protein in the host cells are stabilized, a Japanese encephalitis recombinant adenovirus vaccine is prepared.

[0023] The beneficial effects of the present invention are as follows: The present invention overcomes the deficiencies of the prior art and provides a Japanese encephalitis virus recombinant adenovirus, a shuttle vector, a plasmid and its application in vaccine preparation. The plasmid carrying the target gene is double-digested using restriction endonucleases EcoRI and Xhol to obtain a linearized plasmid. The digested product is subjected to 0.8% agarose gel electrophoresis and the band containing the target gene and pShuttle is cut off for recovery. T4 DNA ligase is used to connect pShuttle to the target gene to obtain a recombinant shuttle plasmid. The recombinant shuttle plasmid and the adenovirus vector system backbone vector pAdEasy-1 are co-transformed into the Escherichia coli BJ5183 strain to obtain a recombinant adenovirus plasmid. The recombinant adenovirus plasmid is transfected into 293T cells to obtain a recombinant adenovirus. SDS-PAGE and Western blot were used to prove the JEV-E protein in the recombinant adenovirus Ad5-JEV-E WT 、Ad5-JEV-E mut Stable expression in the body; after immunizing mice with the recombinant adenovirus, the target gene can be effectively transferred into the body and expressed, and can effectively stimulate the body to produce an immune response. WT , Ad5-JEV-E mutThere was no significant difference in the titer of antibodies induced by immunized mice to neutralize JEV pseudovirus, but the ability to cross-recognize Zika virus (ZIKV)-E protein was significantly reduced, and the antibody-dependent enhancement (ADE) effect of ZIKV infection in vitro was also significantly reduced. Therefore, the recombinant adenovirus vaccine of the present invention is safe and effective, can stimulate the body to produce JEV neutralizing antibodies, and its ADE effect on ZIKV infection is significantly reduced, thus having broad development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is a graph of the experimental results of Western Blot in Example 5 in a specific embodiment of the present invention;

[0025] Figure 2 This is a diagram of the ELISA test results in Example 6 in a specific embodiment of the present invention;

[0026] Figure 3 It is a graph showing the experimental results of the JEV pseudovirus neutralization experiment in Example 7 in a specific embodiment of the present invention;

[0027] Figure 4 This is a graph showing the experimental results of the in vitro ADE experiment in Example 8 in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0028] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0029] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0030] In a specific embodiment of the present invention, the wild-type E protein gene of Japanese encephalitis virus is fused with the JEV-E WT The Japanese encephalitis virus E protein mutant gene has the same meaning as JEV-E mut Have the same meaning; adenovirus and Ad have the same meaning.

[0031] A specific embodiment of the present invention provides a Japanese encephalitis recombinant adenovirus shuttle plasmid and a preparation method thereof. The Japanese encephalitis recombinant adenovirus shuttle plasmid provided by the present invention carries a Japanese encephalitis virus wild-type E protein gene (the encoded amino acid sequence is shown in SEQ ID No.1, and the nucleotide sequence is shown in SEQ ID No.2) or a Japanese encephalitis virus E protein mutant gene (the encoded amino acid sequence is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.4).

[0032] In the shuttle plasmid of Japanese encephalitis recombinant adenovirus provided in a specific embodiment of the present invention, the plasmid vector is pShuttle, the 5' end of the Japanese encephalitis virus wild-type E protein gene or the Japanese encephalitis virus E protein mutant gene is connected to the signal peptide gene, the 5' end of the signal peptide gene is connected to the Kozak sequence, the 5' end of the Kozak sequence is connected to the restriction site 1, the protein sequence encoded by the signal peptide gene is shown in SEQ ID No.5, the nucleotide sequence of the signal peptide gene is shown in SEQ ID No.6, the nucleotide sequence of the Kozak sequence is shown in SEQ ID No.7, and the nucleotide sequence of the restriction site 1 is shown in SEQ ID No.8. The 3' end of the Japanese encephalitis virus wild-type E protein gene or the Japanese encephalitis virus E protein mutant gene is connected to the restriction site 2, and the nucleotide sequence of the restriction site 2 is shown in SEQ ID No.9.

