TWI640628B - hSlu7 TRANSGENIC CELLS FOR PROMOTING INFLUENZA VIRUS REPLICATION - Google Patents

Abstract

The invention discloses a plastid for promoting the proliferation of an influenza virus, wherein the human Slu7 gene fragment carried by the plastid is inserted into the chromosome of the avian cell or the chromosome of the chicken embryonic spinal cord, so that the avian cell or the chicken embryo is infected with the influenza virus. After infection, the human Slu7 gene fragment promotes gene cleavage of influenza virus M mRNA, increases the production of ion channel protein M2, and promotes a large proliferation of influenza virus, which will accelerate the production rate of influenza vaccine and effectively reduce the manufacturing cost of influenza vaccine.

Description

hSlu7 gene transfer cell that promotes influenza virus replication

The invention relates to a human Slu7 ( hSlu7 ) gene vector, a transgenic cell and a vaccine.

Influenza (referred to as influenza) is a respiratory disease caused by an influenza virus. The influenza virus is usually transmitted by mucus such as mouth, sneezing or nasal discharge. The clinical manifestations of severe cases of influenza are early symptoms of acute respiratory infection such as fever, cough and shortness of breath, and then rapidly progress to severe pneumonia, possibly accompanied by acute respiratory distress syndrome, septic shock and multiple organ failure, and mild cases. Clinical manifestations include conjunctivitis and flu-like symptoms.

The influenza virus belongs to the Orthomyxoviridae virus family and is a negative sense single-stranded RNA virus, which can be classified into types A, B and C. Influenza A and B viruses are susceptible to human infection, while influenza C viruses are less prone to pandemics and milder symptoms. As far as the host of the virus is concerned, human beings are the only host of influenza B virus. However, influenza A viruses, which have historically been prone to pandemic and have severe symptoms, can be hosted in humans, or mammals such as horses and pigs, and poultry (such as bird flu). ). The most prevalent influenza A virus of influenza virus has many different subtypes, which are distinguished by a combination of two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of the virus. At present, influenza A virus can be divided into 18 H types and 11 N types, of which H1 ~ H16 subtypes are mainly prevalent in poultry, while H17 and H18 are prevalent in bats. According to the literature, it is currently determined that the subtypes of influenza A virus that have been infected with humans include H1N1, H3N2v, H5N1, H5N6, H6N1, H7N2, H7N3, H7N7, H7N9, H9N2, H10N7 and H10N8, among which H5N1 was first appeared in 1997. Type and the H7N9 subtype found in 2013 caused serious human diseases with a mortality rate of approximately 60% and 30%; conversely, H7N3 and H9N2 subtypes only caused mild or asymptomatic infections in humans.

In the molecular biology study of influenza virus, each viral RNA fragment of the negative single-stranded RNA segment of the influenza virus is contained in the viral ribonucleoprotein complex (vRNP), and vRNP is It is packaged by a viral nucleoprotein (NP) associated with three viral proteins (type A and B influenza viruses are proteins PB1, PB2, and PA; and type C influenza viruses are proteins PB1, PB2, and P3). An RNA-dependent RNA polymerase (RdRp) is formed. For all influenza viruses, after the influenza virus enters the host cell, the incoming vRNP is transported to the nucleus where they are transcribed into viral mRNA and by viral polymerase and host RNA polymerase II and transcriptosome mechanisms. The support is replicated as a new viral RNA. Type A and B influenza viruses have eight viral RNA segments encoding up to 17 and 11 viral proteins, respectively, while influenza C viruses have seven viral RNA segments encoding nine viral proteins. . In the case of influenza A virus, segment 2 encodes the polymerase basic proteins PB1, PB1-F2, and PB1-N40 by using a selective translation initiation site; segment 3 is ribosomal by ribosomal Frameshift) and two additional N-terminally cleaved proteins (PA-N155 and PA-N182) using a selective translation initiation site encoding the polymerase acidic proteins PA and PA-X; segment 7 encoding the matrix protein M1 and Ion channel proteins M2 and M42; segment 8 encodes the non-structural protein NS1, nuclear export proteins NS2/NEP and NS3 by selective mRNA splicing.

Influenza A, which is often administered by doctors to influenza patients, is Influenza® (Tamiflu®, main ingredient is oseltamivir phosphate, oral, Roche Pharmaceuticals Co., Ltd.), Relenza® (Relenza®, main ingredient) It is zenamivir, inhaler, GlaxoSmithKline Pharmaceutical Co., Ltd.), Rapiacta® (main ingredient is peramivir hydrate, intravenous injection, 塩野义制药股份有限公司) and Avigan® (main ingredient is favipiravir, Oral agent, Toyama Chemical Industry Co., Ltd.).

