CN106591370A - Virus vector for treating autoimmune related diseases and diabetes, construction method and applications thereof - Google Patents
Virus vector for treating autoimmune related diseases and diabetes, construction method and applications thereof Download PDFInfo
- Publication number
- CN106591370A CN106591370A CN201510676799.0A CN201510676799A CN106591370A CN 106591370 A CN106591370 A CN 106591370A CN 201510676799 A CN201510676799 A CN 201510676799A CN 106591370 A CN106591370 A CN 106591370A
- Authority
- CN
- China
- Prior art keywords
- diabetes
- mfat
- viral vector
- expression plasmid
- plasmids
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The present invention discloses a virus vector, wherein a lentivirus expression plasmid cloned with an mfat-1 gene or an adeno-associated virus expression plasmid cloned with an mfat-1 gene is transfected into 293FT cells to obtain the virus vector. The invention further discloses a construction method and applications of the virus vector. The invention provides a biological medicine for treating diabetes and autoimmune related diseases, wherein the biological medicine is a safe and non-toxic novel biological medicine with anti-inflammatory activity, the lentivirus and the adeno-associated virus (AAV) are adopted as the vector, the mfat-1 gene is highly expressed by using the gene recombination technology, and the biological medicine can significantly resist autoimmune and inflammations, has a blood glucose lowering effect, and can meet the clinical needs. According to the present invention, the preparation method is simple and easy to operate; and the virus vector has good application prospects.
Description
Technical field
The present invention relates to the medicine of diabetes and autoimmune disease, and in particular to one kind treats diabetes and autoimmune
The virion and its construction method of relevant disease and application.
Background technology
Diabetes be one group as defect of insulin secretion or its biological agent are impaired, or both have concurrently cause with hyperglycemia
The metabolic disease being characterized.Long-standing hyperglycemia, cause various tissues, particularly eye, kidney, heart, blood vessel,
The chronic lesion of nerve, dysfunction.Exist according to China recently《JAMA》With《New England Journal of Medicine》
On deliver epidemiological survey research show[1-2], China there are about 1.4 hundred million diabeticss at present, accounts for total population
11.6%, prediabeteses crowd is up to 50.1%, has been the first big disease for threatening national health.Clinically by sugar
Urine disease is divided into four classes, is type 1 diabetes, type 2 diabetes mellitus, its alloytype and gestational diabetes respectively.Wherein 1 type and 2
Patients with type Ⅰ DM is primary diabetes mellituss, accounts for the overwhelming majority, and usually said diabetes.
Type 1 diabetes are referred to as insulin dependent diabetes mellitus (IDDM) (insulin-dependent diabetes mellitus, IDDM),
For a kind of common organ specific autoimmune's property disease, be seriously jeopardize Children and teenager health chronic disease it
One.According to the assessment of ADA, about 5% diabetes patient is type 1 diabetes, and majority is in child or green grass or young crops
The morbidity of period in year.In child and adolescent patient, T1DM proportions are about 80-90%.Additionally, still there is a class
The Latent Autoimmune Diabetes in aldult (Latent Autoimmune Diabetes in Adults, LADA) of slow morbidity,
Also belong to autoimmune T 1DM in the cause of disease, but due to patient's onset age and clinical manifestation with type 2 diabetes mellitus class
Seemingly, easily by mistaken diagnosis.The absolute number of China type 1 diabetes patient is more than 1,000,000[2].It is whole that type 1 diabetes are fallen ill
During with a series of inflammation and immunologic derangement generation, be mainly shown as autoreactive T cell to beta Cell of islet
Attack and the generation of autoantibody cause islet beta cell function exhaustion, necrosis, cause the absolute of insulin secretion
Deficiency, such as treatment are not good at serious complication, such as blind, renal failure, heart disease, amputation etc. occurring[3]。
Type 2 diabetes mellitus are referred to as non-insulin-depending type (noninsulin-dependent diabetes mellitus, NIDDM),
The overwhelming majority accounted in diabetes.The insulin resistant synergy of different degrees of insulin deficit and tissue is to cause 2
The main pathogenesis of patients with type Ⅰ DM.Once type 2 diabetes mellitus are diagnosed, islet beta cell function progressive is reduced, liver
Glucose is generated and release increases, and causes blood glucose rise.
Either 1 type or type 2 diabetes mellitus, natural history all include the three below stage：1. hyperglycemia early stage, this phase
It is 10 years or so.The existing β cellular immunities destruction of type 1 diabetes, its outstanding feature is that GAD or ICA occur
Deng autoantibody.Type 2 diabetes mellitus are during this period without specific serologic marker, but are often accompanied by hyperinsulinemia and abdomen type
The insulin resistance syndromes such as obesity, islet function show impaired glucose tolerance (impaired glucose tolerance, IGT)
And (or) impaired fasting glucose (IFG) (Impaired fasting glucose, IFG).2. the hyperglycemia phase is on an empty stomach and post-prandial glycemia reaches
To the diagnostic criteria of diabetes.3. chronic complicating diseases phase.