[0033] The method for preparing the aforementioned Japanese encephalitis recombinant adenovirus shuttle plasmid provided in a specific embodiment of the present invention comprises the following steps: S1: using restriction endonucleases EcoRI and Xhol to perform double restriction digestion on pShuttle shuttle plasmid, Japanese encephalitis virus wild-type E protein gene or Japanese encephalitis virus E protein mutant gene;

[0034] S2: subjecting the digestion product of step S1 to 0.8% agarose gel electrophoresis, cutting out the bands of the target gene and pShuttle, respectively, and recovering pShuttle and the target gene Japanese encephalitis virus wild-type E protein gene or Japanese encephalitis virus E protein mutant gene using an agarose gel purification recovery kit;

[0035] S3: Use T4 DNA ligase to connect pShuttle to the Japanese encephalitis virus wild-type E protein gene or the Japanese encephalitis virus E protein mutant gene;

[0036] S4: Add the reaction system after the connection in step S3 to the Stbl3 competent cells, flick to mix, place on ice for 30 minutes, place in a 42°C water bath for 90 seconds, quickly transfer to ice and place on ice for 2 minutes, add LB culture medium without antibiotics to the cells, and shake and culture at 180 rpm / 37°C for 1 hour;

[0037] S5: Centrifuge and remove the supernatant, mix the remaining bacterial solution and inoculate it on LB agar medium containing 50 μg / ml kanamycin, place it upright at 37°C until the liquid is absorbed, and then invert it and culture it at 37°C overnight;

[0038] S6: Select white bacterial clones and add them to 10 ml of LB culture medium (containing 50 μg / ml kanamycin), and culture with shaking at 220 rpm / 37°C for 18 h;

[0039] S7: Extract the recombinant plasmid from the bacterial culture using a plasmid miniprep kit and determine the plasmid concentration.

[0040] The specific embodiment of the present invention also provides a Japanese encephalitis recombinant adenovirus plasmid, which is prepared by the following steps:

[0041] The Japanese encephalitis recombinant adenovirus shuttle plasmid and the human adenovirus type 5 genome backbone plasmid pAdEasy-1 were transformed into Escherichia coli BJ5183 for homologous recombination, and the Japanese encephalitis recombinant adenovirus plasmid containing the exogenous gene Japanese encephalitis virus wild-type E protein gene or Japanese encephalitis virus E protein mutant gene was obtained after screening: Ad5-JEV-E WT or Ad5-JEV-E mut .

[0042] The specific embodiment of the present invention also provides a Japanese encephalitis recombinant adenovirus, and the preparation steps are as follows: the Japanese encephalitis recombinant adenovirus plasmid is transfected into a host cell to obtain a Japanese encephalitis recombinant adenovirus: Ad5-JEV-E WT and / or Ad5-JEV-E mut .

[0043] The specific embodiment of the present invention also provides a use of the aforementioned Japanese encephalitis recombinant adenovirus in the preparation of a Japanese encephalitis recombinant adenovirus vaccine.

[0044] Example 1

[0045] 1. Amplification and extraction of shuttle plasmid pShuttle

[0046] 1.1. Centrifuge a 0.5 μg PShuttle plasmid powder tube at 5000 rpm for 1 min, and add 20 μl ddH2O to dissolve the plasmid;

[0047] 1.2. Take 1 tube of 100 μl Stbl3 competent bacteria and thaw it on ice for 10 minutes to completely melt, add 2 μl (50 ng) plasmid, flick it gently to mix well, and then place it on ice for 30 minutes. Insert the float and heat shock it in a 42℃ water bath for 90 seconds, take it out and place it on ice for another 2 minutes;

[0048] 1.3. Add 900 μl of LB culture medium without antibiotics and culture at 180 rpm / 37°C for 45 min;

[0049] 1.4. Centrifuge at 6000 rpm for 5 min, discard 950 μl of supernatant, resuspend the bacterial pellet with the remaining liquid, and use an inoculation loop to streak the bacteria onto the surface of an LB agar plate containing kanamycin and streptomycin (both 50 μg / ml, applied 30 min in advance);

[0050] 1.5. Incubate the plate in the normal direction for 1 hour, then invert and incubate at 37°C for 14 hours;

[0051] 1.6. Pick a single colony and put it into 20 ml LB culture medium containing antibiotics (50 μg / ml kanamycin, 50 μg / ml streptomycin), culture with shaking at 220 rpm / 37℃ for 14 hours, and collect 10 ml of the bacteria to extract the plasmid.