Because humans have suffered from large-scale influenza viruses (such as the H1N1 influenza A that broke out in 1918), which caused about 50 to 100 million deaths, governments have used flu vaccines to prevent influenza outbreaks. . At present, influenza vaccines are classified into two types: inactivated trivalent influenza vaccine and active attenuated influenza vaccine. The influenza vaccine production method is divided into the use of chicken embryo egg to culture the virus, then deactivated or attenuated to make a vaccine, and the virus is produced by cell culture to prepare a vaccine.

The first method is to implant a virus strain that may cause epidemic into chicken embryo eggs, culture a large amount of virus liquid, and then purify and attenuate the virus liquid to prepare a flu vaccine. This method requires the selection of fertilized eggs from special breeds and strictly reared chickens (about 28 to 35 weeks old), and it is necessary to confirm that the chicken embryo eggs are not contaminated, resistant or low-resistance, so as to avoid the injection of influenza virus. Killed by the chicken embryo's own resistance. A chicken embryo egg can produce about one to three doses of a vaccine. The influenza virus strain needs to be domesticated in the chicken embryo egg to successfully proliferate the virus, and the total process takes about six months. In addition, if the avian flu or other subtype influenza outbreaks that are not predicted, the flu vaccine will take too long to produce a flu vaccine due to the inability of the chicken embryo to supply the flu vaccine. Moreover, bird flu may invade chickens and reduce the source of chicken embryonic eggs, making the flexibility of producing embryo vaccines for chicken embryos to be quite limited. Therefore, the preparation of the influenza vaccine in the first manner will encounter the disadvantages of high production costs, insufficient supply of chicken embryonic eggs and limited vaccine production to cope with the large-scale outbreak of influenza.

The second method is to infect influenza virus strains with mammalian cells, such as MDCK-Darby canine kidney cells and Vero African green monkey kidney epithelial cells, and influenza viruses in large bioreactors (such as stainless steel tanks, In mammalian cells in disposable plastic containers, bags, etc., they are cultured, propagated, and released, and the virus in the cell culture solution is collected, and the vaccine is prepared by inactivation. The second embodiment of the cell culture medium may be a serum-free medium or a higher-risk serum-containing medium. In the second way, compared with the first method, there is no risk that the material (chicken embryo eggs) is not available and the vaccine is not produced, and avoids The allergic problem of ovalbumin, so cell culture production vaccines will become a trend.

In view of the deficiencies in the prior art, the applicant of this case, after careful experimentation and research, and a perseverance spirit, finally conceived the case and can overcome the shortcomings of the prior art. The following is a brief description of the case.

In order to effectively increase the proliferation of influenza virus and accelerate the production rate of influenza vaccine, the present invention will select a human Slu7 ( hSlu7 ) gene fragment which facilitates splicing of influenza virus M mRNA gene into avian cells with a trace amount of influenza virus. Infecting avian cells carrying a human Slu7 gene fragment, which facilitates gene cleavage of influenza virus M mRNA through the human Slu7 gene fragment, thereby increasing the production of ion channel protein M2, which can contribute to the proliferation of influenza virus, which will accelerate The rate of production of influenza vaccines and the effective reduction of the cost of manufacturing influenza vaccines.

Accordingly, the present invention discloses a plastid for promoting the proliferation of influenza virus, comprising: a vector and a human Slu7 gene fragment. The vector includes a CMV promoter and a recombinant target site, and the human Slu7 gene fragment includes an antisense strand and a translation initiation point on the antisense strand, wherein the human Slu7 gene fragment is located between the CMV promoter and the recombination target site, and the translation is performed. The starting point is located downstream of the CMV promoter.

In a specific embodiment, the vector further comprises a first restriction enzyme cleavage site between the CMV promoter and the human Slu7 gene fragment and a second restriction enzyme cleavage site between the human Slu7 gene fragment and the recombinant target site. . In a specific embodiment, the recombination target site is configured to be recognized by the recombinase together with the recombination target site on the chromosome of the cell, and the plastid is inserted into the chromosome. In a specific embodiment, the aforementioned cell is an avian cell, the recombinant target site is a Flp recombination target site, and the recombinase is a Flp recombinase. In a specific embodiment, the human Slu7 gene fragment further comprises a sense strand that is base paired with the antisense strand.

The invention also discloses a system for promoting the proliferation of influenza virus, comprising avian host cells, the aforementioned plastids for promoting the proliferation of influenza viruses, first plastids and second plastids. The avian host cell includes a chromosome and a first plastid inserted into the chromosome, and the first plastid includes a first recombination target site. The aforementioned plastids that promote the proliferation of influenza virus are located in avian host cells and include a human Slu7 gene fragment and a second recombination target site. The second plastid is located in an avian host cell and includes a recombinase gene fragment for expression of the recombinase. Wherein the recombinase is used to perform recombination between the first recombination target site of the first plastid and the second recombination target site of the plastid, such that the plastid and human Slu7 gene segments are inserted into the chromosome, and in the avian host cell After infection with influenza virus, the human Slu7 gene fragment promotes the proliferation of influenza virus.

