CN115960847B - Application of small molecule dependent intein self-splicing system in preparation of attenuated live vaccine - Google Patents
Application of small molecule dependent intein self-splicing system in preparation of attenuated live vaccine Download PDFInfo
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Abstract
The invention belongs to the technical field of preparation of attenuated live vaccines, and particularly relates to application of a small molecule dependent intein self-splicing system in preparation of attenuated live vaccines. The application of the small molecule dependent intein self-splicing system in preparation of attenuated live vaccine is that the small molecule dependent intein self-splicing system is inserted into a virus sequence to prepare the attenuated live vaccine, and the small molecule is used for regulating the intein to carry out self-splicing so as to control the virus replication. The invention has the advantages of simple system, high efficiency and the like, can control virus replication in vivo, and can obtain the immune effect to the greatest extent.
Description
Technical Field
The invention belongs to the technical field of preparation of attenuated live vaccines, and particularly relates to application of a small molecule dependent intein self-splicing system in preparation of attenuated live vaccines.
Background
Prevention and control of pathogenic viruses is one of the global urgent public health problems, and vaccination has proven to be the most effective and powerful method of preventing viral diseases. However, the limited immunoprotection efficacy (Sandor A M,Sturdivant M S,Ting J.Influenza Virus and SARS-CoV-2Vaccines[J].J Immunol,2021,206(11)：2509-2520.). of commercial vaccines against new coronaviruses and influenza viruses maximizes the retention of all antigens of the virus, and the general approach to rapid construction of highly variant attenuated live vaccines has been the goal pursued by vaccine workers.
Most vaccines today rely on inactivation or attenuation techniques, which have been successfully used to treat a number of important animal and human diseases, but both of these techniques suffer from their limitations and associated potential problems. Inactivated vaccines are generally harmless and non-infectious, but produce a short maintenance of immune response, requiring multiple injections to enhance their overall immunogenicity or efficacy. The attenuated live vaccine retains most of the gene sequences of live viruses, has the advantages of strong immunity, long acting time and the like, and still has potential pathogenic risks. There are various strategies for the generation of attenuated live vaccines, including NS1 truncated virus (Pica N,Langlois R A,Krammer F,et al.NS1-truncated live attenuated virus vaccine provides robust protection to aged mice from viral challenge[J].JVirol,2012,86(19)：10293-10301.)、microRNA attenuated virus (Li J,Arevalo M T,Diaz-Arevalo D,et al.Generation of a safe and effective live viral vaccine by virus self-attenuation using species-specific artificial microRNA[J].J Control Release,2015,207：70-76.)、 codon to optimize viral (Si L,Xu H,Zhou X,et al.Generation of influenza A viruses as live but replication-incompetent virus vaccines[J].Science,2016,354(6316)：1170-1173.)、 high interferon sensitive virus (Du Y,Xin L,Shi Y,et al.Genome-wide identification of interferon-sensitive mutations enables influenza vaccine design[J].Science,2018,359(6373)：290-296.)、 protein degradation targeting chimeric virus (PROTAC)(Si L,Shen Q,Li J,et al.Generation of a live attenuated influenza A vaccine by proteolysis targeting[J].Nat Biotechnol,2022.) of virus (Jack B R,Boutz D R,Paff M L,et al.Reduced Protein Expression in a Virus Attenuated by Codon Deoptimization[J].G3(Bethesda),2017,7(9)：2957-2968.)、 carrying premature stop codon (PTC), and the like. Current attenuation strategies are often accompanied by a reduction or loss of safety, effectiveness or productivity. In addition, the immune escape caused by persistent antigen drift and metastasis of seasonal epidemic influenza presents a great challenge to the efficacy of conventional influenza vaccines. Therefore, improving the safety and effectiveness of attenuated live vaccines is an important direction in current vaccine development.
Inteins (inteins) are a very attractive class of molecular switches, which are inserted into a host protein with their corresponding nucleotide sequences chimeric into the corresponding nucleic acid sequences of the host protein, in-frame with the host protein gene, and transcribed and translated synchronously with the host protein gene, whereby the inteins are cleaved from the protein precursor upon translation to form the mature host protein. Natural inteins are not able to be triggered by small molecules to self-cleave. The university of harvard David r.liu teaches the evolution of an intein-based molecular switch that converts the binding of small molecules to the distance between the N and C termini of ER-LBD upon binding of the high affinity synthetic small molecule 4-HT to the active (Buskirk A R,Ong Y C,Gartner Z J,et al.Directed evolution of ligand dependence：small-molecule-activated protein splicing[J].Proc Natl Acad Sci U S A,2004,101(29)：10505-10510.). human estrogen receptor ligand binding region (ER-LBD, residues 304-551) of the target protein. They replaced the nonessential homing endonuclease region in the RecA intein structure with ER-LBD, resulting in a 424 residue N-terminal splice region-ER-LBD-C splice region intein fusion protein. Insertion of the ER-LBD native ligand binding domain into the RecA intein disrupts its self-splicing activity. In order to restore activity in a ligand-dependent manner, they correlated protein splicing with cell survival or fluorescence of Saccharomyces cerevisiae, by repeated mutagenesis and selection, resulting in an intein with strong splicing activity that is highly dependent on 4-HT. And through multi-round protein directed evolution screening, a set of Intein system ：37R3-2(Peck SH,Chen I,Liu DR.Directed evolution of a small-molecule-triggered intein with improved splicing properties in mammalian cells[J].Chem.Biol,2011,18(5):619-630.). which is low in background, rapid in shearing and applicable to mammalian cells is obtained, and the introns of mycobacterium tuberculosis RecA are selected because the introns can be effectively spliced in various environments. Recently, they inserted evolved 37R3-2 into specific positions of Cas9, developing a 4-HT triggered Cas9 nuclease (Davis KM,Pattanayak V,Thompson DB,et al.Small molecule-triggered Cas9 protein with improved genome-editing specificity[J].Nat.Chem.Biol,2015,11(5):316-318).