[0052] 1.7. Take the above bacterial culture, centrifuge at 12000rpm for 1min, and absorb the supernatant as much as possible (if there is a lot of bacterial liquid, the bacterial precipitate can be collected into a centrifuge tube through multiple centrifugation);

[0053] 1.8. Add 250 μl of solution I (with RNase A added) to the centrifuge tube containing the bacterial pellet and use a pipette or vortex oscillator to thoroughly suspend the bacterial pellet;

[0054] 1.9. Add 250 μl of solution II to the centrifuge tube and gently invert it upside down 6-8 times to fully lyse the bacteria;

[0055] 1.10. Add 350 μl of solution III to the centrifuge tube, and immediately and gently turn it upside down 6-8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12000 rpm for 10 min. Use a pipette to carefully transfer the supernatant to another clean centrifuge tube, and try not to suck out the precipitate.

[0056] 1.11. Add the supernatant obtained in the previous step to the adsorption column (the adsorption column is added to the collection tube), place it at room temperature for 2 minutes, centrifuge it at 12000rpm for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0057] 1.12. Add 600 μl of rinse solution I (with anhydrous ethanol added) to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube;

[0058] 1.13. Add 700 μl of rinsing solution II (with anhydrous ethanol added) to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube;

[0059] 1.14. Add 500 μl of rinse solution II to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube;

[0060] 1.15.1 Centrifuge at 2000 rpm for 2 minutes, and place the adsorption column open at room temperature or in a 50°C incubator for several minutes to remove the rinse solution in the adsorption column;

[0061] 1.16. Place the adsorption column in a clean centrifuge tube, drop 50-200 μl of the eluent preheated in a 65°C water bath into the center of the adsorption membrane, let stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute;

[0062] 1.17. In order to increase the recovery efficiency of the plasmid, the obtained eluate can be added back into the adsorption column, placed at room temperature for 2 minutes, and centrifuged at 12000 rpm for 1 minute;

[0063] 1.18. Measure the concentration of the extracted plasmid using a spectrophotometer.

[0064] Example 2

[0065] 2. Construction of the recombinant shuttle plasmid pShuttle

[0066] 2.1. Determination of the concentration of plasmids containing target genes and shuttle plasmid pShuttle

[0067] 2.2. Double enzyme digestion was performed on the exogenous gene-containing plasmid and the shuttle plasmid pShuttle in two sterile 1.5 ml EP tubes, as shown in Table 1;

[0068] Table 1 EcoRI and Xhol double enzyme digestion reaction system

[0069]

[0070]

[0071] Mix well, place in a 37°C water bath for 5-15 minutes, and then heat at 65° for 20 minutes to inactivate

[0072] 2.3. Perform 1% agarose gel electrophoresis on 50 μl of the digested product, cut out the bands containing the exogenous gene and PShuttle, respectively, and place them in two 1.5 ml EP tubes. Weigh them. If the gel weighs 100 mg, it can be regarded as 100 μl (100 mg = 100 μl), and so on;

[0073] 2.4. Add 3 times the volume of GSB solution to the tube, melt the gel in a 55℃ water bath for 6-10 minutes, mix intermittently (2-3 minutes) to ensure that the gel block is completely melted. When the gel is completely melted, observe the color of the solution. If the color is purple, add an appropriate amount of 3M sodium acetate (pH5.2) to adjust the color to the same as GSB (yellow);

[0074] 2.5. After the melted gel solution has cooled to room temperature, add it to the centrifuge column and let it stand for 1 min. Centrifuge at 10,000 g for 1 min and discard the column liquid.

[0075] 2.6. Add 650 μl WB solution, centrifuge at 10,000 g for 1 min, and discard the column liquid;

[0076] 2.7. Centrifuge at 10000g for 1-2 minutes to completely remove residual WB;

[0077] 2.8. Place the centrifuge column in a clean centrifuge tube, open the cover and let it stand for 1 minute to allow the residual ethanol to evaporate. Add 30-50 μl of deionized water (pH>7.0) (preheat the deionized water in a 60-70℃ water bath) to the center of the column and let it stand at room temperature for 1 minute.

[0078] 2.9. Centrifuge at 10000g for 1 min to elute the DNA and store the eluted DNA at -20℃;

[0079] 2.10. Prepare reagents according to Table 2 and carry out ligation reaction;

[0080] Table 2 Shuttle plasmid ligation reaction system

[0081]

[0082]

[0083] The ligation reaction mixture was incubated at 16°C overnight.

