CN104174029A - Application of reovirus dsRNA in preparation of medical endogenous interferon inducer - Google Patents

Abstract

The invention discloses application of reovirus dsRNA in preparation of a medical endogenous interferon inducer. Reov-dsRNAEnII is prepared by using purified reovirus dsRNA as a material, directly acts on a body to induce and generate various endogenous interferons and endows a human body with functions of virus resistance, tumor resistance and constitution enhancement. The reovirus has the advantages that: (A) the reovirus cannot cause remarkable diseases to a human body, does not have tumor derivation, and is remarkably safe; (B) the interferon generated by inducing the human body is complete in types and high in in-vivo concentration, is highly in accordance with the species specificity in human-sourced virus and eukaryotic molecules, does not have immunological rejection or dependence, and has extremely strong bioactivity and pharmacologic effect and high drug effect on a human body; (C) the dsRNA biomaterial is natural and green, and eco-friendly; (D) the resource is endlessly renewable, and is inexhaustible; (E) the special virus dsRNA material can realize high-throughput proliferation, extraction and modern preparation, and is low in cost.

Description

The application of reovirus dsRNA in medical endogenous interferon derivant preparation

Technical field

The invention belongs to nucleic acid pharmaceutical technology field.Reovirus (Reovirus) double-stranded RNA nucleic acid (dsRNA) is at medical endogenous interferon derivant (Endo-Interferon Inducer, EnII) application in preparation refer to utilize modern biotechnology in-vitro multiplication natural, can infinite regeneration, the remarkable Reovirus green resource of safety, high-purity is extracted its dsRNA molecule, and finally prepare in adult with nucleic acid preparation, i.e. medical Reovirus dsRNA endogenous interferon derivant (Reov-dsRNA EnII).This EnII is directly used in human body, the a large amount of various relevant cells of induction human body, as leukocyte, fibroblast, immunological lymphocyte and other relevant cells, the raw A wide selection of colours and designs of high concentration real estate, highly meet species specificity, without immunologic rejection and dependence, extremely strong at people's in vivo bioactivity and pharmacological action, endogenous interferon (the Endo-Interferon of height drug effect, ENIs), thereby give safely and effectively human body antiviral, antitumor and widely diseases prevention, cure the disease and the function such as health invigorating, also can be used for tackling sudden public health event.

Background technology

Owing to thering is the interferon (Interferon of diseases prevention and treatment function widely, IFN) (Dong Changyuan chief editor, " modern Molecular Virology ", publishing house of Wuhan University, in October, 1996) be the best type cytokines that the mankind found so far; Also because endogenous interferon (Endo-Interferon, ENI) has unrivaled advantage compared with exogenous interferon (Exo-Interferon, EXI), and the mankind not yet obtain desirable endogenous interferon derivant (Endo-Interferon Inducer, EnII) so far etc. reason, the mankind are pursuing safety, effective medical EnII always.

1. interferon and classification thereof and characteristic

IFN is the one group of multi-functional soluble glycoprotein that stimulates induction various kinds of cell to produce by interferon inducers (Interferon Inducer).By being combined with cell receptor, IFN can activate more than 300 kind of interferon-stimulated gene (IFN-stimulated genes, ISGs), and performance various biological activity, comprises antivirus action, antitumor action and constitutional opsonic action.

International interferon NK (International Commission on Interferon Nomenclature) regulation, first carries out preliminary classification according to the animal origin of IFN; Be divided into different types according to the difference of the antigenic characteristic of IFN and molecular structure again.In specific type, can be divided into again some hypotypes by aminoacid sequence or composition difference.So, according to the difference of aminoacid sequence and receptor, IFN is divided into I, II and III three types (Richard E.Randall, Stephen G.Interferons and viruses:an interplay between induction, signalling, antiviral responses and virus countermeasures.Jof General Virology, 2008,89:4-6).IFN-α, IFN-β, IFN-г and IFN-ω hypotype are attributed to interferon type Ⅰ, and wherein IFN-α just has more than 20 hypotypes, and IFN-β only has a hypotype; IFN-γ is unique interferon type Ⅱ, and only has 1 hypotype; IFN-λ 1 (IL-29), IFN-λ 2 (IL-28a) and IFN-λ (IL-28b) are attributed to III type interferon; IFN-ω is IFN-α family member, but its structure and size are slightly variant with other IFN-α, and its antigenicity has larger difference. more than classification is only applicable to people and mouse interferon, other animal interferon science accumulation are still few.

According to the way of production, interferon is divided into again natural IFN and non-natural IFN.The former is that cell is accepted interferon inducers stimulation and the direct interferon producing; The latter refers to the interferon of producing by biotechnology.

Natural interferon is of a great variety, molecular weight difference separately, and antigenicity is also different.So far, the disturbance element that people's archeocyte is produced has obtained three kinds of different antigenic components, comprises LeIF antigen (Le), fibroblast interferon's antigen (F) and T lymphocyte interferon antigen (T).In brief, Le antigen interferon is IFN-α, and F antigen interferon is IFN-β, and T-interferon is IFN-γ.

According to source, IFNs can be divided into endogenous interferon (ENI) and exogenous interferon (EXI).EXI applies exogenous interferon inducer (Exo-Interferon Inducer, ExII) to stimulate that Cells In Vitro is produced or biotechnology is synthetic in vitro, then for the IFNs of body; ENI uses endogenous interferon derivant (Endo-Interferon Inducer, EnII) in body, the IFNs that directly induces body to produce.

2. interferon function

Interferon type Ⅰ mainly participates in the effects such as antiviral, antitumor, parasiticide.

Interferon type Ⅱ antivirus action than I type IFN a little less than, except having antiviral, antiproliferative activity, its main biologic activity is immunoregulation effect, main expression and the immunomodulatory effect that participates in induction MHC class antigen.

III type interferon has the aspect effects such as antiviral, antiproliferative and extracorporeal anti-tumor.

Visible, IFN-α, IFN-β, IFN-γ all have antivirus action, and its antivirus action has broad spectrum activity.Its antivirus action to cell is indirectly, (nonspecific) (Richard E.Randall of wide spectrum, Stephen G.Interferons and viruses:an interplay between induction, signalling, antiviral responses and virus countermeasures.J of General Virology, 2008,89:4-6).

In recent years, about the research of induction IFN mechanism of production, IFN antiviral, antitumor and the pharmacology of conditioning human body function and the research of physiological action have obtained gratifying result, have shown that it meets the characteristic of body nature defense function.IFN not only has antiviral and antitumor action, and has constitutional opsonic action; Can treat disease, again can prevent disease; Can resist known viruse, also can anti-unknown virus (comprising potent virus, as SARS-CoV, bird flu virus).