In recent years, immunomodulating and inflammation obtain scientist with the dependency of diabetes and give more sustained attention, and part mechanism is obtained
To illustrate.The occurrence and development of type 1 diabetes have been dominated to the autoimmune response of Islet Antigen.Research shows, Th1
Cell transition polarization is to cause one of key factor of type 1 diabetes[4-6].For a long time, CD4+Responsiveness lymph is thin
Born of the same parents are divided into two big class of Th1 and Th2 always, and the two is mutually restricted by the cytokine secreted by which, in dynamic
State poised state.After certain β cell autoantigens are processed by macrophage or other antigen presenting cells, with MHC
Submission, to antigen presenting cell surface, causes the release of autoimmune signal to II molecules together, so as to activate Th1 cells,
Suppress Th2 cells and its release of cytokines, make Th cells dominant with Th1 cell phenotypes, Th1 cells are further
Activating cytotoxic macrophage, cytotoxic T cell (CD8+T cell) and natural killer cell (NK cells),
Impaired isle β cells, cause insulitis, cause insulin secretion to reduce or lack, finally cause type 1 diabetes[5]。
Likewise, type 2 diabetes mellitus most of patients is with chronic inflammatory disease and shows Insulin resistance, therefore 2 type glycosurias
Disease is also a kind of diseases associated with inflammation[7].For example, inflammatory mediator TNF-α can activate several with suppression insulin signaling
The protein expression of pathway, reduces respond of the body to insulin, induces then insulin resistance[8]。
Danish scientists find, in mice, macrophage invades diabetic pancreas tissue in the early stage of disease.Then,
These inflammatory cells produce a large amount of proinflammatory proteins (cytokine), and direct effect damaging produces insulin β in pancreas is thin
Born of the same parents, cause type 2 diabetes mellitus, and this discovery is published in the Journal of Leukocyte Biology magazines in January, 2014
On[9].Further study show that, the Th1 cells and cd8 t cell in acquired immunity promotes insulin resistant to occur,
And Th2 cells and Treg cells being capable of this effects of antagonism.Therefore, it is also control to control inflammation with modulating T cell functioning
The important target spot of system and treatment type 2 diabetes mellitus[10-11]。
Clinically the conventional medicament such as insulin can not have to the trend that type 2 diabetes mellitus patient's islet beta cell function fails year by year
Effect is prevented or is reversed, and the prevention and treatment to type 1 diabetes equally lack effective means.Clinical research is by injecting islets of langerhans
Element and the medicine of immunologic rejection reaction, prevent T cell and/or inflammatory factor from attacking beta Cell of islet, but current research knot
Fruit does not support the effectiveness of this scheme, because immunosuppressant would generally bring many systemic side effects, including it is all
Such as increase the risk that body occurs tumor and infection, immunosuppressant also results in insulin resistant and β cell functions in itself
Lower[12-13].Additionally, also and pessimistic by the prospect of Treating Type 1 Diabetes by Islets of Langerhans Transplantation.By 2011, entirely
Qiu50Duo Jia medical institutions implement and exceed 1000 islet cell transplantation arts, not good enough yet with islet transplantation long-term effect,
The scarcity of pancreas donor and the reasons such as life-time service immunosuppressant are needed, the clinical islet implemented in the past several years is moved
The quantity of plant has been reduced[14].Monoclonal antibody Anti-CD3 can be directed to the degeneration for mitigating insulin secretion function, and drop
Increase in low new type 1 diabetes 2 years to insulin requirements, played a role in type 1 diabetes early stage, was benefited most
Be type 2 diabetes mellitus patient that is younger or having more remaining β cell functions, but due to exist immunity degradation, heating,
The side reaction such as erythra and anemia, its clinical practice is also in further evaluating[15-16].Therefore, it is correct to understand diabetes inflammation
Disease and autoimmune correlation pathogenesis, exploration can avoid the new method using immunosuppressant, find new opposing inflammation
With autoimmune attack the intracellular factor can more efficient treatment and management of disease, for the body and life meaning of patient
It is great.
In addition to diabetes, other autoimmune diseases such as multiple sclerosis and systemic lupus erythematosus (sle) are also and Th1-
It is closely related with inflammatory challenge that cell-mediated T- cell subsets are unbalance.Block it is this it is unbalance also will to treat these diseases rise
Vital effect.Multiple sclerosis (MS) are a kind of modal nervus centraliss demyelination.Primary disease
Acute active stage nervus centraliss white matter has multiple inflammatory demyelination speckle, and outmoded pathological changes are then formed due to gtelatinous fibre hypertrophy
Calcified plaque, with the characteristics of multiple focus, alleviation, the recurrence course of disease, is apt to occur in optic nerve, spinal cord and brain stem, frequently-occurring disease in
In the blue or green, middle age, women is common compared with male.It is also a kind of very high autoimmune disease based on nerve degeneration of mortality rate.
In the case where the various factors such as inherited genetic factorss, environmental factorss, estrogen level interact, cause T lymphopenia, T
Suppress cell function to reduce, B cell hyperplasia, produce substantial amounts of autoantibody, and with internal corresponding autoantigen
Corresponding immune complex is combined to form, the positions such as skin, joint, thin vessels, glomerule are deposited on.In the ginseng of complement
With under, cause active chronic inflammation and tissue necrosiss (such as lupus nephritises), or antibody directly with histiocyte antigenic action,
Cause cytoclasises, so as to cause the Multisystem damage of body.
Polyunsaturated fatty acid (PUFAs) referred to 18-22 carbon atom, the straight chain fatty containing 2-3 hydrogen bond
Acid, is generally divided into two kinds of ω -3 and ω -6, referred to as ω -3 of the double bond away from carboxyl distalmost end on the 3rd carbon atom reciprocal
PUFAs, on six carbon atom, then referred to as ω -6PUFAs.ω -6 and ω -3PUFAs are by saturated fat
Acid precursors synthesize under different desaturase effects, and vertebra class and mammal lack special desaturation just
Enzyme, therefore ω -6 and ω -3 can not be synthesized in vivo, these unsaturated fatty acids can only be taken in from meals[17]。
ω -3PUFAs are particularly important for normal physiological function is maintained, and it is the key component for constituting cell lipid membrane structures.