The transposon (also known as the jumper) was first discovered in corn in 1947 by Barbara McClintock. Transposables can automatically translocate between genes and insert genes to disrupt the performance of the gene, which in turn affects trait expression. Fish first discovered transposons is Tol2 elements from the Green Qiang fish (medaka fish, Oryzias latipes) of (Tol2 element). At present, although vertebrate chromosomes contain a large number of sequences related to DNA transposons, these DNA transposons are not inherently active, and therefore these DNA transposons are considered to be non-autonomous elements. However, the Tol2 element is an autonomous transposon that has a length of about 4.7 Kb (actually 4682 base pairs) and is capable of self-translating a transposase protein consisting of four exons (exon) ( Transposase protein). The mRNA transcribed from the Tol2 gene can be translated into a protein of 649 amino acids in length, and the transposase protein has a complete function. The Tol2 elements are merged into a single copy by a scraping mechanism, and the Tol2 element does not result in any recombination or modification of the target gene locus.

It has been studied that Tol2 hops containing specific 200 and 150 base pair sequences in the upstream and downstream can be effectively used for translocation, but if the upstream and downstream are less than 150 and 100 base pairs respectively. The plastid will not be used for translocation (Urasaki A. et al., Genetics, 2006, 174: 639-649; herein incorporated by reference).

The Tol2 transposon can be applied to a variety of vertebrates. In the present invention, the Tol2 transposon allows the target gene to be continuously expressed in chicken embryo eggs, unlike the conventional electroporation technique in which the target gene is delivered into chicken embryo eggs for early and transient performance.

Therefore, the present invention also discloses another plastid for promoting the proliferation of influenza virus, including a vector and a human Slu7 gene fragment. The vector includes a first transposon and a second transposon, and the human Slu7 gene fragment includes an antisense strand and a translation starting point on the antisense strand, wherein the human Slu7 gene fragment is located between the first transposon and the second transposon, And the translation starting point is located downstream of the first transposition.

In a specific embodiment, the first transposon and the second transposon are Tol2 transposons, and the nucleotide sequences and lengths of the first transposon and the second transposon are different from each other.

The invention successfully constructs a human Slu7 gene vector capable of translocating a Tol2 transposon, and when the chicken embryo egg is infected by the influenza virus, can express a large amount of influenza virus protein in the chicken embryo egg, thereby promoting the assembly of influenza virus particles. And production system.

The invention also discloses a method for preparing an influenza vaccine, comprising: (a) providing a chicken embryo egg comprising an embryo and a protein, the embryo having a chromosome; and (b) a plastid that promotes the proliferation of the influenza virus (band) There are first and second transposons) and influenza virus injected into the embryo, in which the plastid human Slu7 gene fragment is inserted into the embryo's chromosome; (c) hatching the chicken embryo egg, promoting the influenza with the human Slu7 gene fragment The virus proliferates and is released into the protein; and (d) collects the protein and purifies or deactivates the influenza virus that proliferates in the protein to prepare an influenza vaccine.

In a specific embodiment, step (b) is to inject the plastid and influenza virus into the embryo via microinjection techniques or electroporation techniques.

The invention also discloses the use of a plastid comprising a human Slu7 gene sequence for promoting influenza virus proliferation and for preparing an influenza vaccine using a proliferating influenza virus. The invention also discloses the use of a plastid comprising a human Slu7 gene sequence for promoting influenza virus proliferation. The invention also discloses a plastid for promoting the proliferation of influenza virus, including the human Slu7 gene sequence.

Influenza viruses suitable for use in the influenza virus suspension of the present invention include, but are not limited to, A/WSN/33, A/PR8/34, etc., or modified based on the aforementioned genomic body of the influenza virus. flu virus.

1‧‧‧Chromosomes with human Slu7 gene fragment

2, 3‧‧ ‧ plastid

Fig. 1 is a flow chart showing the preparation of Flp-In-DF-1 cells which can express a human Slu7 gene fragment according to a first embodiment of the present invention.

Fig . 2 is a view showing the results of a plaque assay of Flp-In-DF-1 cells expressing a human Slu7 gene fragment infected with influenza virus in the first embodiment of the present invention.

Figure 3 is a schematic diagram showing the vector carrying the Tol2 transposon and the hSlu7 gene fragment and the promoter CAGGS in the second embodiment of the present invention.

Fig. 4 is a view showing the results of microinjection, electroporation test of chicken embryonic egg plastid DNA, and plaque test after influenza virus infection in the second embodiment of the present invention.