3. the generation of interferon and interferon inducers

Cellular genome password various IFN genes.Because IFN gene suppressor (IFN suppressor) is combined with IFN gene, so in the ordinary course of things, IFN gene is in inhibitory state.The material that can induce IFN to produce is referred to as interferon inducers (Interferon inducer).When interferon inducers acts on cell membrane, just can free the combination of IFN gene suppressor and IFN gene, IFN operon transcriptional start, synthetic mRNA, mRNA is transferred to rapidly endochylema, translates out IFN precursor on ribosome, after excision signal peptide, ripe IFN is secreted into extracellular.

Research shows, after virus infected cell, can induce III type and I type interferon through same mechanism simultaneously, but both expression difference (Osterlund P, Veckman V, Siren J, et a1.Gene expression and antiviral activity of alpha/bela intederons and interleukin-29 in virus-infected human myeloid dendritic cells.J Virol, 2005,79[15]: 9608-9617, Contoli M, Message SD, Laza-Stanca V, et a1.Role of deficient type III interferon-lambda production in asthma exacerbations.Nat Med, 2006,12[9]: 1023-1026, Khaitov MR, Laza-Stanca V, Edwards MR, et a1.Respiratory virus induction of alpha-, beta-and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells.Allergy, 2009, 64[3]: 375-386), organizing of expressing is also different, for example, IFN-λ replys and mainly results from epithelial cell, especially at lung, skin and intestinal, and the cell of nearly all type all can produce I type interferon (Sommereyns C, Paul S, Staeheli P, et a1.IFN-lambda (IFN-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo.PLoS Pathog, 2008, 4[3]: e1000017).Under certain conditions, IFN-λ and I type interferon are not expressed (Doyle SE simultaneously, Schreckhise H, Khuu-Duong K, et a1.Interleukin-29uses a type I interferon-like program to promote antiviral responses in human hepatocytes.Hepatology, 2006,44[4]: 896-906).Nearly all vertebrates (comprising people) cell all can produce IFN, but no matter still external in vivo, must be produced by the induction of IFN derivant.

According to the essence of interferon inducers, can be divided into: viral IFN derivant, double-stranded RNA IFN derivant, metabolite IFN derivant and some pathogen IFN derivants etc.Chlamydia, rickettsia, intracellular toxin, fungal extract etc. also can induce IFN to produce.At present be mainly that the Avian pneumo-encephalitis virus (NDV) of deactivation is as exogenous interferon inducer (ExII). but, the pathogen of living can not be used as interferon inducers, can not serve as especially endogenous interferon derivant (Endo-Interferon inducer, EnII), let alone for human body.

4. the advantage of endogenous interferon and endogenous interferon derivant

Exogenous interferon (EXI) used can produce serial toxic and side effects in the time of clinical use at present, as causes leukopenia, headache, heating, abnormal liver function, cold-like syndrome, bone marrow depression, neurological symptom (as insomnia, anxiety, depression, excitement, irritability, psychosis) etc.In addition, also has rare side reaction, as epilepsy, nephrotic syndrome, interstitial pneumonia and arrhythmia etc.; Also may bring out autoimmune disease, as thyroiditis, thrombocytopenic purpura, hemolytic anemia, rheumatic arthritis, lupus erythematous-like syndrome, vasculitic syndrome and type Ⅰ diabetes mellitus etc.Also because the essence of interferon is an albuminoid polypeptide, so being applied to human body, EXI will inevitably cause the reaction of body allos and dependency.In addition, while being applied to human body due to EXI, kind is single, and medicine and physiological function are poor; And its preparation is complicated, expensive, storage and transport trouble, so, limit a large amount of production and the popularization and application of EXI.

Endogenous interferon (ENI) is to apply endogenous interferon derivant (EnII) in human body, the IFNs that directly induces body to produce.In this case, body has become the compound factory that produces interferon, to produce various interferon, and performance antitumor, infection and opsonic action.ENI is the natural compound interferon being produced by body self, it is LeIF, fibroblast type interferon and immunologic pattern interferon etc., cell induction by number of different types generates (Sang-Myeong L, Susan KS, Steven B.Kleiboeker Porcine reproductive and respiratory syndrome virus field isolates differ in vitro interferon phenotypes Veterinary Veterinary.Immunology and Immunopathology, 2004, 102:217-231).So, can overcome many deficiencies of EXI with the EnII ENI that directly induction produces in human body, as: immunogenicity (being anaphylaxis and rejection) that the IFN A wide selection of colours and designs, the bulk concentration that induce be high, do not have heterologous protein, there is no dependency, without purifying, cheap ... Deng.Also because IFNs has relative species specificity, so antiviral, antitumor and the conditioning functions activity of endogenous interferon are the strongest.

Visible, the IFNs with diseases prevention and treatment function is widely the best type cytokines that the mankind found so far; Endogenous interferon (ENI) has unrivaled advantage compared with exogenous interferon (EXI).But, want to expect ENIs, key is to want obtain can Secure Application in human body, can effectively induce human body produce ENIs, cheap, that be convenient to suitability for industrialized production, free from environmental pollution, resourceful endogenous interferon derivant (EnII).

Once having obtained desirable EnII just can greatly promote the well-being of mankind, be directly used in human body, the a large amount of raw A wide selection of colours and designs of various types of relevant cell high concentration real estates of induction human body, highly meet species specificity, without immunologic rejection and dependence, people's in vivo bioactivity and pharmacological action the strongest, the endogenous interferon (ENIs) of height drug effect, thereby give safely and effectively the extensive antiviral of human body, antitumor and diseases prevention, cure the disease and the function such as health invigorating, also can be used for tackling sudden public health event.

at present for the commercialization EnII of human body be mainly poly (Polyinosinic Polycytidylic Acid, poly[I:C], PIC),for micromolecule double-stranded RNA (the Chadha KC of synthetic, Dembinski WE, Dunn CB, et al.Effect of increasing thiolation of the polycytidylic acid strand of poly I:poly C on the alpha, beta and gamma interferon-inducing properties, antiviral and antipmliferative activities.Antiviral Res, 2004,64[3]: 171-7), domestic in nineteen eighty-three for clinical, use as broad-spectrum antiviral medicament.

but, in recent years find: Poly[I:C] there are a lot of problems, so that be extremely restricted in clinical practice, cannot promote application.poly[I:C] in human body and other primates bodies, easily destroyed, induce by serum nuclease the low (Taniguchi of usefulness that IFN produces, T, Takaoka, A, A weak signal for strong responses:interferon-alpha/beta revisited.Nat.Rev.Mol.Cell.Biol, 2001.2:378 – 386.).More have bibliographical information: Poly[I:C] can cause autoimmune disease, can cause a series of toxic and side effects such as organism fever, hematopoietic function decline, erythropoietin minimizing, leukopenia, liver splenocyte destruction, blood pressure and coagulation function is not normal, transaminase's rising, renal dysfunction, thereby greatly limited Poly[I:C] market and clinical practice (Nie puts into practice, Hu Dongqin, the pyrogen reaction of dislocation double stranded RNA, Chinese biological engineering, 2003,23[2]: 97-98). find and develop the medical EnII of the new second filial generation and be the biomedical family in the whole world the field of contending, people class is being pursued safe and effective medical EnII always.