Substantial amounts of research shows, maintains the proportional balancing method of ω -6/ ω -3PUFAs for health plays an important role.Deliver recently
Two larger scale clinical results of study are shown, take in ω -3PUFAs for a long time and be inversely proportional to the sickness rate of type 1 diabetes：One
Item shows that based on the case control study of 2000 Norway children First Year starts to take rich in ω -3PUFAs from after birth
Cod-liver oil can reduce the onset risk of children with type 1 diabetes mellitus；U.S.'s teenager Autoimmune Diabetes research project
(DAISY) a tracking sex inventory is bright, and the child with type 1 diabetes genetic risk takes in ω -3 by diet
After PUFAs, the risk that islets of langerhans occurs autoimmune inflammation can be reduced.Additionally, the free fatty such as Palmic acid is in vitro
Suppress insulin secretion, and ω -3PUFAs can reverse this phenomenon.ω -3PUFAs are supplemented by diet can strengthen
The level of insulin secretion that glucose stimulates[18-19].But, clinically use the natural fish oils containing ω -3PUFAs to control
The trial for treating diabetes and control body weight is but ended in failure mostly, is traced it to its cause, ω -6PUFAs in the food of intake
Too high levels, it is to be difficult to balance the two ratio only to rely on and supplement natural fish oils.Therefore, we are compiled using fat-1 genes
The omega-3 unsaturated fatty acid desaturase (ω -3fatty acid desaturase) of code promotes internal ω -6 to be changed into ω -3, puts down
Both weighing apparatuses ratio.
From the fat-1 genes of Caenorhabditis elegans (C.elegans), its protein product is that omega-3 unsaturated fatty acid goes
Saturation enzyme, it carries out desaturation reaction by substrate of ω -6PUFAs, generates corresponding ω -3PUFAs, is significantly improving
In animal body while endogenic ω -3PUFAs contents, ω -6PUFAs contents are reduced, change ω -6/ ω -3PUFAs
Ratio.In order that fat-1 preferably can be expressed in mammal, the cDNA of coding fat-1 is fed by we
Newborn animalization (mammalianized), therefore and it is named as mfat-1.Mfat-1 transgenic mice beta Cell of islet
The Anti-G value of cytokine induction can be significantly resisted, and ω -3PUFAs can promote insulin secretion.
List of references：
1.Xu Y,Wang L,He J,et al.Prevalence and control of diabetes in Chinese adults[J].JAmA,
2013,310(9):948-959.
2.Yang W,Lu J,Weng J,et al.Prevalence of diabetes among men and women in China[J].
New England Journal of Medicine,2010,362(12):1090-1101.
3.American Diabetes Association.Diagnosis and classification of diabetes mellitus[J].
Diabetes care,2013,36(Supplement 1):S67-S74.
4.Trembleau S,Penna G,Bosi E,et al.Interleukin 12 administration induces T helper type 1
cells and accelerates autoimmune diabetes in NOD mice[J].The Journal of experimental
medicine,1995,181(2):817-821.
5.Ishigame H,Zenewicz L A,Sanjabi S,et al.Excessive Th1responses due to the absence of
TGF-βsignaling cause autoimmune diabetes and dysregulated Treg cell homeostasis[J].
Proceedings of the National Academy of Sciences,2013,110(17):6961-6966.
6.Axelsson S,Hjorth M,Ludvigsson J,et al.Decreased GAD65‐specific Th1/Tc1
phenotype in children with Type 1diabetes treated with GAD‐alum[J].Diabetic Medicine,
2012,29(10):1272-1278.
7.Donath M Y,Shoelson S E.Type 2 diabetes as an inflammatory disease[J].Nature Reviews
Immunology,2011,11(2):98-107.
8.Stanley T L,Zanni M V,Johnsen S,et al.TNF-αantagonism with etanercept decreases
glucose and increases the proportion of high molecular weight adiponectin in obese subjects
with features of the metabolic syndrome[J].The Journal of Clinical Endocrinology&
Metabolism,2011,96(1):E146-E150.
9.Cucak H,Grunnet L G,Rosendahl A.Accumulation of M1-like macrophages in type 2
diabetic islets is followed by a systemic shift in macrophage polarization[J].Journal of
leukocyte biology,2014,95(1):149-160.
10.Feuerer M,Herrero L,Cipolletta D,et al.Lean,but not obese,fat is enriched for a unique
population of regulatory T cells that affect metabolic parameters[J].Nature medicine,2009,
15(8):930-939.
11.Ilan Y,Maron R,Tukpah A M,et al.Induction of regulatory T cells decreases adipose
inflammation and alleviates insulin resistance in ob/ob mice[J].Proceedings of the National
Academy of Sciences,2010,107(21):9765-9770.
12.Assan R,Bach J F,Czernichow P,et al.Immunosuppressive drugs in diabetes[J].The
Lancet,1986,328(8515):1097.
13.Pereira M J,Palming J,Rizell M,et al.The immunosuppressive agents rapamycin,
cyclosporin A and tacrolimus increase lipolysis,inhibit lipid storage and alter expression of
genes involved in lipid metabolism in human adipose tissue[J].Molecular and cellular
endocrinology,2013,365(2):260-269.