Fig. 5 (a), Fig. 5 (b) and Fig. 5 (c) are respectively the pCAGGS-T2TP plastid, the pT2K-CAGGS-EGFP plastid and the pT2K-CAGGS plastid in the second embodiment of the present invention. schematic diagram.

The inventions set forth in the present invention will be fully understood by the following examples, so that those skilled in the art can do so. However, the implementation of the present invention may not be limited by the following embodiments. Other embodiments may be devised by those skilled in the art in light of the spirit of the embodiments disclosed herein.

First, the system using Flp-In ^TM system that facilitates preparation of influenza virus proliferation:

A first embodiment of the present invention is to construct a Flp-In-DF-1 cell which can express a human Slu7 gene fragment (SEQ ID NO: 8) in influenza virus-infected avian cell Flp-In-DF-1 Later, it can promote the gene cleavage of influenza virus M mRNA, thereby increasing the production of ion channel protein M2, which can help to proliferate a large amount of influenza virus.

Please refer to Fig. 1, which is a flow chart showing the preparation of Flp-In-DF-1 cells which can express the human Slu7 gene fragment according to the first embodiment of the present invention. In Figure 1, the pFRT/lacZeo plastid (with FRT recombination site) linearized by restriction enzyme cleavage was transfected into chicken fibroblast DF-1 and inserted into the chromosome of chicken fibroblasts. The pcDNA5/FRT-hSlu7 plastid with the target gene of interest (human Slu7 gene fragment) and the FRT recombination site, and the commercialized pOG44 plastid are then co-transfected into the aforementioned linearized pFRT/lacZeo plastid. Chicken fibroblasts. Recombination of the FRT recombination site on the pcDNA5/FRT-hSlu7 plastid with the FRT recombination site on the linearized pFRT/lacZeo plastid of chicken fibroblasts through the FLP gene expression on the pOG44 plastid, making human Slu7 The gene fragment is inserted into the chromosome of the chicken fibroblast to form chromosome 1 with the human Slu7 gene fragment. The chicken fibroblast stably exhibits a human Slu7 gene fragment. The following are the experimental methods and results of the first embodiment of the present invention.

(I) Establishment of Flp-In-DF-1-n cells:

1. Transfection test of linearized pFRT/lacZeo plastid:

Containing 10μg pFRT / lacZeo expression vector DNA (Flp-In ^TM system, Invitrogen, USA), 1μL ScaI restriction enzyme, 10 × buffer and added deionized water to a volume of 150μL of the reaction mixture placed in a reaction 37 ℃ 4 hours, The linearized pFRT/lacZeo plastids were purified using a DNA purification/extract kit (DP034-300, Taiwan Jen Micro Biotech).

Next, chicken fibroblast cell line DF-1 (5 × 10 ⁵ cells/well, ATCC® CRL-12203) was inoculated into a 6-well culture dish and cultured overnight in a 37 ° C incubator. When the cell density reached about 90% to 95%, the linearized pFRT/lacZeo plastid was transfected into DF-1 cells with the Lipofectamine 2000 reagent group (Invitrogen, USA). Specifically, 2.5 μg of pFRT/lacZeo plastid was mixed with Opti-MEM medium to a volume of 250 μL, 5 μL of Lipofectamine 2000 reagent and Opti-MEM medium were mixed to a volume of 250 μL, and the mixture of the two tubes was mixed for 5 minutes, and then added to the cavity. Transfection is carried out. After 24 hours of transfection, transfected DF-1 cells were harvested by trypsinization, and transfected DF-1 cells were seeded at 750 cells/well (with 500 μg/mL giomycin). (zeocin) in Dulbecco's Modified Eagle Medium (DMEM) medium was subcultured to a 6-well culture plate, and the culture medium was changed every 3 to 4 days until cell colony formation was observed by the naked eye. Individual cell colonies were selected and expanded for culture, subculture and cryopreservation. The cell line screened by giomycin was named Flp-In-DF-1-n cells, and n was an integer number from 1 to x.

2. Extraction of genomic DNA:

In the charge covered with trypsin in 10 cm culture dishes Flp-In-DF-1- n cells, according QIAamp® DNA mini kit (QIAGEN) Manual extraction per unit of 5 × 10 ⁶ genomic DNA of the cell.