Small molecule dependent intein systems are a very attractive class of molecular switches that can in principle be inserted into any protein of interest, such that protein function depends on activation of the small molecule. The insertion of evolved intein proteins into a variety of unrelated proteins in living cells demonstrated that ligand-dependent activation of protein function is common, rapid, dose-dependent and post-translationally modified. The process does not require specific cellular environment and co-factor involvement and can even be performed (Pinto F,Thornton EL,Wang B.An expanded library of orthogonal split inteins enables modular multi-peptide assemblies[J].Nat.Commun.,2020,11(1):1529.). in vitro however, natural inteins are not able to be triggered by small molecules to self-splice. The intein has wide application prospect in the fields of gene diagnosis and treatment, virus research and the like due to the unique splicing function. For example, when transferring large genes in gene therapy, truong et al found that the dual vector recombinant adeno-associated virus (RAAV) system could invade cells by cleaving inteins by splicing NPU dnae, thus transporting the split-Cas 9 element and recombining Cas9(Truong DJ,Kuhner K,Kuhn R,et al.Development of an intein-mediated split-Cas9 system for gene therapy[J].Nucleic.Acids.Res,2015,43(13):6450-6458).. Furthermore, tornabene et al increased the carrying capacity of AAV vectors by splitting intein-assisted assembly strategies and have been shown to play an important role in viral and vaccine applications in retinal gene therapy (Tornabene P,Trapani I,Minopoli R,et al.Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina[J].Sci.Transl.Med.,2019,11(492))., in order to achieve soluble expression of ebola glycoprotein fusion proteins in e.coli, yang et al developed a fusion protein strategy based on self-splicing intein tags, which probably has an important significance (Ji Y,Lu Y,Yan Y,et al.Design of Fusion Proteins for Efficient and Soluble Production of Immunogenic Ebola Virus Glycoprotein in Escherichia coli[J].Biotechnol J,2018,13(6):e1700627.). for the development of subunit vaccines, suggesting that hepatitis b virus core protein virus-like particles could be modified by intein-mediated trans-splicing, and that trans-splicing of inteins, which showed good antigen delivery potential (Wang Z,Tang S,Yue N,et al.Development of HBc virus-like particles as modular nanocarrier by intein-mediated trans-splicing[J].Biochem.Biophys.Res.Commun.,2021,534:891-895). protein, could also be used to construct cell culture models of SARS-COV2, which would be helpful for the study of viral pathogenesis and the application of the anti-viral mechanism in a new technologies for the development of a live vaccine genome in a biosafety (BSL 2) (Ju X,Zhu Y,Wang Y,et al.A novel cell culture system modeling the SARS-CoV-2life cycle[J].PLoS.Pathog.,2021,17(3):e1009439). -screening technology.
Disclosure of Invention
The invention aims to overcome the defects of the existing attenuated live vaccine technology and provides application of a micromolecular dependent intein self-splicing system in preparation of attenuated live vaccines, wherein the attenuated live vaccines are simple, efficient and safe in system, can control replication of in-vivo viruses, and have remarkable immune effect.
The application of the small molecule dependent intein self-splicing system in preparation of attenuated live vaccine is that the small molecule dependent intein self-splicing system is inserted into a virus sequence to prepare the attenuated live vaccine, and the small molecule is used for regulating the intein to carry out self-splicing so as to control the virus replication.
Preferably, attenuated live vaccines include attenuated live vaccines of influenza virus, novel coronavirus, aids virus, ebola virus, dengue virus, polio virus, encephalitis b virus, zika virus, varicella zoster virus, herpes simplex virus and human cytomegalovirus.
Preferably, the intein self-splicing system is 37R3-2, and the amino acid sequence of the intein self-splicing system is shown in SEQ ID NO: 1.
Preferably, the small molecule is 4-hydroxy tamoxifen (4-HT), and the structural formula is shown in formula (I):
Preferably, the insertion site is located in a structural protein sequence unrelated to the immunogenicity of the virus, in practice, the corresponding insertion site protein should be selected according to the virus type, the influenza virus insertion site may be selected from structural protein polymerase acid Protein (PA), and the new coronavirus insertion site may be selected from S protein.
Further preferably, the insertion site is selected from a cysteine site or a serine, threonine, alanine site located in the linker moiety.
The preparation method of the attenuated live vaccine comprises the following steps:
(1) Performing PCR amplification by taking the intein gene as a template;
(2) Reverse polymerase chain reaction is carried out by taking the protein gene inserted into the site as a template to obtain a linearization carrier;
(3) Inserting intein genes into a linearization vector to obtain recombinant plasmids;
(4) After carrying out cell transfection on the recombinant plasmid, screening out the recombinant plasmid meeting the requirements, transfecting cells with the recombinant plasmid obtained by screening and the normal virus plasmid, and placing the transfected cells in a culture solution containing the small molecules for cell culture to obtain a cell culture solution;
(5) Taking supernatant of the cell culture solution, inoculating the supernatant into the allantoic cavity of the chick embryo containing the small molecules for culture, centrifuging the allantoic fluid of the chick embryo after infection, and taking the supernatant to obtain the attenuated live vaccine.
The concentration of the small molecule is 0.1 to 10. Mu.M, preferably 1. Mu.M.
Preferably, in step (4), the requirements for selection of recombinant plasmids are: ① The activity of the insertion site protein can be obviously inhibited after the intein is inserted, and ② intein has high dependence on the small molecule.
Compared with the prior art, the invention has the following beneficial effects:
1. The invention applies the intein splicing system regulated by small molecules to the preparation of attenuated live vaccines for the first time. Compared with viruses prepared by a non-natural amino acid coding technology and a proteolytic targeting chimeric (PROTAC) technology, the intein self-splicing system used by the invention is a simple, efficient and safe binary regulation system. When the whole genome sequence of the virus is synthesized, the structural protein is inserted into the intein sequence, and after the transcription and translation of the virus genome, because the intein protein sequence is fused, virus particles cannot be assembled normally, when small molecules are added into cells or in vivo, the intein is automatically sheared, the virus structural protein is restored to wild type, the virus can be assembled normally and goes out of cells, a new target cell is infected, the modified virus has an outer shell which is completely the same as the wild type, all protein antigens of the virus are reserved, but the replication of the virus is small molecule dependent, so that the invention has the advantages of simple system, high efficiency and the like, and can control the replication of the virus in vivo to the greatest extent, and obtain the immune effect. Meanwhile, the invention also provides a new technical thought and theoretical guidance for the development of the virus vaccine.
2. The application of the invention can be suitable for preparing various virus vaccines, including influenza virus, new coronavirus, HIV, ebola virus, dengue virus, polio virus, japanese encephalitis virus, zika virus, varicella-zoster virus, herpes simplex virus, human cytomegalovirus and other viruses.
3. The viruses dependent on the small molecules of the invention carry exogenous intein genes so that they can be successfully replicated in MDCK cells and chick embryos, enabling large-scale production without the aid of helper viruses.