14.Shapiro A M J,Lakey J R T,Ryan E A,et al.Islet transplantation in seven patients with
type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen[J].New
England Journal of Medicine,2000,343(4):230-238.
15.Herold K C,Hagopian W,Auger J A,et al.Anti-CD3 monoclonal antibody in new-onset
type 1 diabetes mellitus[J].New England Journal of Medicine,2002,346(22):1692-1698.
16.Aronson R,Gottlieb P A,Christiansen J S,et al.Low-dose otelixizumab anti-CD3
monoclonal antibody DEFEND-1 study:Results of the randomized phase III study in
recent-onset human type 1 diabetes[J].Diabetes care,2014,37(10):2746-2754.
17.Simopoulos A P.Evolutionary aspects of diet:the omega-6/omega-3 ratio and the brain[J].
Molecular neurobiology,2011,44(2):203-215.
18.Stene L C,Joner G.Use of cod liver oil during the first year of life is associated with
lower risk of childhood-onset type 1 diabetes:a large,population-based,case-control study[J].
The American journal of clinical nutrition,2003,78(6):1128-1134.
19.Boucher A.Diabetes in children.Interview by Carol Nichols[J].Australian nursing journal
(July 1993),2008,16(1):33-33.
The content of the invention
The technical problem to be solved, is to provide one kind and treats autoimmunity relevant disease and type 1 diabetes
Viral vector, the viral vector safety non-toxic, and possess therapeutic activity disclosure satisfy that clinical demand.
Present invention technical problem also to be solved, is to provide the construction method of above-mentioned viral vector.
Present invention technical problem finally to be solved, is to provide the feasibility application of above-mentioned slow virus carrier.
To solve above-mentioned technical problem, the technical solution used in the present invention is as follows：
A kind of viral vector, it be by clone have mfat-1 genes slow viruss expression plasmid or clone have mfat-1 genes
Adeno-associated viruses expression plasmid transfect to obtained by 293FT cells, described mfat-1 genes such as ESQ ID No.:1 institute
Show.
The construction method of above-mentioned viral vector, by mfat-1 gene clonings to slow viruss expression plasmid (lentivirus) or gland
Correlated virus expression plasmid (adeno-associated virus), is expanded to 293FT cells packaging by liposome transfection,
Obtain final product.
Wherein, described slow viruss expression plasmid (is purchased from for pLJM1-CMV-hPGK-EGFP plasmids
Addgene (Plasmid#19319)) and pLJM1-CMV-hPGK-mkate2 plasmids.
Wherein, described adeno-associated viruses expression plasmid is pEMBL-AAV-D (+)-CMV-eGFP-SV40 plasmids.
Application of the above-mentioned viral vector in treatment diabetes medicament is prepared is also within protection scope of the present invention.
Wherein, described diabetes are preferably autoimmunity type 1 diabetes.
Application of the above-mentioned viral vector in autoimmune-associated diseases medicine is prepared is also within protection scope of the present invention.
Mfat-1 genes are brought into spontaneous type non-obese glycosuria using viral vector using the method for gene therapy by the present invention
In vivo, compared with control group mice, mfat-1 lentiviral particles can be significantly reduced disease model mice (NOD mices)
Morbidity NOD mouse blood sugar levels, while its insulin level in serum is raised, with significant therapeutic effect.mfat-1
Gene therapy treats diabetes and the mechanism of autoimmune to be included：Mfat-1 is with ω -6PUFAs too high in vivo as bottom
Thing, is translated into ω -3PUFAs, both balances ratio so that the ratio of ω -6/ ω -3 tends to 1：1.These are by ω -6
Endogenouss ω-the 3PUFAs that PUFAs is transformed promote Th cells to break up to Th2 directions, and antagonism ω -6PUFAs
Rush Th1 cell activations effect, reduce IFN-γ, IL-6, TNF-α, the proinflammatory factor secretion level such as IL-17, delay
Solution Th1 cell-mediated inflammatory reaction；Suppress CD8+CTL cells, macrophage are infiltrated in islets of langerhans, alleviate islets of langerhans
The generation of scorching and peripancreatitis；Due to lacking unsaturated fatty acidss acidohydrogenase in mammal body, it is therefore desirable to adopt slow viruss
Mfat-1 is imported internal by carrier or gland relevant viral vector.
The administering mode of above-mentioned viral vector is intravenous injection.
Beneficial effect：Compared with prior art, it is of the invention for treating type 1 diabetes and associated autoimmune disease
Bio-pharmaceutical, be a kind of new bio medicine that safety non-toxic possesses anti-inflammatory activity, with slow viruss as carrier, using base
Because the high expression polyunsaturated fatty acid dehydrogenase of recombinant technique, anti-autoreactivity inflammatory activity are strong, use demand can be met.
Preparation method is simple, easily operates, with good application prospect.
Description of the drawings
Fig. 1 is that pLJM1-mfat-1-EGFP plasmid NheI and EcoRI enzyme action identifies 1% agarose gel electrophoresis figure；
Fig. 2 is the result figure for giving NOD Mus change of blood sugar after slow viruss are treated；
Fig. 3 is the result figure for giving NOD Mus insulin secretions after slow viruss are treated；
Fig. 4 is to give the result figure that NOD Mus insulitis after slow viruss are treated infiltrate development and change；
Fig. 5 is the result figure for giving NOD Mus cytokines change after slow viruss are treated；
Fig. 6 is the result figure for giving NOD Mus onset diabetes rate change after slow viruss are treated.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, and should not also without limitation on described in detail in claims
The present invention.