5.Reovirus dsRNA possesses the potential that develops into desirable medical EnII

The mankind have recognized that: virus has the effect of induction IFN, the dsRNA of synthetic has the effect of induction IFN; So, say in theory should there is from virus genomic dsRNA molecule the potential that develops into EnII.In like manner, more should there is from the dsRNA molecule of people Reovirus the potential that develops into EnII.

Reovirus genome is made up of 10 molecule dsRNAs, and its molecular weight is 0.5 × 10 ⁶-2.7 × 10 ⁶, total molecular weight 14 × 10 ⁶-15 × 10 ⁶, account for 15%-20% of viral weight.Polyacrylate hydrogel electrophoresis (PAGE) technology can be parsed into electrophoresis pattern clearly by 10 molecule dsRNAs, this collection of illustrative plates by Reovirus dsRNAs be divided into greatly (L), in (M), little (S) three slice groups.(Dong Changyuan chief editor, " modern Molecular Virology ", publishing house of Wuhan University, in October, 1996).

Reovirus is containing 10 molecule segmentation dsRNA, with the RNA polymerase of dependenc RNA, therefore the dsRNAs of extraction and purification does not have infectivity; Its genome is double stranded rna molecule, the enzyme of resistance to RNA (RNAase); This virus does not cause mankind's major disease, is dsRNA virus, there is no tumor source property; Be natural viral, its nucleic acid is natural acid, the trouble and worry (Dong Changyuan chief editor, " modern Molecular Virology ", publishing house of Wuhan University, in October, 1996) of the ecological balance of having no effect.Reovirus, because of its special molecular structure, is convenient to vitro proliferation and purification, is conducive to industrialization.These features have been given Reovirus medical and biological engineering value extremely significantly.

In the world, in recent years convincingly demonstrated " safety of dsRNA molecule ".We confirmed Reovirus to human normal cell line if human embryonic lung cell, people's trachea smooth muscle cell etc. are without infectivity; Make median lethal dose(LD 50) mensuration with laboratory animal mice (non-sensitive animal) and not yet search out LD ₅₀, shown the safety of this virus to experiment mice.Nearly ten years, applicant and international counterparts proved again repeatedly: reovirus human (Reovirus, RV) and equal member BTV thereof are fool proof to human body; There is the anti-human cancer effect of targeting; This coe virus has been used to develop targeting oncolytic virus (Oncolytic Virus) (Hu J, Dong CY, Li JK-K, Chen DE and Liang K.Selective in vitro degradation of human cancer cells by Bluetongue virus.Acta Oncologica, 2008,47:124-134; Strong JE, Coffey MC, Tang D, Sabinin P, Lee PWK.The molecular basis of viral oncolysis:usurpation of the Ras signaling pathway by reovirus.EMBO.J, 1998,17 (12): 3351-3362; J.K.-K.Li, Oncolytic Bluetongue viruses:Promise, progress and perspectives, Frontiers in Microbiol., 2011,2:1-13.).

In recent years, applicant is upper at the human archeocyte (normal/tumor cell) of isolated culture again, with current best business-like energy for the interferon inducers Poly[I:C of human body] be contrast,comparative study Reovirus-3 and the exposed dsRNA of Reovirus (Reovirus NdsRNA) safety and as exogenous interferon (Exo-Interferon, EXI) potentiality, result shows: 1. Reovirus-3 is to people source normal cell safety; 2. the human normal cell line safety of Reovirus dsRNA to isolated culture; 3. Reovirus dsRNA can induce the human archeocyte (comprising normal cell and tumor cell) of isolated culture to produce IFN-β effectively; 4. the output of the external evoked IFN-β of Reovirus dsRNA is significantly higher than Poly[I:C] (P<0.01); 5. Reovirus dsRNA can be produced IFN and killed by induction the human cancer cell of isolated culture.

And then we have successfully set up herpes simplex virus I-form (herpes simplex virus tipe1, HSV-1) and have infected Kunming mouse experimental animal model; By this experimental animal model, with current best commercialization interferon inducers Poly[I:C] positive medicine contrast,systematically carry out the research of prevention HSV-1 infectious effect in the effectiveness of safety research in Reovirus dsRNA (Reov-dsRNA) body, induced animal generation IFN and the Research on kinds of the interior induction of body endogenous IFNs thereof, Reov-dsRNA body, the research of the interior anti-HSV-1 treatment of infection effect of body, obtained a series of extremely valuable results of study.

(1) HE dyeing pathological section shows, compared with both not inoculating HSV-1 and also do not apply the blank group of Reov-dsRNA, the cerebral tissue change of HSV-1 infection model group is obvious, has identified HSV-1 infection model and has been successfully established.

(2) mice that Reov-dsRNA processes, without obvious adverse reaction, it is safe pointing out this virus dsRNA in body.

(3) putting Interferon level result in sandwich enzyme-linked immunoadsorbent technics (ELISA) test experience animal serum by dibit shows:

(A) apply merely Reov-dsRNA can be effectively high level induction laboratory animal produce various interferon, wherein, IFN-α (234.45 ± 4.328pg/ml), IFN-β (13.684 ± 0.533ng/ml), IFN-γ (236.846 ± 11.003pg/ml);

(B) taking blank animal as contrast, Reov-dsRNA and poly[I:C] can effectively induce laboratory animal to produce endogenous interferon, as IFN-α, IFN-β, IFN-γ (P<0.05);

(C) in Reov-dsRNA body, induce IFN-α, IFN-β, IFN-γ level significantly higher than poly[I:C] (P<0.05);

(D) with the virus model group comparison of not using Reov-dsRNA, the level that prevention treated animal produces IFN-α, IFN-β, IFN-γ is significantly higher than virus model group, and difference has significance (P1<0.05).

(E) with the virus model group comparison of not using Reov-dsRNA, treatment group animal produces the level of IFN-α, IFN-β, IFN-γ significantly higher than virus model group, and difference has significance (P2<0.05).

(4) statistical analysis Reov-dsRNA prevention HSV-1 infected group and poly[I:C] prevention HSV-1 infected group mouse death rate, result shows: Reov-dsRNA prevention HSV-1 infected group mortality rate is significantly lower than poly[I:C] prevention HSV-1 infected group (P<0.05), prompting Reov-dsRNA has the effect that good prevention HSV-1 infects.

(5) statistical analysis Reov-dsRNA treatment HSV-1 infected group and poly[I:C] treatment HSV-1 infected group mouse death rate, result shows: Reov-dsRNA treatment HSV-1 infected group mortality rate is significantly lower than poly[I:C] treatment HSV-1 infected group (P<0.05), prompting Reov-dsRNA has the effect that good treatment HSV-1 infects.