[0084] 2.11. Take out the Stbl3 competent cells from the -80℃ refrigerator, quickly insert them into an ice box to dissolve them, add 10μl of the ligation product to the EP tube containing 50μl of competent cells, ice bath for 30min, 42° water bath for 90s, gently and quickly transfer to ice and ice bath for 2min. Do not shake the EP tube during this process, add 500μl of antibiotic-free LB culture medium to the centrifuge tube, shake and culture at 180rpm / 37℃ for 60min;

[0085] 2.12. Centrifuge the EP tube at 3000rpm for 5min, discard 450μl of the supernatant, mix the remaining 100μl of the bacterial solution and inoculate it onto LB agar medium containing 50μg / ml kanamycin and 50μg / ml streptomycin, spread it evenly, place it upright at 37℃ until the liquid is absorbed, and then invert it and culture it at 37℃ overnight;

[0086] 2.13. Pick up white colonies from the surface of LB agar medium and add them to 2 ml LB culture medium (containing 50 μg / ml kanamycin), and culture at 220 rpm / 37°C for 18 h;

[0087] 2.14. Plasmid DNA was extracted from the bacterial culture using a plasmid extraction kit. The recombinant shuttle plasmid was digested with restriction endonuclease PmeI, and 5 μl was taken for agarose gel electrophoresis to verify that the plasmid was completely cut to ensure that the linearized shuttle plasmid was obtained.

[0088] Example 3

[0089] 3. Construction of recombinant adenovirus plasmid

[0090] 3.1. Treatment of electric shock cup: Soak in 75% alcohol for 30 minutes, wash with sterile ddH2O 5-10 times, irradiate with ultraviolet light and ventilate in a clean bench for 30 minutes;

[0091] 3.2. Add 1-5 μl of linearized shuttle plasmid to a pre-cooled EP tube containing 50 μl of BJ5183 (padeasy-1) competent medium, mix well, and cool on ice;

[0092] 3.3. Quickly transfer the mixture to a pre-cooled electroporation cup, wipe the surface of the electroporation cup dry, and quickly place it in an electroporation tank for electric shock (1250-1500V / mm, 5ms);

[0093] 3.4. After the electroporation, quickly transfer the electroporation cup to ice, immediately add 1 ml of antibiotic-free LB culture medium, quickly and gently suspend the cells with a pipette and transfer them to an EP tube, shake and culture at 225 rpm / 30°C for 40 min;

[0094] 3.5. Take an appropriate volume of electroporated cell solution and apply it to agar culture plates containing 50 μg / ml kanamycin, place upright for 1 hour, and invert and culture at 37°C for 16-20 hours;

[0095] 3.6. The next day, pick the colonies grown on the plate (select the smallest white colonies), inoculate them into 3 ml of LB culture medium containing 50 μg / ml kanamycin, and culture at 37°C for 10-15 hours;

[0096] 3.7. Use a plasmid extraction kit to extract the plasmid, and perform 0.8% agarose gel electrophoresis. The large plasmid is a possible positive clone, which is further identified by PacI digestion;

[0097] 3.8. If a large fragment (about 30 kb) and a small fragment (about 3.0 kb or 4.5 kb) are shown by single enzyme digestion with PacI and 0.8% agarose gel electrophoresis, it is confirmed to be a recombinant adenovirus plasmid: Ad5-JEV-EWT and Ad5-JEV-E mut ;

[0098] 3.9. The recombinant adenovirus plasmid was transformed into DH5α or Stbl3 competent cells, the bacteria were amplified, and then the plasmid was extracted and identified by PacⅠ single restriction digestion and EcoRⅠ / XhoⅠ double restriction digestion, and finally a large amount of recombinant adenovirus plasmid was obtained.

[0099] Example 4

[0100] 4. Preparation of Recombinant Adenovirus

[0101] 4.1. PacI was used to encode the recombinant adenovirus plasmid Ad5-JEV-E WT and Ad5-JEV-E mut Perform single enzyme digestion to obtain linearized plasmid. The enzyme digestion reaction system is shown in Table 3. After the enzyme digestion system is prepared according to the components in Table 3, pipette and mix evenly, centrifuge instantaneously to collect the liquid on the tube wall to the bottom of the tube, and bathe at 37°C for 30 minutes. After the enzyme digestion is completed, take 5 μl of the enzyme digestion product for electrophoresis to identify whether the plasmid is cut, and the rest is frozen at -20°C for later use;

[0102] Table 3 PacI single restriction enzyme digestion reaction system

[0103]

[0104] 4.2. One day before transfection, cultured 293T cells were plated on a 10 cm culture dish. The cell growth density at the time of transfection was preferably between 70% and 80%;

[0105] 4.3. 4 h before transfection, discard the original culture medium in the culture dish with a pipette and replace it with incomplete MEM culture medium; add 30 μg of linearized Ad5-JEV-E WT or Ad5-JEV-E mut The plasmid was dissolved in physiological saline (total volume 0.5 mL), and then 60 μl of PEI transfection reagent was added and allowed to stand at room temperature for 20 min. The above transfection plasmid was gently added dropwise to 293T cells.