Embodiment 1：The structure of mfat-1 slow viruss expression plasmids.
1. the plasmid of construction expression mfat-1 genes.
First synthesize mfat-1 (such as ESQ ID No.:Shown in 1) gene (Jin Sirui companies), then pass through NheI and EcoRI
Restriction enzyme site sub-clone is obtained to pLJM1-CMV-hPGK-EGFP plasmids (addgene, Plasmid#19319)
PLJM1-CMV-hPGK-EGFP-mfat-1 expression plasmids.Concrete building process is as follows：
1) PCR amplification mfat-1 areas
With the mfat-1 gene orders that synthesize as masterplate, PCR amplifications mfat-1 areas so as to sequence two ends with NheI and
EcoRI restriction enzyme sites：
Primer pair：NheI---mfat-1---EcoRI
mfat-1-F:5'-ATGGTCGCCCACAGCA-3'
--- Kozak sequences：TATTAA
---NheI:GCTAGC
---Final primer:
5'-TATTAAGCTAGCATGGTCGCCCACAGCA-3'
mfat-1-R:5'-TCATCACTTGGCCT-3'
--- Kozak sequences：CAACCG
---EcoRI:GAATTC
---Final primer:
5'-CAACCGGAATTCTCATCACTTGGCCT-3'
PCR amplification system is shown in Table 1.
Table 1
Amplification condition：98 DEG C of degeneration 5min；98 DEG C of 10s, 60 DEG C of 15s, 72 DEG C of 2min, totally 30 circulations；Finally
72 DEG C of extension 10min.Target DNA fragment is reclaimed with 1.5% agarose gel electrophoresiies, using Agarose Gel DNA
Extraction Kit obtain the target DNA fragment of purification.
2) enzyme action
DNA fragmentation the restricted enzyme NheI and EcoR of amplification out mfat-1 are scaled off, with corresponding interior
Enzyme cutting carries out enzyme action to pLJM1-EGFP zero load shuttle plasmids.
Table 2
Reaction condition：37 DEG C of water-bath 2h.Above-mentioned digestion products separate purpose fragment with 1% agarose gel electrophoresiies, use
Agarose Gel DNA Extraction Kit obtain the target DNA fragment of purification.
3) connect
Destination gene expression fragment is connected with pLJM1 carriers.
Table 3
4) convert
The competence antibacterial that connection product conversion is used is DH5 α escherichia coli, using CaCl2Prepared by method, preparation method
And follow-up conversion operation reference《Molecular cloning》The second edition, it is specific as follows：
The competent preparations of A：
I adds 1ml saturations bacterium solution in 100ml LB, shakes bacterium, and 37 DEG C, 280rpm, 2-3h are thin until reflecting
Optical density OD600 of strain density is between 0.4-0.6；
Ii is proceeded in the 50ml polypropylene tube of ice pre-cooling, is placed in 10min in ice bath；
4 DEG C of iii, 1000g, are centrifuged 10min；
Iv abandons supernatant, and pipe is inverted 1min, flows to end liquid, adds the 0.1mol/l CaCl of 10ml ice pre-coolings2Weight
Outstanding precipitation, is placed in 30min in ice bath；
4 DEG C of v, 1000g, are centrifuged 10min；
Vi abandons supernatant, adds the 0.1mol/l CaCl of 2ml ice pre-coolings2Resuspended precipitation, adds the sterilizing of 0.5ml 75%
Glycerol (uses 0.1mol/l CaCl2Prepare), mix, 100 μ l/ pipes subpackages into 1.5ml centrifuge tubes, in -80 DEG C of refrigerators
Half a year can be preserved.
Vii transformation efficiencies are identified
The conversion of B connection products：
I takes 1 pipe competent cell, is placed in and melts on ice, adds 3 μ l connection products, mixes, ice bath 30min；
42 DEG C of water-baths of ii, stand 90sec.It is transferred quickly in ice, ice bath 1-2min；
Iii adds 900 μ l LB, 37 DEG C of water-bath 10min；
Iv antibacterials expand, 37 DEG C, 210rpm, 45min；
V coated plates, the 100 μ l above-mentioned antibacterial for having converted is coated on the agar plate containing 100 μ g/ml ampicillin；
Vi is inverted flat board, 37 DEG C of culture 16-20h.
5) Fig. 1 is shown in positive colony screening (enzyme action identification, sequencing identification).
2. pack, expand and purification lenti-mfat-1 lentiviral particles.
The incasing cellss of slow viruss are 293FT cell strains, and growth medium is DMEM (containing 10%FBS).It is adherent thin
Born of the same parents Jing culture growing multiplications form cell monolayer.
(1) virus amplification：
1) 24h before transfecting, with the 293FT cells of trypsinization exponential phase, with the culture containing 10% serum
The whole cell density of keynote is 105 cells of 1x/ml, is inoculated in six orifice plates, 37 DEG C, cultivates in 5%CO2 incubators.
Can be used to transfect after 24h when cell density is up to 70%～80%.Cell state is most important for virus packaging, because
This needs to ensure good cell state and less passage number.
2) before transfecting, cell culture medium is replaced by fresh complete medium by 1h.
3) following plasmid reagent (1.5 μ g of PLJM1 carriers, psPAX2 carriers are added in 1.5ml EP pipes
1.125 μ g of Addgene (Plasmid#12260), pMD2.G carrier Addgene (Plasmid#12259) 0.375 μ g), with
100 μ l OPTI-MEM mix homogeneously, are incubated at room temperature 5 minutes.