(6) statistical analysis Reov-dsRNA prevention HSV-1 infected group and Reov-dsRNA treatment HSV-1 infected group mouse death rate, result shows: Reov-dsRNA treatment HSV-1 infected group mortality rate is higher than Reov-dsRNA prevention HSV-1 infected group (P<0.05), and the effect that prompting Reov-dsRNA prevention HSV-1 infects is better than the effect that Reov-dsRNA treatment HSV-1 infects.

By upper, can draw to draw a conclusion:

(1) Reov-dsRNA is safe in experimental animals;

(2) Reov-dsRNA can efficiently induce IFN-α, IFN-β, IFN-γ in experimental animals, and enhancing human body immunity function is brought into play the effect of good prevention herpes simplex infections;

(3) Reov-dsRNA can efficiently induce IFN-α, IFN-β, IFN-γ in experimental animals, and enhancing human body immunity function is brought into play the effect of good treatment herpes simplex infections;

(4) preventive effect that Reov-dsRNA infects HSV-1 is better than poly[I:C];

(5) therapeutic effect that Reov-dsRNA infects HSV-1 is better than poly[I:C];

(6) effect that seemingly, Reov-dsRNA prevention HSV-1 infects is better than the effect that Reov-dsRNA treatment HSV-1 infects.

Above result of study clearly points out applicant: Reov-dsRNA to possess to develop into the potential of desirable medical endogenous interferon derivant (EnII), has the potential that develops into significantly novel antiviral medicine.

applicant studies Reov-dsRNA as EnII, and no matter body is interior or external, all taking Poly[I:C] be contrast; No matter resonable in opinion, still in vivo, in results of in vitro studies, Reov-dsRNA EnII is all better than Poly[I:C].applicant's consideration, its reason may be (A) Poly[I:C] be the dsRNA of synthetic, Reovirus dsRNA is natural dsRNA; (B) Poly[I:C] be compared with micromolecule dsRNA, Reovirus dsRNA is macromole dsRNA; (C) Reov-dsRNA EnII is from Reovirus, and as previously mentioned, the specific conditions such as its molecular size and architectural feature (10 molecule dsRNAs, tri-slice groups of L, M and S, G+C content) are closely related.

It is very ripe that 6.dsRNA molecule inducing cell produces the cellular and molecular mechanisms of IFNs

Above, we with regard to the generation of interferon, interferon inducers, IFNs and the various important physiological function of IFNs ... Deng have been introduced.Relevant its cellular and molecular mechanisms is also studied very clearly: be first, interferon inducers is as part, act on specific cell receptor, communicate information to nucleus by cell signal path, with the various IFN genes of activation of human genome encoding, express various interferon, and be discharged into extracellular.Next is, is discharged into extracellular interferon as part, is combined with target cell specific receptor, induces target cell to produce the biological function effects such as antiviral by signal transducting system.

Reov-dsRNA and Poly[I:C] essence be all dsRNA molecule, all can induce IFN produce.In recent years confirm, TLR3 (Toll like receptor3) and RIG-I/MDA5 intracellular signal transduction pathway are the important molecule that identification dsRNA, induction I type IFN express.TLR3 is mainly expressed in immunocyte, be positioned pattern recognition receptors (the pattern recognition receptor on cellular inclusion (endosome) film, PRR), identification enters the dsRNA molecule of cell vesicle through endocytosis, and induction I type IFN expresses.RIG-I/MDA5 is positioned intracytoplasmic PRR, and dsRNA in identification kytoplasm induces IFN synthetic by not relying on the mode of TLR3.(Alexopoulou?L,Holt?AC，Medzhitov?R，et?al.，Recognition?of?double-stranded?RNA?and?activation?of?NF-kappa?B?by?toll-like?receptor3.Nature，2001,413[6857]:732-738)。Two approach mediation TLRs (Toll like receptors) signal transductions are found that there is at present, one is marrow sample differentiation factor 88 (myeloid differentiation factor 88MyD88) dependent pathway (Akashi S, Shimazu R, Ogata H, et al., Cutting edge:cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages.J.Immunol.2000, 164[7]: 3471-3475), another be MyD88 dependent/non-dependent approach (Kawai T, Adachi O, Ogawa T, et al., Unresponsiveness of MyD88-deficient mice to endotoxin.Immunity, 1999,11[1]: 115-122).MyD88 molecule is the linkers in most of TLRs signal transductions, and still, MyD88 afunction can not stop all TLRs signals completely.So must also there is the non-dependence signal pathway of MyD88 in TLRs signal transduction.TLRs is single transmembrane receptor, and extracellular region has LRRs (leucine-rich repeats) domain, identifies specific pathogen-associated molecular pattern (PAMP) part, as pathogen component, pathogen product, dsRNA etc.; Intracellular region is the TLR core parts of transmission of signal downstream.The diversity of TLRs family member's extracellular region structure is larger, is the structure basis in conjunction with different ligands.In 11 known TLRs, only have TLR3 identification external source dsRNA, synthetic (the Alexopoulou L of induction I type IFN, Holt AC, Medzhitov R, et al., Recognition of double-stranded RNA and activation of NF-kappa B by toll-like receptor3.Nature, 2001,413[6857]: 732-738).

tretinoin induced gene protein I (RIG-I)also be a kind of receptor of the viral dsRNA of identification in cell, belong to the dsRNA unwindase family member containing DexD/H box.The C end of RIG-I is the territory of untwisting, can be by unwindase domain in conjunction with dsRNA synthetic or virus, and the mode relying on ATP enzyme is untied dsRNA.N end is that the caspase of 2 series connection is raised domain (CARD), and induction IFN-β expresses and needs RIG-I.But RIG-I does not participate in signal path (Kato H, the Sato S in IFN-β downstream, Yoneyama M, et al., Cell type-specific involvement of RIG-I in antiviral response.Immunity, 2005,23[1]: 19～28). mDA5 is again that an energy is in conjunction with dsRNA, activate IRF-3 (IFN regulatory factor3), induction IFN express signaling molecule (Kata H, Takeuchi O, Sato S, et al., Differential roles of MDA5and RIG-I helicases in the recognition of RNA viruses.Nature, 2006,441[7089]: 101～105).

Interferon type Ⅰ is the crucial hinge molecule that connects the natural immunity and acquired immunity.It is not identical that different TLRs inductions produces interferon type Ⅰ ability, and the ability of different DC cell subsets generation interferon type Ⅰs is also different.