[0106] 4.4. 4 hours after transfection, discard the cell supernatant, add 8 mL of MEM complete culture medium, and culture in a 37°C / 5% CO2 incubator;

[0107] 4.5. When the cells show obvious pathological changes, collect the cells and supernatant, freeze and thaw repeatedly between -80℃ and 37℃ for 4 times, centrifuge at 4000rpm for 10min, collect the virus supernatant, and inoculate the supernatant into the 293T cells cultured the day before. Repeat the above 3 rounds, collect the pathological cells and supernatant, freeze and thaw repeatedly between -80℃ and 37℃ for 4 times, centrifuge at 4000rpm for 10min, collect the virus supernatant;

[0108] 4.6. Centrifuge the virus supernatant at 10,000 rpm for 30 min, keep the supernatant, ultracentrifuge at 50,000 rpm for 2 h, suspend the virus precipitate with PBS, and measure the absorbance of the sample at 260 nm and 280 nm using a UV spectrophotometer. PBS is used as a blank control. If the OD 260 The value is between 0.1 and 1, OD 260 / OD 280 Between 1.2-1.4, then, the number of recombinant adenovirus particles (VP) / mL = OD 260 ×10 12 OD 260 If the value exceeds 1, corresponding dilution is required. The number of recombinant adenovirus particles (VP) / mL = OD 260 × dilution factor × 10 12 .

[0109] Example 5

[0110] 5. Infection of cells with recombinant adenovirus

[0111] 5.1. One day before infection, cultured 293T cells were plated in a 24-well cell culture plate. The cell growth density at the time of infection was preferably between 70% and 80%;

[0112] 5.2. The resulting recombinant adenovirus Ad5-JEV-E WT or Ad5-JEV-E mut 293T cells (MOI = 1) were added and infected at 37°C for 2 h, the virus solution was discarded, MEM complete culture medium was supplemented, and cultured in a 37°C / 5% CO2 incubator for 2 days;

[0113] 5.3. Collect the cell supernatant into a 1.5 ml EP tube, centrifuge at 8000 rpm for 5 min, and transfer the supernatant into a new 1.5 ml EP tube for Western Blot experiment.

[0114] 6. Western Blot assay to detect target protein

[0115] 6.1. Prepare 15% SDS-PAGE gel;

[0116] 6.2. Take 48μl of the cell supernatant and add 12μl of 5× protein loading buffer, mix well and boil at 100℃ for 5min. Take 20μl and add it to the spotting well. Electrophoresis at 100V for about 40min. When the sample is electrophoresed between the upper and lower gel layers, increase the voltage to 120V and electrophoresis for another 120min.

[0117] 6.3. After the electrophoresis is completed, cut out the PVDF membrane according to the size of the gel, put the membrane into the methanol solution for activation for 1 minute, and then transfer it into the 1× electrotransfer buffer for equilibrium for 5 minutes. Make a sandwich in the order of sponge, filter paper, gel, PVDF membrane, filter paper, and sponge, and put the sandwich into the electrophoresis tank with the membrane facing the positive electrode and the gel facing the negative electrode. Add 1× electrotransfer buffer to cover the sponge, put the electrophoresis tank into the ice-water mixture, and electrotransfer at 100V for 2 hours;

[0118] 6.4. After electroporation, remove the PVDF membrane, wash it once with PBS, place it on a shaker, and block it with 5% skim milk powder at room temperature for 3.5 hours (70 rpm);

[0119] 6.5. After blocking, wash the membrane with 0.1% PBST (PBS containing 0.1% Tween), 5 min each time, for a total of 3 times. Add primary antibody diluent at a ratio of primary antibody (JEV-E protein immune mouse serum): 0.1% PBST = 1:2000, and incubate overnight at 4°C;

[0120] 6.6. Discard the primary antibody mixture and wash the membrane with 0.1% PBST for 5 minutes each time, for a total of 5 times. Add the secondary antibody diluent at a ratio of 1:5000 (HRP-labeled goat anti-mouse IgG monoclonal antibody): 0.1% PBST, and incubate at room temperature for 1 hour;