4) first add 100 μ l OPTI-MEM in another 1.5ml EP pipe, add 3 μ l Lip2000 transfection reagents,
Mixing is flicked, is incubated at room temperature 5 minutes.
5) DNA after dilution is mixed with the Lipofectamine2000 transfection reagents after dilution, is gently mixed,
Incubate 15 minutes at room temperature, to form the transfection composite of DNA and Lipofectamine2000 transfection reagents.
6) 200 μ l mentioned reagents are instilled in 293FT cell culture mediums, is gently shaken up, in 37 DEG C, 5%CO2Carefully
Cultivate in born of the same parents' incubator.
(2) viral purification：
1) culture medium containing transfection mixture is discarded after cultivating 24h, adds 2ml fresh complete mediums to continue culture.
2) cell conditioned medium being collected respectively at 48h and 72h, collecting supernatant mixing twice, 1250rpm centrifugation 5min go
Except possible 293FT cells, then filtered with 0.45 μm of filter.Virus can short-term (<4 DEG C are stored in 3d), it is long-term to protect
Be stored in -80 DEG C it is standby, subpackage avoids multigelation.
(2) virus titer is determined
Virus titer is determined using TCID50 methods.Compare amplification, purification and the titer determination of naked virus ibid.
Embodiment 2：Lenti-mfat-1 lentiviral particles treat the effect of type 1 diabetes.
(1) the continuous two weeks NOD mices more than 11.1mmol/L of random blood sugar are divided into two groups, matched group (Con) and
Slow viruss treatment group (Lenti-mfat-1), 5 per group, treatment group is 10 according to virus titer8The concentrated solution of TU/mL
10 are pressed according to Mouse Weight9After TU/Kg carries out tail vein injection slow viruss, the change (Fig. 2) of mice random blood sugar is detected.
(2) the general physiological conditions of NOD mices after slow viruss are treated are received
After giving treatment group's mouse tail vein injection mfat-1 lentiviral particles, compared with matched group, mice ingests drinking-water feelings
Condition is normal, and after treating 2 weeks, treatment group's proportion of weight to height is higher than matched group, but not statistically signigicant.
(3) receive the monitoring (Fig. 2) of NOD mice random blood glucose levels after slow viruss are treated.
The continuous two weeks NOD mices more than 11.1mmol/L of random blood sugar are divided into two groups, matched group (Lenti-con) and
Slow viruss treatment group (Lenti-mfat-1), 4 per group, treatment group is according to 109It is slow that TU/ml/Kg carries out tail vein injection
After virus, the change of mice random blood sugar is detected weekly, and compared with matched group, slow viruss treatment group NOD mices is random
Blood glucose starts substantially to lower in second week, and by the 6th week, blood sugar level declined and maintained normal level, reaches
To 6.3mmol/L.Illustrate that Lenti-mfat-1 lentiviral particles have significant therapeutic effect (Fig. 3-6) to NOD mices.
After viral therapy is given 9 weeks, mouse peripheral blood polyunsaturated fatty acid change level is detected, finds to give
Lenti-mfat-1 mice EPA ratios increase, and illustrate carrying mfat-1 slow viruss successful operations (table 4) for giving.
Table 4 gives the change of mouse blood polyunsaturated fatty acid after slow viruss treatment
Every kind of ω -3and omega 6 polyunsaturated fatty acids are represented using relative percentage, wherein the institute gone out by gas chromatographic detection
There is the peak area of fatty acid for absolutely, the content of purpose fatty acid then goes out peak area by which and sentences total peak area and meter
Draw.Every group of Data duplication three times, is counted using oneway-ANOVA, the * P compared with matched group<0.05, NP:Not
Detect.
Claims (10)
1. a kind of viral vector, it is characterised in that it be slow viruss expression plasmid that clone is had into mfat-1 genes or gram
The grand adeno-associated viruses expression plasmid for having mfat-1 genes is transfected to obtained by 293FT cells, and described mfat-1 genes are such as
ESQ ID No.:Shown in 1.
2. viral vector according to claim 1, it is characterised in that described slow viruss expression plasmid is
PLJM1-CMV-hPGK-EGFP plasmids and pLJM1-CMV-hPGK-mkate2 plasmids.
3. viral vector according to claim 1, it is characterised in that described adeno-associated viruses expression plasmid is
PEMBL-AAV-D (+)-CMV-eGFP-SV40 plasmids.
4. the construction method of the viral vector described in claim 1, it is characterised in that by mfat-1 gene clonings to slow
Viral expression plasmids or adeno-associated viruses expression plasmid, are expanded to 293FT cells packaging by liposome transfection, are obtained final product.
5. the construction method of viral vector according to claim 4, it is characterised in that described slow viruss expression
Plasmid is pLJM1-CMV-hPGK-EGFP plasmids and pLJM1-CMV-hPGK-mkate2 plasmids.
6. the construction method of viral vector according to claim 4, it is characterised in that described adeno-associated viruses
Expression plasmid is pEMBL-AAV-D (+)-CMV-eGFP-SV40 plasmids.
7. application of the viral vector described in claim 1 in treatment diabetes medicament is prepared.
8. application according to claim 7, it is characterised in that described diabetes are 1 type of autoimmunity sugar
Urine disease.
9. application of the viral vector described in claim 1 in autoimmune-associated diseases medicine is prepared.