Drawings
FIG. 1, schematic representation of the production of 4-HT dependent H1N1 influenza virus, PA: polymerase acid protein, PB1: polymerase basic protein 1, pb2: polymerase basic protein 2, np: nucleoprotein, M: matrix protein, NS: nonstructural proteins, HA: hemagglutinin, NA: neuraminidase;
FIG. 2, western-Blot selection of optimal insertion sites for inteins in PA, (A) insertion of inteins to replace each colored residue, (B) transfection of HEK-293T cells with wild-type PA and with an intein-PA mutant expression plasmid pair, 6h later with 4-HT or DMSO containing 4. Mu.M/mL, and 24h later with cell harvest for immunoblotting, (C) quantification of spliced protein bands by densitometry to calculate the percentage of spliced protein, protein splice percentage for each PA mutant expressed as change in expression of wild-type PA protein (PA/GAPDH);
FIG. 3, cytotoxicity of 4-HT at different concentrations in HEK293T and MDCK cells, (A) cytotoxicity of 4-HT on HEK293T cells at 24h, (B) cytotoxicity of 4-HT on HEK293T cells at 48h, (C) cytotoxicity of 4-HT MDCK cells at 24h, (D) cytotoxicity of 4-HT MDCK cells at 48h, (E) cytotoxicity of 4-HT MDCK cells at 72h, <0.05, <0.01, < P, <0.01vs.Control group, < P,;
FIG. 4, preparation and characterization of 4-HT dependent H1N1 influenza virus, (A) virus produced in the presence or absence of 4-HT, cell morphology observed under microscope after 48H, scale 100 μm, (B) relative packaging efficiency of recombinant influenza virus, relative packaging efficiency indicating ratio of recombinant influenza virus to wild-type PR8 virus reaching-100% CPE at 48H (n=3), (C) identification of infectivity and replication capacity of progeny virus in HEK293T cells by CPE method, (D) comparison of morphology of 4-HT dependent virus and wild-type virus, scale 100nm, (E) multicycle replication kinetics curve of S218 virus in HEK293T cells (n=3), (F) growth characteristics of recombinant influenza virus in 10 day old chick embryos (n=3);
FIG. 5, antiviral innate immune response of 4-HT dependent viral infected human nasal mucosal epithelial cells (hNEC); (A-B) qRT-PCR detection of expression of cell pattern recognition receptors such as RIG-I (A) and TLR-3 (B) in hNEC after 24h of infection of WT and S218 viruses; (C-E) qRT-PCR detection of expression of IFN- β (C) IRF-7 (D) MXA (E) and STAT-1 (F) and other natural immune genes in human nerves hNEC 24h after infection with the WT and S218 viruses; (G-J) hNEC-induced chemokine responses of WT and S218 infection, collecting apical and basal cell culture supernatants 24H after WT and S218 virus infection, and analyzing the expression levels of IL-8 (G and H) and IP-10 (I and G) by ELISA; all data are expressed as expression changes relative to uninfected cultures, < P0.05, < P <0.01, < P <0.001VS.Control group,n = 3;
FIG. 6, safety assessment of 4-HT dependent H1N1 influenza virus in mice, (A) effect of 14 days after 4-HT intraperitoneal injection in mice on pathological changes in each critical organ, (B) survival and weight changes after 2 weeks of nasal vaccination of BALB/C mice (n=6) with the above virus or vaccine, (C) effect of virus titer in lung tissue (n=3) on day 3 after 10 2TCID50 WT and S218 virus infection in mice, (D) effect of 4-HT on plaque phenotype (n=3) of mice WT and S218 virus;
FIG. 7, lung tissue morphology 3 days after virus infection of mice;
FIG. 8, levels of IgG antibodies and subtypes thereof, IFN-. Gamma., IL-4 and sIgA antibodies in alveolar lavage fluid in mouse serum;
Fig. 9, evaluation of challenge protection in mice.
Detailed Description
The invention is further described below with reference to examples.
All the raw materials used in the examples were commercially available except for the specific descriptions.
Example 1
In this example, the small molecule 4-HT dependent intein self-splicing system 37R3-2 was used in the preparation of live attenuated influenza A (H1N 1) vaccines by the following method:
(1) PCR amplification is carried out by taking the intein gene 37R3-2 as a template;
(2) Reverse polymerase chain reaction is carried out by taking a protein gene of an insertion site as a template, wherein the insertion site selects a cysteine site on structural protein polymerase acidic Protein (PA) and serine, threonine and alanine sites positioned on a connecting part, and the specific site selects Thr200, ala215, ser218, ser224, ser225, ala231, ser247, ser250 and Ala255, so that the amino acid sequence of the corresponding linearization carrier PHW2000-PA-T200、PHW2000-Pa-a215、PHW2000-PA-S218、PHW2000-PA-S224、PHW2000-PA-S225、PHW2000-Pa-a231、PHW2000-PA-S247、PHW2000-PA-S250、PHW2000-Pa-a255,PA protein is respectively obtained as shown in SEQ ID NO:2 is shown in the figure;
(3) Inserting the amplified intein genes into linearization vectors respectively, and substituting amino acids at corresponding sites of the linearization vectors with the intein genes 37R3-2 to obtain recombinant plasmids;
(4) After cell transfection is carried out on the recombinant plasmid, the cell is transfected by the recombinant plasmid obtained by screening out the plasmid which is inserted with Intein and remarkably inhibits the activity of PA protein and has high dependence on 4-HT, namely, intin-PA-T200, intin-PA-A 215 and Intin-PA-S218, and the recombinant plasmid obtained by screening and the normal viral plasmid are transfected, and are placed in a culture solution containing 1 mu M4-HT for cell culture, so as to obtain a cell culture solution;
(5) Taking supernatant of the cell culture solution, inoculating the supernatant into a chick embryo allantoic cavity containing 1 mu M4-HT for culture, centrifuging the infected chick embryo allantoic fluid, and taking the supernatant to obtain the attenuated live vaccine.
The specific preparation process and safety and immunogenicity of the influenza a (H1N 1) attenuated live vaccine were evaluated as follows:
1. materials and methods
1. Material
1.1 Materials and reagents
4-HT (S7827, selleck); all primers were synthesized by Shanghai Ind; the 8 plasmid influenza a virus/polis/8/1934H 1N1 (PR 8) virus packaging system is purchased from department of biotechnology, inc. In guangzhou; seamless cloning kit was purchased from Beyotime Biotech; DMEM (8121382) and fetal bovine serum (FBS, 16000-044 years) were purchased from Gibco.
2. Method of
2.1 Cell culture
2.1.1 Cell resuscitation
One HEK293T or MDCK cell is taken out from a refrigerator with the temperature of minus 150 ℃, is quickly transferred to a constant temperature water bath kettle with the temperature of 37 ℃ to be continuously rocked to be melted, the melted cell suspension is transferred to a 15mL sterile centrifuge tube, 6mL of 10% FBS-DMEM culture medium is added, the mixture is centrifuged for 3min at the room temperature of 1500rpm, the supernatant is discarded, 8mL of 10% FBS-DMEM resuspended cells are added, and the mixture is transferred to a 100mm cell culture dish and cultured at the temperature of 37 ℃ and 5% CO 2.