7. interferon receptors and signalling channel thereof

Product IFN disengages outside born of the same parents, as part, be combined with target cell specific receptor, activate " Janus " kinases (the Janus kinases being connected with receptor endochylema end, JAKs), JAKs is phosphorylation activation signal transducer and the tyrosine residue of transcribing in exciting son (signal transducers and activators of transcription, STATs) again, and signal is amplified.The Src homology region 2 (SH2) of the STATs tyrosine residue of phosphorylation activation and phosphorylated tyrosine forms homodimer and heterodimer.Heterodimer and homodimer all can be combined with IRF, form different trimers.The adjusting sequence (IFN stimulated regulatory elements, ISREs) that different trimers and IFN activate is in conjunction with the gene expression to drive respectively the regulation and control of IFN-α/β or the gamma regulated gene expression of IFN-.In recent years research work is got clear, dsRNA dependent kinases (double-stranded RNA-dependent Protein Kinase, PKR) and 2 '-5 ' oligo-adenylate synthetase (OAS) participates in antiviral response (the Smith E J of IFN mediation, Marie I, Prakash A, et al.IRF3and IRF7phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or Ikappa B kinase but is blocked by Vaccinia virus E3L protein.J Biol Chem, 2001, 276[12]: 8951～8957).

The research achievement of the intracellular signal transduction pathway that dsRNA molecule and inducing interferon thereof produce is also for safety is applied Reov-dsRNA EnII induction human body endogenous IFNs effectively, give human body inherently safe antiviral, antitumor effectively, diseases prevention widely, cure the disease, functions such as health invigorating and provide reliable theoretical foundation for tackling sudden public health event.

8. brief summary

Applicant's application basic theory research in early stage achievement is that solid theoretical basis and technical conditions have been established in the patent application of the present invention that applicant proposes in conjunction with the fundamental research achievement of above-mentioned series.A kind of so brand-new Poly[I:C that is better than just provided by the invention] Reov-dsRNA EnII.

Summary of the invention

The present invention just based on: IFNs is the best type cytokines that the mankind found so far, has diseases prevention and treatment function widely; Endogenous interferon (ENI) has unrivaled advantage compared with exogenous interferon (EXI); Want to expect ENI, key be to obtain can Secure Application in human body, can effectively induce human body to produce ENIs, cheap, that be convenient to suitability for industrialized production, free from environmental pollution, resourceful EnII; The current commercialization EnII for human body, Poly[I:C], have a lot of problems, so that be extremely restricted in clinical practice; Find and develop the medical EnII of the new second filial generation and be the biomedical family in the whole world the field of contending, the mankind are pursuing safe and effective medical EnII newly always.

The present invention is applied to reovirus double-stranded RNA (Reovirus dsRNA) to prepare endogenous interferon derivant (Endo-Interferon Inducer, EnII), forms Reov-dsRNA EnII.This Reov-dsRNA EnII has potential applicability in clinical practice, direct injection human body, in human body, follow under the condition of self-defense reactive mode, induction human body produces various endogenous interferon (Endo-Interferon, ENIs), give the treatment of human body viral diseases, viral diseases prevention, antineoplastic treatment and therapeutic alliance and diseases prevention widely, cure the disease and constitutional function, and for tackling the important function such as sudden public health event.This patent provides this brand-new Poly[I:C that is better than] Reov-dsRNA EnII not only have height safety and high efficiency, be applicable to the mankind's urgent need, and cheap, be convenient to suitability for industrialized production, free from environmental pollution, aboundresources.

This EnII has important medical function like this, is mainly its natural molecule structure.As long as we can effectively breed efficiently Reovirus, efficient high-purity and extract its dsRNA nucleic acid, and finally prepare in adult with nucleic acid preparation etc., can become brand-new, safer, more effective, natural, infinitely industrialization, that the mankind are badly in need of EnII, promote the well-being of mankind.

Technical essential of the present invention:

(1) by cell in vitro culture systems propagation Reovirus;

(2) extract highly purified Reovirus dsRNA molecule;

(3) be prepared into the Reov-dsRNA EnII that can be directly used in human body.

All energy meet the technical method that obtains high-purity Reovirus dsRNA and be prepared into Reov-dsRNA EnII all can reach this object.For example, applicant has taked following technical measures to realize target of the present invention early stage, and its basic step is:

1. cell used and cultivation thereof: taking world health organisation recommendations as producing the desirable cell of vaccine for man,----African green monkey kidney cell (Vero cell), Chinese hamster ovary cell (Chinese hamster ovary, CHO), young hamster kidney passage cell (BHK-21) they are virus amplification cultured cell.Cell is at 37 DEG C, 5%CO ₂, 10% calf serum, DMEM culture medium cellar culture.

2.Reovirus cultivates propagation: in the time that cell grows to 80%-90% and converges, abandon culture fluid, with the 1 × PBS washing attached cell of pH7.4 1-2 time, inoculation Reovirus suspension 1.0ml is in culture bottle, 37 DEG C of absorption are after 1-2 hour, abandon virus liquid, with containing the interpolation DMEM culture medium of 2% calf serum at 37 DEG C, 5% CO ₂maintain under condition.Once, there is obvious cytopathic effect (CPE) to cell in every 12 hours subsequently light Microscopic observations, the cultured cell of the Reovirus of results propagation in the time that CPE reaches 90%, discards culture fluid.

The first extraction of 3.Reovirus and the purification of Reovirus dsRNA:

Valve rubber broom scrapes the band poison cell that discards culture fluid, is collected in sterile test tube; Add 10 times of volume DEPC water, fully dissolve band poison cell;-20 DEG C of multigelations 3 times, 12000-15000rpm, centrifugal 10-15min, collects supernatant; The centrifugal 30min of 4000rpm after saturated ammonium sulphate supernatant, containing the standby dsRNA purification of being used as of virus precipitation.

Adopt TRIzol method to extract preparation Reovirus dsRNA.Equal-volume PBS (pH7.4) dissolves containing virus precipitation, dress bag filter; DEPC water dialysis, proceeds to EP pipe after thoroughly removing ammonium sulphate; Add 9.5 times of volume Trizol, lytic virus; Add 0.2 times of volume chloroform to lytic virus liquid; 12000g, centrifugal 12-15min, gets upper strata water and collects Reovirus RNA; Equal-volume isopropanol precipitating Reovirus RNA; Abandon supernatant, 75% washing with alcohol precipitation, air-dry; DEPC water dissolution Reovirus RNA ,-20 DEG C store for future use.

4. extract the qualification of Reovirus genome dsRNA:

(1) adopt agarose gel electrophoresis technology and technique of polyacrylamide gel electrophoresis, analytical electrophoresis collection of illustrative plates qualification Reovirus genome dsRNA.

(2) extract Reovirus dsRNA Purity: combination (A) employing ultraviolet spectrophotometry (according to OD260/OD280 ratio) and (B) technique of polyacrylamide gel electrophoresis are in conjunction with the purity of the running gel background protein staining technology judgement Reovirus dsRNA that extracts.

(3) adopt determined by ultraviolet spectrophotometry dsRNA content, according to dsRNA concentration=OD ₂₆₀× extension rate × 50 (μ g/ml) formula calculates Reovirus dsRNA concentration.

5. normal saline is prepared into Reov-dsRNA EnII injection type, uses through intramuscular injection.