10. the application according to any one in claim 7～9, it is characterised in that administering mode is intravenous injection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510676799.0A CN106591370A (en) | 2015-10-19 | 2015-10-19 | Virus vector for treating autoimmune related diseases and diabetes, construction method and applications thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510676799.0A CN106591370A (en) | 2015-10-19 | 2015-10-19 | Virus vector for treating autoimmune related diseases and diabetes, construction method and applications thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106591370A true CN106591370A (en) | 2017-04-26 |
Family
ID=58554096
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510676799.0A Pending CN106591370A (en) | 2015-10-19 | 2015-10-19 | Virus vector for treating autoimmune related diseases and diabetes, construction method and applications thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106591370A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103058A (en) * | 2017-05-12 | 2018-06-01 | 北京五加和分子医学研究所有限公司 | A kind of gene therapy medicament of type-1 diabetes mellitus |
| CN108103103A (en) * | 2017-05-10 | 2018-06-01 | 北京五加和分子医学研究所有限公司 | A kind of gene therapy medicament of hyperlipidemia |
| WO2018177376A1 (en) * | 2017-04-01 | 2018-10-04 | 广州华真医药科技有限公司 | Viral vector for treating autoimmune disease and diabetes and construction method and application thereof |
| CN111500636B (en) * | 2017-04-01 | 2024-04-26 | 广州华真医药科技有限公司 | Virus vector for treating autoimmune related diseases and construction method and application thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1322840A (en) * | 2001-06-20 | 2001-11-21 | 中国医学科学院基础医学研究所 | Glandular associated viral vector and its prepn and use |
| WO2002024234A2 (en) * | 2000-09-20 | 2002-03-28 | The Regents Of The University Of California | Use of recombinant gene delivery vectors for treating or preventing diseases of the eye |
| WO2002059296A3 (en) * | 2001-01-25 | 2003-05-01 | Evolva Biotech As | Concatemers of differentially expressed multiple genes |
| CN101503706A (en) * | 2009-03-09 | 2009-08-12 | 刘红林 | Method for obtaining transgenic chicken by lentivirus vector mediated in vivo transfection of spermatogonial stem cell |
| CN102168061A (en) * | 2010-12-21 | 2011-08-31 | 南京医科大学 | Human lung cell BEAS-2B/CYP2A13, and preparation method and application thereof |
| CN102268453B (en) * | 2007-04-23 | 2013-04-03 | 深圳益世康宁生物科技有限公司 | LMP-1 recombinant adeno-associated virus (rAAV) vector and construction method as well as application thereof |
| CN103074347A (en) * | 2013-01-23 | 2013-05-01 | 南京华贞生物医药科技有限公司 | Humanized mfat1 gene for mammals to express and application of same |
| CN103173495A (en) * | 2013-02-25 | 2013-06-26 | 中国疾病预防控制中心病毒病预防控制所 | Novel rabies virus fake virus system as well as preparation and application thereof |
| CN103446188A (en) * | 2013-09-02 | 2013-12-18 | 中国人民解放军第三军医大学第一附属医院 | Applications of TRADD (Tumor necrosis factor receptor associated death domain protein) gene over-expressed lentivirus for inhibiting formation of skin scars |
| CN104694575A (en) * | 2015-01-12 | 2015-06-10 | 深圳市中美康士生物科技有限公司 | Application of promoter optimized lentiviral genetically modified T cells in oncotherapy |
-
2015
- 2015-10-19 CN CN201510676799.0A patent/CN106591370A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002024234A2 (en) * | 2000-09-20 | 2002-03-28 | The Regents Of The University Of California | Use of recombinant gene delivery vectors for treating or preventing diseases of the eye |
| WO2002059296A3 (en) * | 2001-01-25 | 2003-05-01 | Evolva Biotech As | Concatemers of differentially expressed multiple genes |
| CN1322840A (en) * | 2001-06-20 | 2001-11-21 | 中国医学科学院基础医学研究所 | Glandular associated viral vector and its prepn and use |
| CN102268453B (en) * | 2007-04-23 | 2013-04-03 | 深圳益世康宁生物科技有限公司 | LMP-1 recombinant adeno-associated virus (rAAV) vector and construction method as well as application thereof |
| CN101503706A (en) * | 2009-03-09 | 2009-08-12 | 刘红林 | Method for obtaining transgenic chicken by lentivirus vector mediated in vivo transfection of spermatogonial stem cell |
| CN102168061A (en) * | 2010-12-21 | 2011-08-31 | 南京医科大学 | Human lung cell BEAS-2B/CYP2A13, and preparation method and application thereof |
| CN103074347A (en) * | 2013-01-23 | 2013-05-01 | 南京华贞生物医药科技有限公司 | Humanized mfat1 gene for mammals to express and application of same |
| CN103173495A (en) * | 2013-02-25 | 2013-06-26 | 中国疾病预防控制中心病毒病预防控制所 | Novel rabies virus fake virus system as well as preparation and application thereof |
| CN103446188A (en) * | 2013-09-02 | 2013-12-18 | 中国人民解放军第三军医大学第一附属医院 | Applications of TRADD (Tumor necrosis factor receptor associated death domain protein) gene over-expressed lentivirus for inhibiting formation of skin scars |
| CN104694575A (en) * | 2015-01-12 | 2015-06-10 | 深圳市中美康士生物科技有限公司 | Application of promoter optimized lentiviral genetically modified T cells in oncotherapy |
Non-Patent Citations (2)
| Title |
|---|
| 周军智 等: "含PGK 启动子慢病毒表达质粒的构建与鉴定", 《安徽师范大学学报(自然科学版)》 * |