3. The FRT and lacZ region sequences of pFRT/lacZeo plastids were amplified by polymerase chain reaction to confirm that the pFRT/lacZeo plastid was inserted into the chromosome of Flp-In-DF-1-n cells:

The test was carried out by polymerase chain reaction (PCR) and KOD-Plus-PCR reagent set of Taiwan Baili Biotechnology Co., Ltd. 1 μL of template (pFRT/lacZeo plastid), 1.5 μL of FRT-Forward primer (5'-TCA CCG TCA TCA CCG AAA CG-3'; SEQ ID NO: 1; 10 pmol/μL), 1.5 μL FRT-Reverse primer ( 5'-GTAACG CCA GGG TTT TCC CA-3'; SEQ ID NO: 2; 10 pmol/μL), 5 μL dNTP (2 mM), 2 μL MgSO ₄ (25 mM), 5 μL 10×KOD-Plus-buffer, 1 μL KOD- The reaction mixture of plus-(1.0 U/μL) and 33 μL of deionized water was denatured at 94 ° C for 2 minutes, and then reacted at 94 ° C for 15 seconds, 55 ° C for 30 seconds, and 68 ° C for 30 seconds (35 cycles total), and finally The FRT and lacZ region sequences of the pFRT/lacZeo plastids were amplified by a program extending at 68 °C for 5 minutes. The PCR amplification product was analyzed by 1.5% agarose gel electrophoresis, and it was confirmed that a band of about 500 bp was amplified (results not shown), which was consistent with the experimentally designed 496 bp FRT and lacZ region length, and pFRT/lacZeo plastid insertion was confirmed. The chromosome of Flp-In-DF-1-n cells.

4. Analysis of β-galactosidase activity:

In this test, β-galactosidase activity analysis was performed according to the β-Gal test kit (K1455-01, US Invitrogen) operating manual. In detail, 10 μL of cell lysate was added to sterile water to a volume of 30 μL, and then 70 μL of ortho-nitrophenyl-β-galactoside (ONPG) and 2-mercaptoethanol were added. (β-mercaptoethanol) 200 μL of cleavage buffer was applied at 37 ° C for 30 minutes. If β-galactosidase is present, it will appear yellowish. 500 μL of Stop Buffer was added to stop the reaction. The absorbance was read at a wavelength of 420 nm, and the sample containing ONPG and the cleavage buffer was used as a blank test group, and the undissolved cell lysate was used as a control group. The specific activity of the cell lysate is calculated by the formula "hydrolyzed ONPG (nmole) / t / protein content (mg)", where t is the action time of 37 ° C, that is, 30 minutes; the hydrolyzed ONPG (nmole) is given the formula "(420 nm) Absorbance value x (8 x 10 ⁵ nL) / (4500 nL / nmole-cm) (1 cm)", where 4500 is the extinction coefficient; protein content (mg) is determined by protein quantification method well known in the art. According to the β-galactosidase activity level and cell type (results not shown), Flp-In-DF-1-19, Flp-In-DF-1-16, Flp-In-DF-12-22 and Flp-In-DF-1-8 cells were subjected to subsequent experiments.

(2) Establishment of a Flp-In-DF-1 cell line expressing a human Slu7 gene fragment:

1. Establishment of a human Slu7 gene fragment with BamHI and NotI restriction enzyme cleavage:

The test was carried out by PCR and the KOD-Plus-PCR reagent set of Taiwan Baili Biotechnology Co., Ltd. 1 μL of template (human Slu7 gene fragment; SEQ ID NO: 8), 1.5 μL hSlu7-BamHI-Forward primer (5'-CGG GAT CCA TGT CAG CCA CAG TTG-3'; SEQ ID NO: 3; 10 pmol/μL; Sequence 5'-GGATCC-3' with BamHI restriction enzyme cleavage, 1.5 μL hSlu7-NotI-Reverse primer (5'-TTT TCC TTT TGC GGC CGC CTA CTG TCC AAG-3, SEQ ID NO: 4; 10 pmol/μL; sequence 5'-GCGGCCGC-3' with NotI restriction enzyme cleavage, 5 μL dNTP (2 mM), 2 μL MgSO ₄ (25 mM), 5 μL 10×KOD-Plus-buffer, 1 μL KOD-plus- The reaction mixture of (1.0 U/μL) and 33 μL of deionized water was denatured at 94 ° C for 2 minutes, and further reacted at 94 ° C for 15 seconds, 53 ° C for 60 seconds, 68 ° C for 150 seconds (35 cycles total), and finally at 68 ° C. The 5 minute extension program amplifies sequences with BamHI and NotI restriction enzyme cleavage sites before and after the human Slu7 gene fragment. The PCR amplification product was analyzed by 1.5% agarose gel electrophoresis, and the PCR amplification product was purified by DNA purification/extraction kit (DP034-300, Taiwan Jenmark Biotech Co., Ltd.).