Compared with prior art, tool of the present invention has the following advantages and beneficial effect:

(1) Reov-dsRNA EnII is better than best business-like Poly[I:C used at present significantly], can apply to very safely human body.Reovirus is dsRNA virus, to human security, does not cause any major disease of the mankind; This virus is containing segmentation dsRNA, with the RNA polymerase of dependenc RNA, therefore the Reovirus dsRNAs of extraction and purification does not have infectivity; Reovirus is dsRNA virus, there is no tumor source property; In recent years human research's great achievement conclusion especially, dsRNA molecule is to human security, and macromole dsRNA can inducing interferon, and micromolecule dsRNA induction RNA interferes (RNAi).So brand-new Reov-dsRNA EnII of the present invention can be applied to human body very safely.

(2) clinical effectiveness of Reov-dsRNA EnII of the present invention is better than best business-like Poly[I:C used at present significantly].Poly[I:C] be the small fragment dsRNA molecule of synthetic, the ability that induces IFN is low, with many side effect, and expensive, market is subject to great restriction. the present invention in research Reov-dsRNA EnII process, no matter in body still external, all taking Poly[I:C] be contrast; No matter in theory, still in vivo, in outer experimental result, Reov-dsRNA EnII all be better than Poly[I:C].

(3) Reov-dsRNA EnII is new desirable natural acid medicine, has medicine effect widely and physiological regulation function.IFNs is the best type cytokines that the mankind found so far, not only has antiviral and antitumor action, and has constitutional opsonic action.Reov-dsRNA EnII prepared by the present invention is medical endogenous interferon derivant, directly applies to human body, and induction body produces the endogenous interferon (ENIs) of A wide selection of colours and designs.In this case, body has become the compound factory that produces interferon, to produce compound interferon, and performance antitumor, infection and opsonic action.This ENIs is natural interferon, so, in human body, directly induce the ENIs producing can overcome many deficiencies of exogenous interferon with EnII.Immunogenicity (being anaphylaxis and rejection) that IFN A wide selection of colours and designs, bulk concentration that Reov-dsRNA EnII induces in human body be high, do not have heterologous protein, there is no dependency; Can be used as medicine for treatment, also can be used as prophylactic; Both can be used for viral diseases treatment, also can be used for viral diseases prevention; Can resist known viruse, also can anti-unknown virus (comprising potent virus, as SARS-CoV, bird flu virus); Can antiviral therapy, also can antineoplaston (comprising associating antineoplaston), there is diseases prevention widely, cure the disease and constitutional function, and for tackling the important function of sudden public health event.Also because IFNs has relative species specificity, so ENIs antiviral, antitumor and the conditioning functions activity of being induced in vivo by Reov-dsRNA EnII are the strongest.EnII prepared by the present invention follows the defense reaction rule of body generation interferon, the generation that can more effectively induce ENIs completely for human body.

(4) Reov-dsRNA EnII also can be used as Reov-dsRNA exogenous interferon inducer (ExII) in the nature of things.

(5) brand-new theoretical basis.Due in recent years about dsRNA molecule as part and target cell specific receptor, as TLR3, the achievement accumulation of interaction and the research of cell signal path thereof, make the consequence of dsRNA and neuron target cell interaction and the maturation of cellular and molecular mechanisms thereof, for direct application Reov-dsRNA EnII is in human body, induce variously to produce IFNs (being ENIs) in somatic cell and established theoretical basis.In Reov-dsRNA EnII body, the mechanism of action of induction ENIs is that application Reov-dsRNA molecule is by the expression of above-mentioned mechanism direct regulation and control human archeocyte interferon gene.This is a brand-new theoretical system, has disclosed exposed virus genome dsRNA and need not directly be applied to human body by the mode of injection by transgenic technology, and effectively induced body to produce various ENIs safely, brings into play its pharmacology and physiological action.

(6) there is significant resources advantage.Reovirus dsRNA is a kind of brand-new natural green bio material that can infinite regeneration to people's safety, inexhaustible.Reov-dsRNA EnII just taking extract the natural endogenous interferon derivant of preparation as resource.

(7) prepare natural dsRNA EnII with Reovirus resource, be convenient to vitro proliferation and purification, without purification, cheap, be conducive to industrialization, production cost is low, is conducive to the market supply.

(8) because the speed of inducing ENIs to produce in Reov-dsRNA EnII body is fast, safe, efficient, cheap, so, can be used for routine prevention and the treatment of clinical disease, also can be used for tackling sudden public health event.

(9) the EnII market that prepared by the present invention is very large.Medical value and the market prospect of desirable EnII shown in applicant's previous work.Reov-dsRNA EnII has the physiological function that induction IFN produces, and have extensive antiviral and anticancer effect, and this two classes disease of viral disease and neoplastic disease is all at present human health and life to be threatened to maximum disease.From the appearance of (as: AIDS, SARS, bird flu, tuberculosis etc.) such as in recent years new virosiss and popular, the prevention of new infectious disease seem particular importance, the as can be seen here ample scope for abilities of Reov-dsRNA EnII.So Reov-dsRNA EnII both had wide market in new, the old prevention of infectious disease and treatment, have again the great value of tackling sudden public health event, such as it was the first medicine of resistance of hepatitis B, has some idea of.Because ENI has unrivaled advantage compared with EXI, so desirable Reov-dsRNA EnII will occupy IFN and produce relevant very big market, and can greatly expand existing IFN market.

Brief description of the drawings

Fig. 1. reovirus (Reovirus) structural representation.Virus is spherical in shape, double capsid, and genome segmentation is arranged in monolayer inner capsid.

Fig. 2. propagation infects SV 59 virus (Reovirus-3) the ultra micro section Electronic Speculum figure of vero cell.

Fig. 3. In vitro culture Reovirus dsRNA genome 10%PAGE collection of illustrative plates.A is the vero cell extract of uninfecting virus; B & C is the vero cell extract that infects Reovirus, shows 10 molecule dsRNA bands; D is 1.0kb DNA marker (purchased from Canadian Fermentas company).

Fig. 4. do not infect the Vero cell of HSV-1.Under fluorescence microscope, the Vero cell that does not infect HSV-1 has no green fluorescence, and under ultraviolet light, visible cell is fusiformis, queueing discipline, and riotous growth, has no cytopathic effect (CPE).

Fig. 5. the herpes simplex virus type 1 (HSV-1) of Green Fluorescent Protein infects vero cell.Experiment infects by the HSV-1 propagation of Green Fluorescent Protein the Vero cell of cultivating, and sends strong green fluorescence.This image also obviously presents: infect HSV-1 vero cellular contraction, become the cytopathic effect (CPE) such as justify, come off.

The virus multiplication image of the inoculation Vero of Fig. 6 .HSV-1 infecting mouse model brain tissue homogenate cell.Get the inoculation Vero of HSV-1 infecting mouse model brain tissue homogenate cell, experimental cell sends green fluorescence clearly, illustrates: HSV-1 infecting mouse model is successfully established, and is successfully separated to HSV-1 from animal pattern cerebral tissue.This image also obviously presents: infect HSV-1 vero cellular contraction, become the cytopathic effect (CPE) such as justify, come off.