2.1.2 Passage and culture of cells
When the abundance of cells reached 95%, the original medium was discarded, washed with 2mL of PBS, removed, digested with 2mL of pancreatin at 37℃for 1min until the cells had fallen, stopped with 6mL of 10% FBS-DMEM medium and the cells were blown down with a pipette, centrifuged at 1500rpm at room temperature for 3min, the supernatant was discarded, 6mL of 10% FBS-DMEM was added to resuspend the cells, and the cells were passaged to a cell culture dish at 1 pass 3, supplemented with 6mL of 10% FBS-DMEM, and cultured at 37℃with 5% CO 2.
2.2 Plasmid construction
2.2.1 Primer design: the primer was designed with Snapgene software based on the insertion site and the vector sequence.
TABLE 1 primer sequences for PCR
2.2.2PCR (polymerase chain reaction)
PCR amplification procedure: pre-denaturation at 95℃for 3min, denaturation at 95℃for 10s, annealing at 65℃for 15s, denaturation and annealing cycles for 30 times, extension at 72℃for 77s and final extension at 72℃for 5min.
PCR amplification system: 1. Mu.L of template, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 2X LongHiFi PCR MasterMix 12.5.5. Mu.L, 10.5. Mu.L of ddH 2 O, and 25. Mu.L of Total.
2.2.3 Agarose electrophoresis
1) And (3) glue preparation: firstly, weighing 0.5g agarose by an electronic balance, adding the agarose into a heat-resistant conical flask, measuring 50mL of 1 xTAE working solution by using a measuring cylinder, shaking and uniformly mixing the agarose and the working solution, heating the mixture in a high-temperature range by a microwave oven for 4min to dissolve the agarose until the agarose is completely transparent, adding 5 mu L of 10000 x STARSTAIN GREEN nucleic acid dye when the temperature of the agarose is reduced to 45-55 ℃, selecting a proper rubber plate and a proper comb, pouring the rubber plate and waiting for solidification at room temperature;
2) Loading and running glue: samples were spotted into agarose wells and run at GenStar D5000,5000 as an indicator Marker,130v for 20 min.
2.2.4 Glue recovery
And (3) photoresist: and (3) placing the running agarose gel into a gel imager, cutting the agarose gel at the position of the target strip by using a clean blade if the size of the strip is as large as that of the target strip, and recycling the gel according to the instruction of the kit.
The method comprises the following basic steps of sol-adsorption column adsorption, cleaning and elution:
1) Adding sol buffer, and placing in a metal bath at 65 ℃ to completely melt the gel block, wherein the solution is light yellow, clear and transparent;
2) Adding the DNA into the activated adsorption column to enable the DNA to be adsorbed on a silica gel film;
3) Adding a Washing buffer to clean impurities;
4) Elution was performed by adding ddH 2 O preheated at 65 ℃.
2.2.5 Homologous recombination ligation
Adding the required reagent into the EP pipe according to the connection system, uniformly mixing, and standing at 37 ℃ for 10min.
The connection system is as follows: pHW-NP linearized vector 30ng, intin amplified fragment 90ng,2X Seamless Cloning Mix5. Mu.L, nucleic FREE WATER To 10. Mu.L.
2.2.6 Conversion
5 Mu L of the ligation product and 50 mu L of DH5 alpha E.coli competent cells were added to a 1.5mL EP tube, the mixture was allowed to stand on ice for 30min, heat-shocked at 42℃for 90s, and then again allowed to stand on ice for 2min, 200 mu L of the non-antibiotic LB liquid medium was added, and the mixture was placed on a double-layer insulating shaker at 37℃for 45-60min under shaking at 200 rpm. And (3) coating the bacterial liquid after the transformation on an ampicillin-resistant LB culture plate, putting the bacterial liquid into a 37 ℃ incubator for inversion, and culturing the bacterial liquid after the surface of a culture medium is dried for 14-16 hours after inversion.
2.2.7 Shaking
Single colonies on the plates were picked with a 10. Mu.L small tip and placed in a culture tube containing 2mL of ampicillin-resistant medium and shake cultured for 14-16h.
2.2.8 Bacterial liquid PCR
PCR amplification procedure: pre-denaturation at 94℃for 5min, denaturation at 94℃for 10s, annealing at 56℃for 10s, extension at 72℃for 130s, denaturation, annealing and extension cycles for 30 times, final extension at 72℃for 5min.
PCR amplification system: 1. Mu.L of bacterial liquid, 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 10. Mu.L of 2X LIGHTING TAG PCR Mix, 8. Mu.L of ddH 2 O and 20. Mu.L of Total.
2.2.9 Agarose electrophoresis (ref 2.2.3)
2.2.10 Sequencing
If agarose electrophoresis is consistent with the size of the target band, 200 mu L of bacterial liquid is sucked and sent to a biological company for sequencing, and the rest bacterial liquid is stored to-80 ℃ by glycerol. And if the sequencing result is completely correct, extracting plasmids, and if the sequencing result is incorrect, searching for a reason and reconstructing.
2.3 Extraction from endotoxinfree plasmids
Since endotoxin is removed from generating toxicity to cells, the kit is extracted from the endotoxin-removed plasmid. The bacterial liquid is cultured for 12-15mL and 14-16h in advance. The subsequent plasmid extracting step is carried out according to the specification, and the basic steps include collecting thalli, re-suspending thalli, cracking thalli, neutralizing and cracking, filtering, isopropanol precipitating protein, column balancing, column adsorbing, cleaning, eluting, measuring concentration and preserving plasmid at-20 ℃.
2.4 Cell harvesting and preparation
2.4.1 Cell harvesting
After the medium was discarded, 1mL of PBS was added for washing once, the 293T cells were directly and completely blown down by adding 1mL of PBS, PBS in the petri dish was transferred to a 1.5mL EP tube, centrifuged at 3000rpm for 3min, and the waste liquid was discarded. Residual cells in the dish were washed with 1mLPBS and transferred to the EP tube, collected by centrifugation, and repeated 3 times.
2.4.2 Sample treatment
After the cells were resuspended in 100. Mu.L of PBS, 100. Mu.L of SDS lysate was added (after boiling the water, the sample was boiled in water for 10min, after the sample was boiled, centrifuged at 13000rpm for 10min at 4℃and the sample was stored at-80 ℃.