2. Adhere the human Slu7 gene fragment with BamHHI and NotI restriction enzyme sites to pcDNA5/FRT plastid DNA:

The human Slu7 gene fragment with BamHI and NotI restriction enzyme cleavage sites and pcDNA5/FRT plastids were performed using restriction enzymes, T4 adhesion enzyme products and an operating manual sold by New England Biolabs (NEB). DNA cutting and bonding. Will contain 5 μg of DNA (human Slu7 gene fragment with BamHI and NotI restriction enzyme sites and pcDNA5/FRT plastid DNA, respectively), 5 μL of 10×3 buffer, 1 μL of BamHI, 1 μL of NotI and supplemented with deionized water volume to 50 μL of the reaction mixture was reacted at 37 ° C for 24 hours, and each DNA fragment was separated by electrophoresis on a 1% agar gel. The colloid of the desired fragment size was cut under a UV lamp, and then the QIAquick® gel extraction kit (28704, QIAGEN) was used. The desired DNA fragment is purified by colloid. The human Slu7 gene fragment with BamHI and NotI binding ends was mixed with pcDNA5/FRT plastid DNA in a ratio of 1:3 to form a mixture, and the binding reaction was carried out with T4 binder at room temperature for 2 hours. Contains pcDNA5/FRT-hSlu7 plastid DNA.

3. plastid transformation and expression in Escherichia coli DH5α:

The mixture after completion of the adhesion reaction was added to DH5α competent cells for plastid transformation, and then uniformly coated on the surface of agar medium containing 100 μg/mL ampicillin. After culture, several single colonies were selected for small-scale cultivation. The plastid purification kit (DP01MD-100, Taiwan Jenmark Biotech) was used to extract a small amount of plastid DNA from a single colony, and DNA sequencing analysis was performed to identify pcDNA5/FRT-hSlu7 plastids in a single colony. DNA. The primers for DNA sequence analysis were CMV-Forward primer (5'-AGA CGC CAT CCA CGC TG-3'; SEQ ID NO: 5) and BGH pA-Reverse primer (5'-CAA CAG ATG GCT GGC AAC-3'; SEQ ID NO: 6). The pcDNA5/FRT-hSlu7 plastid DNA was retransformed into DH5α competent cells, and a large amount of pcDNA5/FRT-hSlu7 plastid DNA was extracted and extracted.

4. pcDNA5/FRT-hSlu7 plastid DNA and Flp recombinase expression vector pOG44 were co-transfected into Flp-In-DF-1 cell line:

First, Flp-In-DF-1-n cells (5 × 10 ⁵ cells/well) were inoculated to a 6-well culture dish, and cultured overnight in a 37 ° C incubator. When the cell density reached about 90% to 95%, the pcDNA5/FRT-hSlu7 plastid DNA and the Flp recombinase expression vector pOG44 were expressed in Lipofectamine 2000 reagent group (Invitrogen, USA) (the ratio was 1:9 (w/w), A total of 3 μg) was co-transfected into Flp-In-DF-1-n cells. Specifically, 0.3 μg of pcDNA5/FRT-hSlu7 plastid DNA, 2.7 μg of Flp recombinase expression vector pOG44 and Opti-MEM medium were mixed to a volume of 250 μL, and 5 μL of Lipofectamine 2000 reagent and Opti-MEM medium were mixed to a volume of 250 μL. After mixing the above two tube mixtures for 5 minutes, they were added to the holes for transfection. After 24 to 48 hours of co-transfection, co-transfected cells were harvested by trypsin and co-transfected cells were seeded at 750 cells/well (with 400 μg/mL wetmycin (Hygromycin) The DMEM medium of B) was subcultured to a 6-well culture plate, and the culture solution was changed every 3 to 4 days until cell colony formation was observed by the naked eye. Individual cell colonies were selected and expanded for culture, subculture and cryopreservation. The cell line screened by hygromycin was named Flp-In-DF-1-hSlu7-n cells, and n was an integer number from 1 to x.

5. Identification of Flp-In-DF-1-hSlu7-n cell line:

Flp-In-DF-1-hSlu7-n cells (1.25 × 10 ⁵ cells/well) were inoculated into a 6-well culture dish and cultured overnight in a 37 ° C incubator. On the second day, the culture solution was changed to a DMEM medium containing 500 μg/mL of giomycin, and the culture solution was changed every 3 to 4 days to observe the cell survival rate. Finally, several strains of Zeocin positive and hygromycin B positive were screened, and Flp-In-DF-1-hSlu7-16 cells were used (exposure book is Flp-In-DF- 1-hSlu7-16-4 cells are representative.

(3) plaque test:

Chicken fibroblast cell line DF-1 (control group), Flp-In-DF-1-16 (control group, giomycin-positive) and Flp-In-DF-1-hSlu7-16 cells (experimental group) The cells were seeded in a 6-well culture plate at a cell volume of 5 × 10 ⁵ cells/well, and cultured overnight in a 37 ° C incubator. When the cell density reaches about 90% to 95%, the culture solution is aspirated and the cells are washed twice with 1× phosphate buffer (PBS), and then vaccinated with a multiplicity of infection (MOI) of 0.001 PFU/cell. Virus suspension (containing influenza virus A/WSN/33). After the influenza virus was adsorbed to the cells at 37 ° C for 1 hour, the cells were washed once with 1 × PBS, and serum-free cell culture medium was added thereto, and cultured in a 37 ° C incubator. The supernatant was collected after 24, 48 and 72 hours of culture, and the viral power was quantified by a plaque assay well known in the art.