Fig. 7 .HSV-1 propagation infects vero cell micro observation result.Infected vero cell sends green fluorescence; The density of green fluorescence and intensity direct reaction HSV-1 copy the amount of propagation.The lower right corner purple figure of this Fig. 7 is the In vitro culture vero cell (seeing Fig. 4) of not inoculating HSV-1; 3 figure are the In vitro culture vero cell that has infected HSV-1 above; The lower left corner and middle 2 figure are that mouse model brain tissue homogenate is inoculated in Vero cell below, and green fluorescence shows that a large amount of HSV-1 are at Vero cell proliferation.

Fig. 8. normal cerebral tissue's paraffin section, optical microphotograph Microscopic observation image after HE dyeing.Normal cerebral tissue as can be seen from Fig. 8, layer of structure is clear, without hyperemia or inflammatory cell infiltration.(amplification is shown in scale in figure)

Fig. 9 .HSV-1 Mice Inoculated cerebral lesion histopathology optical microscope photograph 1, shows that HSV-1 infecting mouse model is successfully established.Visible in Fig. 9: a large amount of core is assembled and sticks in blood vessel wall by the monokaryon inflammatory cell of engrain, and present the tendency that tissue spreads towards periphery.(amplification is shown in scale in figure)

Figure 10 .HSV-1 Mice Inoculated cerebral lesion histopathology optical microscope photograph 2, shows that HSV-1 infecting mouse model is successfully established.Visible in Figure 10: the monokaryon inflammatory cell of a large amount of nuclear hyperchromatisms is gathered in cerebral tissue, form kitchen range shape.(amplification is shown in scale in figure)

Figure 11 .HSV-1 Mice Inoculated cerebral lesion histopathology optical microscope photograph 3, shows that HSV-1 infecting mouse model is successfully established.Visible in Figure 11: large stretch of cerebral tissue necrosis, brain cell cracking; Screened stock disengages, and cell boundaries is unclear, dyes redness, fuzzy a slice; Karyon engrain, concentrated, dispersion of fragment are in red Cytoplasm region.(amplification is shown in scale in figure)

Figure 12 .Reov-dsRNA EnII induction 3 kinds of interferon (IFN-α, IFN-β, IFN-γ) effect result of study statistical analysis figure.In Figure 12: 1 is normal saline (NS) group; 2 is Reov-dsRNA EnII group; 3 is Poly[I:C] group.From Figure 12: taking the interferon of blank (NS) group as background, with NS contrast, Reov-dsRNA EnII and Poly[I:C] all can effectively induce 3 kinds of IFNs, various IFNs levels are significantly raised; With Poly[I:C] contrast, Reov-dsRNA EnII induces various IFNs effects to be significantly higher than Poly[I:C], point out Reov-dsRNA EnII to apply greatly potential.

The statistical analysis figure of Reov-dsRNA EnII induction 3 kinds of interferon (IFN-α, IFN-β, IFN-γ) effect in the research of Figure 13 .Reov-dsRNA EnII prevention HSV-1 infection experiment.In Figure 13: 1 is normal saline (NS) group; 2 is Reov-dsRNA+HSV-1 group; 3 is Poly[I:C]+HSV-1 group.From Figure 13: taking the interferon of blank (NS) group as background, Reov-dsRNA+HSV-1 group and Poly[I:C]+HSV-1 group express 3 kinds of IFNs generation significantly higher than NS group.This just shows, Reov-dsRNA and Poly[I:C] can effectively induce the expression of various interferon, various IFNs levels are significantly raised; And, with Poly[I:C] and contrast, Reov-dsRNA induces the effect of various IFNs to be significantly higher than Poly[I:C].This just for setting up the immunologic mechanism of viral infection resisting before poisoning intrusion with Reov-dsRNA EnII, opposing poisoning intrusion causes a disease laboratory theoretical foundation is provided; Also provide laboratory theoretical foundation for using Reov-dsRNA EnII to tackle sudden public health event.

The statistical analysis figure of Reov-dsRNA EnII induction 3 kinds of interferon (IFN-α, IFN-β, IFN-γ) effect in the research of Figure 14 .Reov-dsRNA EnII treatment HSV-1 infection experiment.In Figure 14: 1 for only using HSV-1 infected group; 2 is HSV-1+Reov-dsRNA group; 3 is HSV-1+Poly[I:C] group.From Figure 14: with the group 1 only infecting with HSV-1 relatively, first cause after infection again by the group 2 (HSV-1+Reov-dsRNA group) of Reov-dsRNA treatment with after first causing infection with HSV-1 and use again Poly[I:C with HSV-1] output of the group 3 for the treatment of (HSV-1+Poly[I:C] group) 3 kinds of IFNs of expression increases very significantly.This just shows, Reov-dsRNA and Poly[I:C] can effectively infect rear expression of inducing various interferon at HSV-1, various IFNs levels are significantly raised; And, with Poly[I:C] and contrast, Reov-dsRNA induces the effect of various IFNs to be significantly higher than Poly[I:C].This,, with regard to for application Reov-dsRNA EnII after infecting at poisoning intrusion treats, realizes viral diseases laboratory theoretical foundation is provided; Also provide laboratory theoretical foundation for using Reov-dsRNA EnII treatment unknown virus to infect.

Figure 15. apply respectively Reov-dsRNA EnII, Poly[I:C], the comparative study statistical analysis figure of 3 kinds of interferon (IFN-α, IFN-β, IFN-γ) output in laboratory animal serum after HSV-1.In Figure 15: 1 is that HSV-1 set of applications, 2 is for Poly[I:C] set of applications, 3 is Reov-dsRNA EnII set of applications.Minimum from the effect of Figure 15: HSV-1 3 kinds of interferon of induction (IFN-α, IFN-β, IFN-γ); Poly[I:C] induce the effect of 3 kinds of interferon (IFN-α, IFN-β, IFN-γ) significantly strong than HSV-1; The effect of Reov-dsRNA EnII 3 kinds of interferon of induction (IFN-α, IFN-β, IFN-γ) is the strongest, has significantly exceeded Poly[I:C].This has just significantly pointed out: novelty, scientific value and the significant clinical value of Reov-dsRNA EnII.