2.5Western-Blot
2.5.1 Glue preparation
SDS-PAGE gels were prepared according to the formulations in the following table, with the separation gel in the lower layer and the concentrated gel in the upper layer. A gel was prepared at a concentration of 10% according to the desired protein molecular weight.
TABLE 2SDS-PAGE gel formulations
2.5.2 Electrophoresis
The gel plate was fixed on a rack, and the prepared Western-Blot electrophoresis buffer was added, and samples and Maker were added to the wells as required. And (3) running to be separated by a Maker by using an 80V constant voltage electrophoresis mode, and regulating the voltage to be 120V constant voltage to continue running until the running is finished.
2.5.3 Semi-dry transfer film
Soaking the sponge cushion in a quick film transferring liquid, placing the PVDF film in the film transferring liquid after soaking the sponge cushion in a methanol solution for about 1min for 5min, then prying off a rubber plate, cutting off concentrated rubber and redundant rubber around, and removing the remained separating rubber, wherein the soaked sponge cushion and PVDF film are as follows from bottom to top: the double-layer foam cushion, PVDF membrane, separating gel and double-layer foam cushion are placed on a film transferring instrument in sequence, and excessive film transferring liquid and bubbles are extruded out by using rollers. After the film transfer instrument is covered, the film is transferred for 10min at a constant pressure of 25 v.
2.5.4 Closure
With 5% milk (0.5 g milk per 10mL TBST), at 40rpm, at room temperature for 1h or overnight at 4 ℃.
2.5.5 First antibody incubation
And (3) placing the PVDF membrane which is closed and cut according to the molecular weight into an antibody incubation box, adding the prepared primary antibody, and shaking the box overnight at 4 ℃.
2.5.6 Washing film
Collecting primary antibody, storing at-20deg.C, and recycling. TBST working solution is added, the rotating speed is 100rpm, and the room temperature is 5min. Repeated three times.
2.5.7 Secondary antibody incubation
The prepared secondary murine antibody is added into an antibody incubation box, and incubated at the rotating speed of 40rpm for 40-60min at room temperature.
2.5.8 Washing film
Collecting the secondary antibody, storing at-20deg.C, and recycling. TBST working solution was added at 100rpm for 5min at room temperature and repeated three times.
2.5.9 Development
Preparing developing solution according to the ratio of 1:1, placing a PVDF film on a plastic packaging film, sucking the liquid remained on the surface, uniformly coating the developing solution on the PVDF film, then placing the film into a developing instrument for imaging, and carrying out band density analysis by using imageJ software.
2.6 Packaging of wild-type and 4-HT dependent H1N1 influenza Virus
Preparation and propagation of wild-type influenza virus and 4-HT dependent virus containing intron genes in the genome. To obtain PR8-WT influenza virus, HEK293T cells were cultured in a medium containing DMEM, 10% fetal bovine serum and 1% penicillin/streptomycin at 37℃in a 5% carbon dioxide humid environment. When the cell fusion reached 80-90%, the cells were transfected with Sinofection transfection reagent using a mixture of 3 μg of each of the 8 plasmids according to the instructions for the transfection reagent in Yizhushenzhou. After 6h the transfection solution was replaced with DMEM containing 5% foetal calf serum, 1% penicillin/streptomycin and 2. Mu.g/mL TPCK-TREATED TRYPSI. The cells were further cultured at 37℃in 5% carbon dioxide. After 48h, the supernatant was collected and centrifuged at 1000 Xg for 10min to remove contaminating cells. In addition, to obtain 4-HT dependent viruses, the experimental procedure was as above, but the changes were as follows: the plasmid of wild type PA was replaced with the corresponding mutant PA-Intin plasmid and 1. Mu.M 4-HT was added to the medium. Supernatant was collected and infected with 9 day old chick embryo eggs at 37 ℃. Allantoic fluid containing virus was collected 72h after infection and stored at-80 ℃.
2.7 Cytopathic Effect (CPE) experiments
An appropriate amount of MDCK cells were inoculated in a 6-well plate or a 96-well plate, and when the cell density reached about 90%, influenza virus was added to a 1% FBS-DMEM medium containing 2. Mu.g/mL of TPCK pancreatin, and mixed culture was carried out at 37℃for 1 hour. After 1h, the culture solution was taken out and replaced with fresh culture solution for further culture. After 3d incubation, the CPE percentage of the cells was observed and recorded under a microscope.
2.8 Evaluation of replication kinetics of the virus
To determine the kinetics of virus growth in vitro, MDCK cells were seeded into DMEM containing 10% fetal bovine serum, 2×10 5 cells per well. After 24h incubation, the moi=0.01 virus was infected at 37 ℃ for 1h with gentle shaking of the 6-well plate every 15 min. After 1h, the virus-containing DMEM solution was removed, and the cells were washed 3 times with PBS, and then further cultured in DMEM containing 1% FBS and 2. Mu.g.mL - 1 TPCK-treated pancreatin. Cell supernatants were collected at various time points (24, 48, 72, 96 h) post infection and assayed for viral titres by TCID 50.
2.9 Cell immunoassay
Differentiated human nasal mucosal epithelial cells (hNECs) were seeded at 5X 10 4 cells per well in 24 well Transwell plates containing icell primary epithelial cell basal broth (icell Bioscience, primed-icell-001). When the cells reached 80-90% confluence, WT and S218 viruses were inoculated at 10TCID 50/mL for 1h. The inoculum was removed and the root tips and basolateral 3 times were rinsed with PBS. Fresh 150. Mu.L and 500. Mu.L of culture medium were then added to the apical and basal sides, respectively. After 24h of culture, the cell culture fluid of the cell culture fluid at the top and the outside of the substrate is collected for cytokine analysis, and total RNA of the cells is extracted for qRT-PCR detection.
2.10 Animal experiments
Safety, immunogenicity and protective effects of the vaccine were evaluated with BALB/c mice 6-8 weeks old without specific pathogens. The feeding conditions are as follows: the light/dark period is 12 hours, the temperature is 24 ℃, and the humidity is 50%. LD 50 of wild-type PR8 virus against BALB/c mice was 10 -3.6/50. Mu.L.
2.10.1 Evaluation of safety of Virus
(1) Experimental grouping: ① PBS control (-4 ht, n=6); ② PBS control (+4ht, n=6); ③ WT group (-4 ht,10 2 TCID 50/v, n=9); ④ WT group (+4ht, 10 2TCID50/WT, n=9)
⑤ S218 group (-4 ht,10 2TCID50/r, n=9); ⑥ S218 group (+4ht, 10 2TCID50/n=9 only) ⑦; s218 group (10 3TCID50/piece, n=6); ⑧ S218 group (10 4TCID50/piece, n=6).