[0121] 6.7. Discard the secondary antibody mixture and wash the membrane with 0.1% PBST for 5 minutes each time, for a total of 5 times. Mix the ECL luminescent solution in the ratio of A solution: B solution = 1:1 and evenly drip it onto the PVDF membrane. After incubation for 1 minute, expose and take pictures for preservation;

[0122] 6.8. Figure 1 As shown, the recombinant adenovirus Ad5-JEV-E WT and Ad5-JEV-E mut After infecting 293T cells, the target proteins were expressed. Figure 1 Band 1 in the sample is Ad5-JEV-E WT Supernatant of infected 293T cells, band 2 is Ad5-JEV-E mut Supernatant of infected 293T cells, band M is Blue Plus IV Protein Marker (10KDa-190KDa).

[0123] Example 6

[0124] 7. Immunization of mice with recombinant adenovirus

[0125] 7.1. Fifteen 6-week-old male C57BL / 6 mice were divided into three groups (Ad5-JEV-E WTGroup, Ad5-JEV-E mut group and PBS group, 5 mice in each group);

[0126] 7.2. Each mouse was subcutaneously injected with 1×10 8 vp Ad5-JEV-E WT or Ad5-JEV-E mut Or the same volume of PBS as the control group; boost immunization once 14 days after immunization;

[0127] 7.3. Two weeks after the second immunization, mice were killed, and blood was collected from the heart to separate serum. The serum was bathed in a 56°C water bath for 30 minutes to inactivate complement and stored at -20°C for later use.

[0128] 8.ELISA to detect antibodies in mouse serum

[0129] 8.1. Coating antigen: Add 20 μg / ml JEV-E to each well of the high-affinity ELISA plate. 406 or ZIKV-E 410 (coating buffer dilution, 100 μl / well), incubate at 37 degrees for 1.5 hours. Discard the liquid in each well, add 200 μl PBS to each well, shake for 10 seconds and then discard the liquid in each well;

[0130] 8.2. Block the plate: add 150 μl of 5% skim milk powder (dissolved in PBS) to each well and incubate at 37 degrees for 1.5 hours. Discard the liquid in each well, add 200 μl of PBS to each well, shake for 10 seconds and then discard the liquid in each well;

[0131] 8.3. Add the serum to be tested: add 100 μl of three-fold diluted PBS group mouse serum or recombinant adenovirus immunized mouse serum to each well and incubate at 37 degrees for 1 hour. Discard the liquid in each well and wash the plate 4 times with 0.5% PBST (PBS containing 0.5% Tween) (add 200 μl 0.5% PBST to each well, shake for 10 seconds and then discard the liquid in each well);

[0132] 8.4. Add secondary antibody: add 100 μl HRP-labeled goat anti-mouse IgG secondary antibody (1:1000 dilution, PBS) to each well, incubate at 37 degrees for 1 hour, discard the liquid in each well, and wash the plate 4 times with 0.5% PBST (add 200 μl 0.5% PBST to each well, shake for 10 seconds and then discard the liquid in each well);

[0133] 8.5. Add colorimetric solution: add 40 μl TMB colorimetric solution (20 μl each of solution A and solution B) to each well, react at room temperature for 5 min, and add 20 μl stop solution to each well;

[0134] 8.6. Determine OD value: Use a microplate reader to measure the OD450 value of each well;

[0135] 8.7. The results are as follows Figure 2 As shown in A, compared with Ad5-JEV-E WT , Ad5-JEV-E mut The serum antibodies induced by immunization of mice recognized the Japanese encephalitis virus antigen (JEV-E 406 ) is slightly reduced; e.g. Figure 2 As shown in B, compared with Ad5-JEV-E WT , Ad5-JEV-E mut The serum antibodies induced by immunization of mice cross-recognized Zika virus antigens (ZIKV-E 410 )’s ability to function has been significantly reduced. Figure 2 A is coated with JEV-E 406 The results of ELISA test of protein, Figure 2 B is coated with ZIKV-E 410 ELISA results of proteins.