Please refer to Fig. 2, which is a result of a plaque assay of Flp-In-DF-1 cells expressing a human Slu7 gene fragment infected with influenza virus in the first embodiment of the present invention. The viral power of Flp-In-DF-1-hSlu7-16 cells infected with influenza virus A/WSN/33 48 and 72 was higher than that of influenza virus A/WSN/33 infected with Flp-In-DF-1- 16 cells (control group), indicating that the transgenic cells carrying the human Slu7 gene fragment of the present invention can promote influenza virus replication, and the transgenic cells carrying the human Slu7 gene can serve as a platform for promoting the proliferation of influenza viruses, and the proliferating influenza virus Further manufactured into a flu vaccine.

Second, the use of Tol2 jumper to prepare a system to promote the proliferation of influenza virus:

(I) Preparation of plastids:

The pCAGGS-T2TP plastid (as shown in plastid 2 in Figure 3), pT2K-CAGGS-EGFP plastid (selective) and pT2K-CAGGS-hSlu7 plastid (as shown in plastid 3 in Figure 3) The mixture was mixed at a weight ratio of 1:2:2, and pCAGGS-T2TP plastid, pT2K-CAGGS-EGFP plastid (selective) and pT2K-CAGGS plastid were mixed at a weight ratio of 1:2:2. That is, 100 μg, 200 μg, and 200 μg of the above plastid DNA were mixed with a conventional Tris-EDTA (TE) buffer to a final volume of 500 μL, and then 1/10 volume of 50 μL of 3 M sodium acetate (pH 5.2) was added. And 2.5 times the volume of 1250 μL of 100% ethanol, placed at -80 ° C for 1 hour to overnight. Thereafter, the cells were centrifuged at 13,000 × g for 10 minutes at room temperature, and the plasmid DNA was washed with 2 mL of a 70% ethanol solution, and then centrifuged at 13,000 × g for 10 minutes at room temperature. The 70% ethanol solution was aspirated, and the pellet in the microcentrifuge tube was air-dried at room temperature for 5 to 10 minutes. Finally with 4μL (5%) A 200 μL Hank's balanced salt solution (HBSS) of fast green was used to suspend plastid DNA.

Among them, pCAGGS-T2TP plastid (as shown in Figure 5 (a), SEQ ID NO: 7, Kawakami et al., Genetics, 2004, 166 (2): 895-899) and pT2K-CAGGS-EGFP plastid (As shown in Figure 5(b), Sato et al., Genomes & Developmental Control, 2007, 305(2): 616-624) is from Dr. Kawakami, Kyoto University, Japan (see also http:// Kawakami.lab.nig.ac.jp/trans.html), pT2K-CAGGS-hSlu7myc plastids were constructed using the pT2K-CAGGS plastids of the above laboratory. The pT2K-CAGGS plastid (as shown in Figure 5(c)) was used as a control group with only the Tol2 element at the left and right ends and the promoter CAGGS located between the two Tol2 elements (Niwa et al., Gene, 1991, 108: 193-199), without any target genes to be expressed. The pT2K-CAGGS-hSlu7myc plastid was constructed by binding a human Slu7 gene fragment (SEQ ID NO: 9) having a BglII restriction enzyme cleavage to a pT2K-CAGGS plastid having a BglII restriction enzyme cleavage site. The foregoing plastids can be prepared by conventional molecular biology techniques known to those skilled in the art, or obtained from the aforementioned laboratories and papers, without the need for registration of biological materials. The aforementioned plastids and related literature are incorporated herein by reference.

(II) Microinjection and electroporation tests:

In this experiment, the plastid DNA was microinjected into the spinal cord of chicken embryo eggs for 2 days (2 days old), and the probability of plastid DNA being sent to chicken embryo cells was increased by electroporation. First, the chicken embryo eggs are placed horizontally so that the chicken embryos in the chicken embryo eggs are in an upward position, and the local eggshells are removed. A sterile 18G needle was attached to a 10 mL syringe, and the needle was inserted into the chicken embryo egg at an oblique downward angle, and about 5-10 mL of albumin (protein fraction) was withdrawn, but no part of the yolk was removed. 5 μL of the plastid DNA mixture was injected into the neural tube chamber of the spinal cord of each chicken embryo with a glass needle. The glass needle was removed and the electrodes were placed on the left and right sides of the chicken embryo. A shock of 50 milliseconds (msec), a pause of 950 milliseconds, exchange of positive and negative electrodes, and then electric shock, pause, exchange of electrodes (a total of 5 shocks), each shock applied 20 volts to the chicken embryo. The chicken embryo eggs are then covered with cling film and the chicken embryo eggs are continuously hatched. On the 9th day of fertilization (9 days old), the flu is known In the course of the vaccine process, the influenza virus is injected into the chicken embryo, and the influenza virus is collected on the 11th day of fertilization (11 days old) in the conventional influenza vaccine process.