(2) Dose of 4HT administration: 4-HT intraperitoneal injection (20 mg/kg), once daily
(3) The animal experiment steps: to detect the replication of the virus in mice, 3 mice were sacrificed on day 3 post-inoculation, 3 mice were taken from each ③④⑤⑥ group and their lungs were titrated for virus shift, and a few lung tissues were fixed with paraformaldehyde for pathology assessment. The remaining groups were monitored daily for weight loss and mortality for each group for 14 days.
Evaluation of immunity and protection of 2.10.2S218 vaccine
(1) Experimental grouping: female mice at 6 weeks of age were randomized into: ① PBS control group (nasal inoculation 50. Mu.L); ② S218 virus vaccine group (nasal inoculation 10 3TCID50/ μl); ③ Tetravalent influenza split vaccine group (intramuscular injection 0.1mL, 30. Mu.g/mL); each group of 15.
(2) The animal experiment steps: on day 21 post immunization, 5 animal serum and bronchoalveolar lavage samples were collected from each group and serum immunoglobulins IgG, igG 1, igG 2a antibodies, IL-4 and IFN-gamma cytokines were detected for sIgA antibodies in the alveolar lavage. The remaining 10 mice were lightly anesthetized on a normal small animal anesthesia machine and then challenged with 10LD 50 PR8-WT virus. On day 3 after challenge, 5 mice were sacrificed for each group, and lung tissue was taken for virus titration, HE staining and qRT-PCR detection of inflammatory factors TNF- α, IL-6, IFN- γ. The remaining mice were monitored for weight loss and mortality for 14 days.
2.11 Enzyme-Linked immunosorbent assay (ELISA)
IL-8 and IP-10 in root tips and basolateral culture supernatants were analyzed using a commercial kit (ml 028580; ml 038327). Detecting total immunoglobulins Ig G, ig G1 and Ig G2a in mouse serum using a mouse Ig G/Ig G1/Ig G2a kit (ml 625210; ml1244589V; ml 102258V); IL-4 and IFN-gamma concentrations were detected using the mouse IL-4/IFN-gamma kit (ml 064310V; ml 002277V). The concentration of sIgA in the mouse bronchoalveolar lavage fluid was determined using the mouse sIgA ELISA kit (ml 001917V).
2.11qRT–PCR
To determine the expression of RIG-I, TLR-3, IFN- β and IRF-7, mxA, STAT-I, hNEC cells were collected. In addition, to detect mRNA expression of inflammatory-related cytokines in mouse lung tissue following influenza virus challenge, lung tissue was collected for homogenization, total RNA was extracted by a universal total RNA kit (GeneBetter, R013) and quantified by a Nanone uv spectrophotometer. Then, 1. Mu.g of each sample was reverse transcribed into cDNA using a reverse transcription kit (GeneBetter, P710), and a polymerase chain reaction was performed using a 2X Universal SYBR GREEN QPCR Mix (GeneBettr, P600). Relative expression of mRNAs was measured by the method of 2 -ΔΔCT, which was first normalized to GAPDH or beta-actin, then normalized to uninfected cultures, and shown as fold-change in gene expression. The primers for qRT-PCR are shown in Table 3.
TABLE 3qRT-PCR primer sequences
| Target | Gene | Forward Primer sequence(5’to 3’) | Reverse Primer sequence(5’to 3’) |
| Human | GAPDH | GAAGGTGAAGGTCGGAGTC | GAAGATGGTGATGGGATTTC |
| Human | RIG-I | TGTGCTCCTACAGGTTGTGGA | CACTGGGATCTGATTCGCAAAA |
| Human | TLR-3 | TTGCCTTGTATCTACTTTTGGGG | TCAACACTGTTATGTTTGTGGGT |
| Human | IFN-β | GCTTGGATTCCTACAAAGAAGCA | ATAGATGGTCAATGCGGCGTC |
| Human | IRF-7 | CCCACGCTATACCATCTACCT | GATGTCGTCATAGAGGCTGTTG |
| Human | MxA | GTTTCCGAAGTGGACATCGCA | CTGCACAGGTTGTTCTCAGC |
| Human | STAT-I | CAGCTTGACTCAAAATTCCTGGA | TGAAGATTACGCTTGCTTTTCCT |
| Mouse | β-actin | ACGGCCAGGTCATCACTATTG | CAAGAAGGAAGGCTGGAAAAG |
| Mouse | TNF-α | CCTGTAGCCCACGTCGTAG | GGGAGTAGACAAGGTACAACCC |
| Mouse | IL-6 | CTGCAAGAGACTTCCATCCAG | AGTGGTATAGACAGGTCTGTTGG |
| Mouse | IFN-γ | ATGAACGCTACACACTGCATC | CCATCCTTTTGCCAGTTCCTC |
2.13 Histological examination of mouse lung tissue
Lung tissue from influenza virus infected mice was collected, fixed in 4% paraformaldehyde for 3 days, and embedded with paraffin blocks. Then, the tissue sections were cut into sections 3 μm thick, and lung injury was assessed using hematoxylin-eosin (HE) staining. Representative images were captured using a fluorescence microscope (NEXCOPE).
2.14 Plaque assay to detect viral titres
MDCK cells were seeded in 12-well plates until confluent monolayers were produced. Next, the cells were washed with PBS. The virus was then inoculated into cells after 10-fold dilution with DMEM gradient and incubated for 1h at 37 ℃ in a 5% co 2 cell incubator. After 1h, the virus inoculation solution was removed and washed with PBS, then immediately 1.6mL DMEM medium containing 1.5% low melting agarose (SIGMA ALDRICH, A5431) and 2. Mu.g mL -1 TPCK treated trypsin was added. After 10min of horizontal placement, the cells were incubated with 5% CO 2 at 37℃for 3-5 days in an inverted position. Finally, cells were fixed with 4% paraformaldehyde, stained with crystal violet (Sigma-Aldrich, C6158), and plaques were observed and counted.
2.15 CCK-8 experiment
Each well was seeded with 5 x 10 3 HEK293T and MDCK cells in 96-well plates. After 24h incubation, the medium was removed and replaced with 200. Mu.L of fresh medium containing different concentrations of 4-HT (0, 0.5, 1,2, 4, 8, 16, 32. Mu.M). At the indicated times after incubation (24, 48 and 72 h), 10. Mu.L of CCK-8 reagent (TargetMol, USA) was added to each well and incubated for 1.5h at 37 ℃. Absorbance was measured at 450nm using a microplate reader (BIO-TEK, H1M).