[0136] Example 7

[0137] 9.JEV pseudovirus neutralization experiment

[0138] 9.1 Vero cells were plated in 96-well light-proof cell culture plates (2.5×10 4 / well), cultured in a 37°C, 5% CO2 incubator for 24 h;

[0139] 9.2 The serum to be tested was diluted three-fold in a U-shaped 96-well cell culture plate (50 μl / well, PBS dilution), and serum-free wells (50 μl PBS / well) and background wells (50 μl PBS / well) were set up. Except for the background wells, 50 μl JEV pseudovirus (containing 500 pseudovirus particles, VSVΔG backbone and luciferase gene) was added to each well and incubated at 37 degrees for 1 hour;

[0140] 9.3 Discard the supernatant of each well of Vero cells, add the mixed solution to each well of the U-shaped bottom 96-well plate to the Vero cells, and incubate at 37 degrees for 1 hour. Discard the liquid in each well, wash each well once with PBS. Add 150μl RPMI-1640 complete culture medium (10% FBS) to each well, and culture in a 37 degree / 5% CO2 incubator for 24 hours;

[0141] 9.4 Discard the liquid in each well, add 50 μl of lysis buffer to each well and lyse for 2 min at room temperature, add 50 μl of luciferase substrate to each well, and immediately measure the fluorescence value using a multifunctional microplate reader;

[0142] 9.5 The calculation formula for the percentage of neutralization of JEV pseudovirus by each serum dilution is: [(fluorescence value of serum-free well-fluorescence value of serum well) / (fluorescence value of serum-free well-fluorescence value of background well)]×100%;

[0143] 9.6 The results are as follows Figure 3 As shown, compared with Ad5-JEV-E WT , Ad5-JEV-E mut There was no significant difference in the titer of serum antibodies induced by immunized mice against JEV pseudovirus.

[0144] Example 8

[0145] 10. In vitro ADE assay

[0146] 10.1 K562 cells were plated in a U-shaped 96-well cell culture plate (2×10 4 / well), cultured in a 37°C, 5% CO2 incubator for 24 h;

[0147] 10.2 In a common 96-well plate, the test serum was diluted three-fold (50 μl / well, PBS dilution), and serum-free wells (50 μl PBS / well) and background wells (50 μl PBS / well) were set up. Except for the background wells, 50 μl ZIKV live virus (containing 1000 virus particles) was added to each well and incubated at 37 degrees for 1 hour;

[0148] 10.3 Centrifuge the K562 cell culture plate at 2000rpm for 10min, discard the supernatant in each well, add the mixed liquid in each well of the ordinary 96-well plate to the K562 cells, incubate at 37 degrees for 1h, centrifuge the K562 cell culture plate at 2000rpm for 10min, discard the liquid in each well, wash each well once with PBS. Add 150μl RPMI-1640 complete culture medium to each well, and culture in a 37 degree / 5% CO2 incubator for 24h;

[0149] 10.4 Centrifuge the K562 cell culture plate at 2000 rpm for 10 minutes, discard the liquid in each well, add 100 μl of 4% paraformaldehyde liquid to each well, and fix at room temperature for 40 minutes;

[0150] 10.5 Centrifuge the K562 cell culture plate at 2000 rpm for 10 minutes, discard the liquid in each well, add 100 μl of 0.5% Triton (prepared in PBS) to each well, and permeabilize the membrane at room temperature for 40 minutes;

[0151] 10.6 Centrifuge the K562 cell culture plate at 2000 rpm for 10 min, discard the liquid in each well, and add 100 μl ZIKV-E to each well. 410 immune mouse serum (1:200 dilution, PBS), incubated at room temperature for 1 h;

[0152] 10.7 Centrifuge the K562 cell culture plate at 2000 rpm for 10 min, discard the liquid in each well, and wash 4 times with PBST;

[0153] 10.8 Add 100 μl FITC-labeled goat anti-mouse IgG flow cytometry monoclonal antibody (1:1000 dilution, PBS) to each well and incubate at room temperature for 1 hour;

[0154] 10.9 Centrifuge the K562 cell culture plate at 2000rpm for 10min, discard the liquid in each well, wash 4 times with 0.5% PBST (add 200μl 0.5% PBST to each well, shake for 10 seconds and discard the liquid in each well), add 400μl PBS to each well to suspend the cells.

[0155] 10.10 Measure the percentage of FITC-positive cells in each well using flow cytometry;

[0156] 10.11 The formula for calculating the enhancement factor of ZIKV virus infection at each serum dilution is: (percentage of FITC-positive cells in serum-well cells-percentage of FITC-positive cells in serum-free well cells) / (percentage of FITC-positive cells in serum-free well cells-percentage of FITC-positive cells in background well cells);

[0157] 10.12 The results are as follows Figure 4 As shown, compared with Ad5-JEV-E WT , Ad5-JEV-E mut The antibody-dependent enhancement (ADE) effect of serum antibodies induced by immunized mice on ZIKV infection was significantly reduced.