| 李斌 等: "mfat-1 小鼠胆固醇代谢特点及相关机制", 《中国动脉硬化杂志》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018177376A1 (en) * | 2017-04-01 | 2018-10-04 | 广州华真医药科技有限公司 | Viral vector for treating autoimmune disease and diabetes and construction method and application thereof |
| KR20190135516A (en) * | 2017-04-01 | 2019-12-06 | 광저우 후아젠 파마슈티컬 컴퍼니 리미티드 | Viral vector for the treatment of autoimmune disease and diabetes and method and application thereof |
| CN111500636A (en) * | 2017-04-01 | 2020-08-07 | 广州华真医药科技有限公司 | Virus vector for treating autoimmune related diseases and construction method and application thereof |
| KR102383866B1 (en) * | 2017-04-01 | 2022-04-06 | 광저우 후아젠 파마슈티컬 컴퍼니 리미티드 | Viral vectors for the treatment of autoimmune diseases and diabetes and methods for their preparation and applications |
| CN111500636B (en) * | 2017-04-01 | 2024-04-26 | 广州华真医药科技有限公司 | Virus vector for treating autoimmune related diseases and construction method and application thereof |
| CN108103103A (en) * | 2017-05-10 | 2018-06-01 | 北京五加和分子医学研究所有限公司 | A kind of gene therapy medicament of hyperlipidemia |
| CN108103103B (en) * | 2017-05-10 | 2021-06-25 | 北京锦篮基因科技有限公司 | Gene therapy medicine for hyperlipemia |
| CN108103058A (en) * | 2017-05-12 | 2018-06-01 | 北京五加和分子医学研究所有限公司 | A kind of gene therapy medicament of type-1 diabetes mellitus |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cho et al. | A review of clinical trials: mesenchymal stem cell transplant therapy in type 1 and type 2 diabetes mellitus | |
| Lu et al. | Adipose tissue-resident immune cells in obesity and type 2 diabetes | |
| Faunce et al. | Cutting edge: in vitro-generated tolerogenic APC induce CD8+ T regulatory cells that can suppress ongoing experimental autoimmune encephalomyelitis | |
| Jeffery et al. | Clinical potential of regulatory T cell therapy in liver diseases: an overview and current perspectives | |
| Li et al. | IL-10 and its related cytokines for treatment of inflammatory bowel disease | |
| CN106167789B (en) | Hypoxia-treated mesenchymal stem cells and application thereof | |
| US20230364201A1 (en) | Pharmaceutical composition containing insulin-like growth factor-2 and use thereof | |
| Aharoni et al. | Immunomodulatory therapeutic effect of glatiramer acetate on several murine models of inflammatory bowel disease | |
| Kwak et al. | Insights into myeloid-derived suppressor cells in inflammatory diseases | |
| CN105934155A (en) | Methods of using adipose tissue-derived cells in the modulation of pain and/or fibrosis | |
| CN106591370A (en) | Virus vector for treating autoimmune related diseases and diabetes, construction method and applications thereof | |
| Zhang et al. | The characteristics of regulatory macrophages and their roles in transplantation | |
| Bharath et al. | Adaptive immunity and metabolic health: harmony becomes dissonant in obesity and aging | |
| Swart et al. | Changing winds in refractory autoimmune disease in children: clearing the road for tolerance with cellular therapies | |
| AU2022200653A1 (en) | Viral vector for treating autoimmune disease and diabetes and construction method and application thereof | |
| Liu et al. | Treatment effect of low-intensity pulsed ultrasound on benzene-and cyclophosphamide-induced aplastic anemia in rabbits | |
| WO2005004789A2 (en) | Method for treating diseases associated with changes of qualitative and/ quantitative composition of blood extracellular dna | |
| Kimura et al. | Temporary resolution of insulin requirement in acquired partial lipodystrophy associated with chronic graft‐versus‐host disease | |
| Varian et al. | Consuming cholera toxin counteracts age-associated obesity | |
| Liu et al. | Fecal microbiota transplantation for the management of autoimmune diseases: Potential mechanisms and challenges | |
| Oosman et al. | Treatment of obesity and diabetes in mice by transplant of gut cells engineered to produce leptin | |
| Elsas et al. | The effects of allergen and anti-allergic drugs on murine hemopoietic cells: moving targets, unusual mechanisms, and changing paradigms | |
| Sydora et al. | Neonatal exposure to fecal antigens reduces intestinal inflammation | |
| Luo et al. | Targeting IL-12 family cytokines: A potential strategy for type 1 and type 2 diabetes mellitus | |
| Venzin | Th1-LIKE HBV-SPECIFIC CD4+ T CELLS ARE ABLE TO REVERT THE CD8+ T CELL DYSFUNCTION INDUCED BY HEPATOCELLULAR PRIMING |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhao Zijian Inventor after: Li Fanghong Inventor after: Bi Xinyun Inventor after: Li Xiaoxi Inventor after: Lin Yan Inventor before: Bi Xinyun Inventor before: Li Xiaoxi Inventor before: Zhao Zijian Inventor before: Lin Yan Inventor before: Li Fanghong |
|
| CB03 | Change of inventor or designer information | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180411 Address after: 510660 Science City Scientific City, Guangzhou high tech Industrial Development Zone, Guangdong Province, No. 3 Applicant after: Guangzhou true China Medical Technology Co., Ltd. Address before: 211100, No. 118 East Tianyuan Road, Jiangning District, Jiangsu, Nanjing Applicant before: Nanjing SinoGen Biomedical Technology Co., Ltd. |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170426 |
|
| RJ01 | Rejection of invention patent application after publication |