2.16 Statistical analysis
All experiments in this study were repeated at least twice. Data are shown as mean ± s.d. Two sets were compared using the Two-measured student t test; comparison of multiple groups was analyzed by one-way anova and Dunnet test. P <0.05 is considered statistically significant. All statistical analyses were performed using GRAPHPAD PRISM 8.0.2.
2. Results and analysis
1. Screening of optimal insertion site for influenza A polymerase acid Protein (PA) -intein
To achieve this, we first inserted small molecule 4-HT dependent inteins at 9 positions of the PA protein (Thr 200, ala215, ser218, ser224, ser225, ala231, ser247, ser250, ala 255). Given the small impact of the linking moiety on protein function, this moiety becomes the preferred site for intein insertion. Depending on the splicing mechanism of the intein, splicing itself leaves a cysteine residue to join the ends of the extein, so that all the cysteine sites in the gene sequence and serine, threonine, alanine sites in the joining portion similar to the cysteine structure are selected for insertion into the intein gene sequence, as shown in FIG. 2A. 9 recombinant Intin-PA mutant vectors were introduced into HEK293T cells. 6h after transfection, cells were treated with or without 1. Mu.M 4-HT and after 24h Western-Blot (WB) assay was performed. As shown in FIG. 2B, the expression of wild-type (WT) PA protein was not affected by 4-HT, indicating that 4-HT did not affect PA expression. Inteins, on the other hand, are present in mature PA proteins slightly higher than 100kDa, probably due to fusion of PA with inteins, since PA is 82kDa in size, whereas inteins are about 41kDa in size. Of these 5 mutants, corresponding to intein insertions Thr200, ala215, ser218, ala231 and Ser247, showed 4-HT dependent PA expression (fig. 2C), consistent with the 4-HT triggered PA activity (fig. 2B) results. Furthermore, there were four intelin-PA mutants (Ser 224, ser225, ser250 and Ala 255) that produced large amounts of PA splice products whether 4-HT was present or not (fig. 2B), indicating that insertion of inteins at these sites did not significantly inhibit PA protein activity or lost dependence on 4-HT due to the high background of Intein splicing. WB results indicate that it is feasible to design mutants by inserting inteins at selected PA sites, as they can tolerate 413 amino acid insertions. Based on the result that the PA mutant showed high splicing activity in the presence of 4-HT and low activity in the absence of 4-HT, three Intin-PA mutant plasmids, thr200, ala215 and Ser218, were finally selected for subsequent experiments.
Preparation and characterization of 2.4-HT dependent H1N1 influenza virus
The most commonly used influenza strain A/PR/8/34 (H1N 1) in the laboratory was selected and the PHW2000 plasmid containing 8 influenza A/PR/8/34 virus fragments was used to rescue the H1N1 influenza virus. HEK293T cells were co-transfected with 3 candidate mutant (Intin-PA-T200, A215 and S218) plasmids and the remaining 7 plasmids, and 4-HT dependent H1N1 influenza virus was successfully rescued in vitro. First we evaluated the toxic effect of 4-HT on cells, no cytotoxicity was observed in HEK293T and MDCK cells treated with 0.5, 1, 2. Mu.M 4-HT for 24-48h or 24h-72h (FIG. 3) and finally 1. Mu.M 4-HT was selected for subsequent experiments. Cytopathic effects (CPE) demonstrated the production of 4-HT dependent viruses, with Intin-PA-T200 and S218 producing 4-HT dependent CPE effects (FIGS. 4A-B). While A215 loses regulation of 4-HT, probably due to the self-splicing of inteins at this position during viral packaging and the loss of response to 4-HT. In addition, since the titer of virus in cell supernatant was low, we proliferated the virus through chick embryo. Allantoic fluid containing wild-type (WT) and mutant (T200 and S218) viruses was collected to infect HEK293T cells. The results showed that 4-HT did not affect the infection of HEK-293T cells by WT virus (FIG. 4C), whereas the CPE effect was observed by the T200 and S218 viruses only in the presence of 4-HT (FIG. 4C), indicating that replication of the viral mutant was regulated by 4-HT. The virus morphology was observed by transmission electron microscopy, and it was seen that the S218 virus was approximately 100nm in diameter and length, complete in shape, and clearly visible in envelope structure, and was a typical spherical influenza virus particle similar in size and shape to the parental strain (FIG. 4D). However, the T200 strain has incomplete surface coating structure, and is different from the parent strain, so that S218 is finally selected as a representative strain for subsequent experiments. The safety of the S218 virus as a candidate vaccine is largely dependent on its degree of attenuation within the cell. Thus, we also compared the replicative capacity of WT and S218 in HEK293T cells and 9 day old SPF chicken embryos. As shown in fig. 4E, the replication kinetics of S218 virus in HEK293T cells was similar to that of WT virus, and S218 virus had a significant attenuation effect in HEK293T (fig. 4E). By examining the titer of S218 virus in chick embryos 3 days after inoculation of the chick embryos with S218 virus and WT virus, it was found that replication of S218 virus in chick embryos was significantly reduced (fig. 4F). Thus, the S218 virus achieves attenuation at both the cellular level and in the chick embryo.
Cellular immune evaluation of 4-HT dependent H1N1 influenza Virus
Mucosal immunity is the first line of defense against invasion by 31 respiratory viruses including influenza a virus. Human nasal mucosal epithelial cells (hNEC) play a key role in influenza virus and vaccine infection, as they are not only the primary cells that are infected, but also the key cells that initiate innate immune responses. To compare the innate immune response of S218 and WT viruses, we selected a hNEC culture model of primary differentiation and examined the expression of host cell pattern recognition receptors (PRRs, including TLR3 and RIG-I) and the antiviral genes IFN- β, interferon regulatory factor 7 (IRF-7), STAT-I and myxovirus resistance gene A (MXA) using qRT-PCR.
The results show that: the expression levels of RIG-I, TLR-3, IFN-. Beta., IRF-7, STAT-I and MXA genes were significantly higher in hNEC infected with S218 and WT than in uninfected hNEC (FIG. 5A-F), and there was no significant difference between the expression levels of the two groups (FIG. 5A-F). To determine if S218 and WT infected hNEC differ in external cell signaling, we compared the expression of chemokines IL-8 and IP-10 in cell supernatants. After infection with both viruses, the expression of IL8 and IP-10 was significantly up-regulated in the apical and basal side culture supernatants (FIG. 5G-J), and the differences were not statistically significant (FIG. 5G-J). hNEC suggesting that S218 infection has similar innate immunity gene and chemokine expression as WT infection. Thus, the S218 virus can stimulate a strong natural immune response in hNEC, which is likely to become a candidate attenuated live vaccine.