[0158] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0159] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A Japanese encephalitis recombinant adenovirus shuttle plasmid, characterized in that: The Japanese encephalitis recombinant adenovirus shuttle plasmid carries a Japanese encephalitis virus E protein mutant gene, and the amino acid sequence encoded by the Japanese encephalitis virus E protein mutant gene is shown in SEQ ID No.

3.

2. The Japanese encephalitis recombinant adenovirus shuttle plasmid according to claim 1, characterized in that: The nucleotide sequence of the Japanese encephalitis virus E protein mutant gene is shown in SEQ ID No.

4.

3. The Japanese encephalitis recombinant adenovirus shuttle plasmid according to claim 1 or 2, characterized in that: The 5' end of the Japanese encephalitis virus E protein mutant gene is connected to the signal peptide gene, the 5' end of the signal peptide gene is connected to a Kozak sequence, the 5' end of the Kozak sequence is connected to an enzyme cleavage site one, the protein sequence encoded by the signal peptide gene is shown in SEQ ID No.5, the nucleotide sequence of the signal peptide gene is shown in SEQ ID No.6, the specific nucleotide sequence of the Kozak sequence is: gccacc, and the specific nucleotide sequence of the enzyme cleavage site one is: gaattc.

4. The Japanese encephalitis recombinant adenovirus shuttle plasmid according to claim 1 or 2, characterized in that: The 3' end of the Japanese encephalitis virus E protein mutant gene is connected with a second restriction site, and the specific nucleotide sequence of the second restriction site is: ctcgag.

5. The Japanese encephalitis recombinant adenovirus shuttle plasmid according to claim 1 or 2, characterized in that: The vector of the Japanese encephalitis recombinant adenovirus shuttle plasmid is pShuttle.

6. A method for preparing a Japanese encephalitis recombinant adenovirus shuttle plasmid, characterized in that: The following steps are involved: S1: Double digestion of pShuttle shuttle plasmid and Japanese encephalitis virus E protein mutant gene shown in SEQ ID No.4 with restriction endonucleases EcoRI and Xhol; S2: The digested product of step S1 is subjected to 0.8% agarose gel electrophoresis, the bands of the target gene and pShuttle are cut out respectively, and pShuttle and the target gene Japanese encephalitis virus E protein mutant gene are recovered using an agarose gel purification recovery kit; S3: Use T4 DNA ligase to connect pShuttle to the Japanese encephalitis virus E protein mutant gene; S4: Add the reaction system connected in step S3 to the Stbl3 competent cells, flick to mix, place on ice for 30 min, place in a 42°C water bath for 90 s, quickly transfer to ice and place on ice for 2 min, add LB culture medium without antibiotics to the cells, and culture at 180 rpm / 37°C for 1 h; S5: Centrifuge and remove the supernatant, mix the remaining bacterial liquid and inoculate it on LB agar medium containing 50 μg / ml kanamycin, place it upright at 37℃ until the liquid is absorbed, and then invert it and culture it at 37℃ overnight; S6: Select white bacterial clones and add them to 10 ml LB culture medium containing 50 μg / ml kanamycin, and culture with shaking at 220 rpm / 37℃ for 18 h; S7: Extract the recombinant plasmid from the bacterial culture using a plasmid miniprep kit and determine the plasmid concentration.

7. A Japanese encephalitis recombinant adenovirus plasmid, characterized in that: The plasmid carries the foreign gene Japanese encephalitis virus E protein mutant gene, and is obtained by transforming the Japanese encephalitis recombinant adenovirus shuttle plasmid described in any one of claims 1 to 5 and the human adenovirus type 5 genome backbone plasmid pAdEasy-1 into Escherichia coli BJ5183 for homologous recombination and then screening.

8. A Japanese encephalitis recombinant adenovirus, characterized in that: The virus is obtained by transfecting the Japanese encephalitis recombinant adenovirus plasmid as claimed in claim 7 into host cells.

9. Use of the Japanese encephalitis recombinant adenovirus according to claim 8 in the preparation of a Japanese encephalitis recombinant adenovirus vaccine.

10. The use according to claim 9, characterized in that The method comprises the following steps: inoculating the Japanese encephalitis recombinant adenovirus described in claim 8 into host cells and then performing continuous passages, and preparing the Japanese encephalitis recombinant adenovirus vaccine after the transcription and expression of the recombinant adenovirus E protein in the host cells are stable.

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