Please refer to FIG. 4, which is a microinjection, electroporation test, and plaque test of influenza virus after infection of chicken embryo eggs in the second embodiment of the present invention. In Figure 4, the experimental group (pT2K-CAGGS-hSlu7 plastid) has a higher flu virus price than the illuminating group (pT2K-CAGGS7 plastid), indicating that the vector carrying the human Slu7 gene and the Tol2 transposon will promote Chicken embryo eggs are infected with influenza virus and produce a large amount of influenza virus, so that these large amounts of influenza virus can be further prepared as influenza vaccine.

In addition, in another specific embodiment, after microinjection and electroporation test of 2 day old chicken embryo eggs, the expression of human Slu7 protein can be observed in 9-day-old chicken embryo eggs after electroporation for 7 days (results not show). Therefore, the present invention can perform microinjection and electroporation on the insemination days of 2 days of age, so that the future chicken embryo eggs can express human Slu7 protein. Since the chicken embryo egg can express the human Slu7 protein, the chicken embryo egg is infected by the influenza virus, and the human Slu7 gene can help the gene cleavage of the influenza virus M mRNA, increase the production of the ion channel protein M2, and contribute to the proliferation. Influenza virus and speed up the production of influenza vaccines.

The present invention utilizes the advantages of the Tol2 transposon ( jumper ): 1) translocation in vertebrates; 2) translocation in developing embryos; 3) adaptation to 37-38 ° C in chicken embryo hatching; 4) Random insertion into any site of the chromosome; 5) Inserting the gene in a single copy and not modifying and recombining at the insertion site, allowing the human Slu7 gene to promote gene cleavage and increase of the influenza virus M mRNA The production of channel protein M2 helps to proliferate influenza virus in chicken embryo eggs and accelerate the production rate of influenza vaccine.

The invention is a difficult and innovative invention, and has profound industrial value, and is submitted in accordance with the law. In addition, the present invention may be modified by those skilled in the art without departing from the scope of the appended claims.

<110> Chang Gung University

<120> hSlu7 gene transfer cell promoting influenza virus replication

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<221> FRT-Forward primer

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<221> FRT-Reverse primer

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<221> hSlu7-BamHI-Forward primer

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<221> hSlu7-NotI-Reverse primer

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<221> BGH pA-Reverse primer

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<221> pCAGGS-T2TP plastid

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<221> Human Slu7 gene fragment

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Claims (7)

A system for promoting the proliferation of an influenza virus, comprising: a bird host cell comprising a chromosome and a first plastid inserted into the chromosome, the first plastid comprising a first recombinant target site; a Slu7 recombination a plastid in the avian host cell and comprising a human Slu7 gene fragment, a promoter operably linked to the human Slu7 gene fragment, and a second recombination target site; and a second plastid And a recombinant enzyme gene fragment for expressing a recombinase, wherein the recombinase is used to perform a first recombination target site of the first plastid and a second recombination target site of the Slu7 recombinant plastid Recombination between the spots , the human Slu7 gene fragment and the promoter in the Slu7 recombinant plastid are inserted into the chromosome, and the human Slu7 gene fragment promotes the epidemic after the avian host cell is infected with the influenza virus The proliferation of cold viruses. The system of claim 1, wherein the recombination target sites are Flp recombination target sites, and the recombinase is Flp recombinase. A method for preparing an influenza vaccine, comprising: (a) providing a chicken embryo egg comprising an embryo and a protein having a chromosome; (b) delivering a Slu7 translocation plastid and an influenza virus into the embryo, wherein the indexing Slu7 plastid transposon comprises a first, a second and a human Slu7 transposon gene fragment, the human gene fragment Slu7 Slu7 translocation plastid is transmitted through the first transposon And transposing the second transposon into the chromosome of the embryo; (c) incubating the chicken embryo egg to release the human Slu7 gene fragment to promote the proliferation of the influenza virus and releasing the protein into the protein; (d) collecting the protein and purifying or deactivating the influenza virus proliferating in the protein to prepare the influenza vaccine. The method of claim 3, wherein the first transposon and the second transposon are Tol2 transposons, and the nucleotide sequences and lengths of the first transposon and the second transposon are mutually different. The method of claim 3, wherein the step (b) is to deliver the Slu7 translocation plastid and the influenza virus into the embryo by microinjection technique or electroporation technique. A use of a plastid comprising a human Slu7 gene sequence for promoting influenza virus proliferation and for preparing an influenza vaccine using a proliferating influenza virus. A use of a plastid comprising a human Slu7 gene sequence for promoting influenza virus proliferation.