4.4-HT-dependent H1N1 influenza Virus safety evaluation in mice
BALB/c mice are commonly used to study the safety and efficacy of influenza vaccines, and therefore BALB/c mice were selected to study S218 virus safety in vivo. Safety experiments were performed by selecting PR8-WT (10 2TCID50) and three different doses of S218 (10 2,103,104TCID50) virus. HE staining results indicated that 4-HT had no toxic effect on mice (fig. 6A-B). Compared to the control group, there was no significant effect on survival, body weight (FIG. 6B) and pulmonary viral titres (FIG. 6C-D) of mice after infection with WT virus, whether or not 4-HT was added, indicating that 4-HT did not affect WT virus replication in vivo. In mice infected with S218 virus, no death, weight loss, or other influenza-like clinical symptoms were observed even at the dose of 10 4TCID50 (fig. 6B). In contrast to the WT virus, no plaque phenotype was observed in the S218 (-4-HT) infected mice lungs (FIG. 6D), indicating that the S218 virus does not resume virulence in vivo, while plaque phenotype was observed in the S218 (+4HT) group of mice lungs (FIG. 6D), indicating that the S218 virus is regulated by 4-HT in vivo. Although there was no significant difference in intrapulmonary viral titers from WT in S218 (+4ht) infected mice (fig. 6C), both mortality and weight loss were lower in mice vaccinated with S218 (+4ht) virus than in WT mice, and lung lesions were lighter in S218 mice than in WT mice (fig. 7), which also further indicated that S218 virus was attenuated in vivo. So the use of 4-HT dependent intein regulatory mechanisms can achieve a high degree of attenuation of influenza virus in vivo. The results show that the 4-HT dependent H1N1 influenza virus has good safety in mice.
5.4-HT-dependent H1N1 influenza virus immunogenicity evaluation
To test for immunogenicity of S218, a commercially available tetravalent inactivated split influenza vaccine (QISIV) was selected for comparison. Mice were vaccinated nasally with 50 μl of 10 3TCID50 S218 or PBS, or injected intramuscularly with 100 μl QISIV (30 μg/mL). Virus-specific antibodies and cytokine levels were detected in serum 3 weeks after immunization. As shown in FIG. 8A, both S218 and QISIV induced a strong serum IgG antibody response. To further verify Th bias of IgG humoral immune response, we also examined IgG subtypes in serum. The IgG1 and IgG2a antibody levels were elevated for both S218 and QISIV groups (fig. 8B-C), with the IgG2a antibody level for S218 group being significantly higher than for IgG1 group (fig. 8C), and vice versa for QISIV group. Thus, S218 induced predominantly a Th 1-type immune response, while QISIV induced predominantly a Th 2-type immune response. In addition, S218 was able to induce high levels of sIgA antibodies (FIG. 8D), while QISIV vaccine was unable, while the S218 and QISIV immunized mice had significantly higher IL-4 and IFN-gamma levels than the PBS group (FIG. 8E-F). In summary, the S218 virus vaccine induces a strong humoral, mucosal and cellular immune response in the body.
Protective assessment of homologous Virus attack by 4-HT dependent H1N1 influenza Virus
To test whether the S218 vaccine provides protection against homologous virus challenge, comparison was made with QISIV and PBS. When S218, QISIV or PBS immunized mice were 3 weeks post inoculation, they were challenged with 10×ld 50 WT virus. The results indicated that PBS group mice all had decreased body weight and died on day 5 after challenge, while S218 and QISIV immunized groups mice all survived without significant weight loss (fig. 9A-B). In addition, influenza virus infection can trigger an innate immune response. Thus, we examined IFN- β, IL-6 and TNF- α levels in lung tissue 3 days after WT virus challenge. The results show that the expression level of IFN-beta, IL-6 and TNF-alpha genes in lung tissues of mice in S218 and QISIV immunized groups is significantly lower than that in PBS group (FIG. 9C-E), suggesting that the S218 vaccine can significantly reduce inflammatory response triggered by pulmonary virus infection of mice.
In summary, the present invention successfully developed a 4-HT dependent H1N1 attenuated live vaccine using a small molecule dependent intein splicing mechanism to control viral protein activity, the outer shell of the virus is similar to the wild type, retaining all protein antigens of the virus, but replication of the virus is highly dependent on 4-HT. In this way, the virulence of the virus can be better controlled while retaining a strong immunogenicity to ensure effectiveness. The experimental result proves that the 4-HT dependent virus has high attenuation effect in vitro and in vivo, can stimulate human nasal mucosa cells in vitro to generate strong innate immune response, can induce same strong humoral, mucosal and cellular immunity in mice, and provides complete protection for the attack of homologous viruses.
Claims (4)
1. An attenuated live vaccine, characterized in that: the attenuated live vaccine is prepared by inserting a micromolecular dependent intein into a viral gene sequence from a splicing system, and the micromolecular controlled intein is subjected to self-splicing to control viral replication;
the attenuated live vaccine is used for treating influenza A and H1N1 virus;
The intein self-splicing system is 37R3-2, and the amino acid sequence of the intein self-splicing system is shown in SEQ ID NO:1 is shown in the specification;
the small molecule is 4-hydroxy tamoxifen, and the structural formula is shown as formula (I):
(Ⅰ)；
The insertion site is positioned at Ser218 site of polymerase acid protein PA of H1N1 virus, and the amino acid sequence of the polymerase acid protein PA is shown as SEQ ID NO: 2.
2. A method for preparing the attenuated live vaccine of claim 1, wherein the method comprises the steps of: the method comprises the following steps:
(1) Performing PCR amplification by taking the intein gene as a template;
(2) Reverse polymerase chain reaction is carried out by taking the protein gene inserted into the site as a template to obtain a linearization carrier;
(3) Inserting intein genes into a linearization vector to obtain recombinant plasmids;
(4) After carrying out cell transfection on the recombinant plasmid, screening out the recombinant plasmid meeting the requirements, transfecting cells with the recombinant plasmid obtained by screening and the normal virus plasmid, and placing the transfected cells in a culture solution containing the small molecules for cell culture to obtain a cell culture solution;
(5) Taking supernatant of the cell culture solution, inoculating the supernatant into the allantoic cavity of the chick embryo containing the small molecules for culture, centrifuging the allantoic fluid of the chick embryo after infection, and taking the supernatant to obtain the attenuated live vaccine.
3. The method for preparing attenuated live vaccine according to claim 2, wherein: the concentration of small molecules is 0.1-10 mu M.
4. A method of preparing a live attenuated vaccine according to claim 3, wherein: the concentration of small molecules was 1. Mu.M.
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| CN112708605A (en) * | 2021-01-14 | 2021-04-27 | 中山大学 | Proteome obtained by splitting Cas9 protein and application thereof |
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