Figure 16. make blank taking HSV-1 as background, Poly[I:C] doing positive drug contrast, research Reov-dsRNA EnII prevention HSV-1 induces laboratory animal to produce the statistical analysis figure of IFN-α, IFN-β, IFN-γ effect while infection.In Figure 16: 1 for only using HSV-1 processed group; 2 is Poly[I:C]+HSV-1 processed group, first use Poly[I:C] process the rear HSV-1 counteracting toxic substances of using; 3 is Reov-dsRNA+HSV-1 processed group, first processes the rear HSV-1 counteracting toxic substances of using with Reov-dsRNA EnII.This research is intended to inquire into the relation that on the basis of Reov-dsRNA EnII prevention HSV-1 infection effect, further research Reov-dsRNA EnII induces 3 kinds of interferon (IFN-α, IFN-β, IFN-γ) generation and its prevention HSV-1 to infect.There is the effect of 3 kinds of interferon of induction (IFN-α, IFN-β, IFN-γ) in certain body from Figure 16: HSV-1; Make blank taking HSV-1 as background, further obtain Poly[I:C] in body the effect of 3 kinds of interferon of induction remarkable stronger than HSV-1; Poly[I:C] contrast of the positive medicine of+HSV-1, then the effect that further obtains 3 kinds of interferon of induction in Reov-dsRNA EnII body has significantly exceeded Poly[I:C], in three, in body, induce the effect of 3 kinds of interferon the strongest.This has just further pointed out: novelty, scientific value and the significant clinical value of Reov-dsRNA EnII.

Figure 17. make blank taking HSV-1 as background, Poly[I:C] doing positive drug contrast, research Reov-dsRNA EnII treatment HSV-1 induces laboratory animal to produce the statistical analysis figure of IFN-α, IFN-β, IFN-γ effect while infection.In Figure 17: 1 for only using HSV-1 processed group; 2 is HSV-1+Poly[I:C] processed group, first use HSV-1 infection animal, rear with Poly[I:C] treatment; 3 is HSV-1+Reov-dsRNA EnII processed group, first uses HSV-1 infection animal, rear with Reov-dsRNA EnII treatment.This research is intended to inquire on the basis of Reov-dsRNA EnII treatment HSV-1 infection effect further research Reov-dsRNA EnII 3 kinds of interferon of induction (IFN-α, IFN-β, IFN-γ) and produces and the relation of its treatment HSV-1 infection effect.There is the effect of 3 kinds of interferon of induction (IFN-α, IFN-β, IFN-γ) in certain body from Figure 17: HSV-1; Make blank taking HSV-1 as background, further obtain Poly[I:C] in body the effect of 3 kinds of interferon of induction remarkable stronger than HSV-1; HSV-1+Poly[I:C] contrast of positive medicine, then the effect that further obtains 3 kinds of interferon of induction in Reov-dsRNA EnII body has significantly exceeded Poly[I:C], in three, in body, induce the effect of 3 kinds of interferon the strongest.Similarly, this research has further been pointed out: novelty, scientific value and the clinical value of Reov-dsRNA EnII.

Figure 18. 3 kinds of interferon of different seminar (IFN-α, IFN-β, IFN-γ) expression comparative study block diagram.In Figure 18: ^*represent each seminar and the comparison of blank group, P < 0.05; ^*represent each seminar and the comparison of HSV-1 infection model group, P < 0.05; △ represents Reov-dsRNA+HSV-1 group and Poly[I:C]+HSV-1 organizes relatively, P < 0.05; △ △ represents HSV-1+Reov-dsRNA group and HSV-1+Poly[I:C] organize relatively P < 0.05.

Detailed description of the invention

Requiring based on patent of the present invention is " application of reovirus dsRNA (Reovirus dsRNA) in medical endogenous interferon derivant (EnII) preparation ", this Reov-dsRNA EnII will directly apply to human body by injection, induction human body produces various endogenous interferon (Endo-Interferons, ENIs), thereby give human body antiviral, antitumor and diseases prevention widely, cure the disease and constitutional function, also can be used for tackling sudden public health event.So this section will further be set forth the present invention with specific embodiment.At this, we emphasize that following embodiment is only for further illustrating the present invention, but not for limiting the protection domain of requirement of the present invention.

Recovery and the subculture (taking vero cell as example) of the subculture cell line of vero cell and other sensitive cellss for embodiment 1. reoviruses (Reovirus) amplification

1.1Vero cell culture and propagation

Adopt DMEM culture medium, add 10% calf serum, adjust pH to 7.0-7.4, put 37 DEG C, 5%CO ₂under condition, cultivate, propagation Vero cell, step is as follows.

1.1.1 from liquid nitrogen container, take out African green monkey kidney cell (Vero cell) cryopreservation tube, be placed in rapidly 37 DEG C of water water-baths that mix up in advance temperature, rock gently cryopreservation tube, make to melt completely rapidly in the cell suspension 1min in cryopreservation tube.

1.1.2 after melting, cryopreservation tube is carried out centrifugal, cryopreservation tube is put into centrifuge, 800-1000rpm, 10-20min, precipitates cell completely; Supernatant is abandoned in suction, adds 1.5mL DMEM culture fluid, and piping and druming makes to precipitate Eddy diffusion.

1.1.3 again that cryopreservation tube is centrifugal, 800-1000rpm, 10-20min, precipitates cell completely; Supernatant is abandoned in suction, adds the DEME culture fluid containing 10 ﹪ (v/v) calf serum, piping and druming precipitation cell dispersion; With same culture fluid adjusting cell suspension, making final concentration is (0.8-1.2) × 10 ^3-4cell/mL.

1.1.4 by every 8-12mL cell suspension inoculation one culture bottle, supply culture fluid, 37 DEG C, 5 ﹪ CO ₂(v/v) under condition, leave standstill and cultivate; After 24 hours, change DMEM culture fluid, to remove not adherent cell and cell debris.

1.1.5 the variation of bore hole observation every day culture fluid, Microscopic observation cultured cell growth conditions, changes and states DMEM culture fluid once for every 1～2 day.

1.1.6 treat more than 80% can carry out passage cultivation at the bottom of Growth of Cells is paved with bottle, from incubator, take out Tissue Culture Flask, discard culture fluid, use without calcium magnesium phosphate balance buffer (1 × PBS, pH7.4) and wash cell 2-3 time; Add 0.25% (w/v) trypsin solution 6-10 to drip; Rotate culture bottle, make trypsin solution can infiltrate whole cell face; Be placed in 37 DEG C of incubator incubation numbers minute, under optical microscope, observe cellular contraction and become after circle, carefully discard trypsin solution.

1.1.7 add the DMEM culture medium containing 10% (v/v) calf serum, blow and beat gently the cell being attached on bottle wall with suction pipe, make it to be all shed in culture fluid, form cell suspension.

1.1.8 in cell suspension, add the DMEM culture medium that contains 10% (v/v) calf serum to final volume 15-25mL, average subpackage cell suspension is in 2-3 new culture bottle.

The cultivation of 1.2 cells after going down to posterity

Adopt the DMEM culture fluid containing 10% (v/v) calf serum, in 37 DEG C, 5% (v/v) CO ₂under condition, continue to cultivate, be and go down to posterity.

Embodiment 2.Reovirus (seeing Fig. 1, Fig. 2) propagation

2.1 choose vero cell in good condition, converge rawly when reaching 80%-90% and converging until cell, inhale and abandon culture fluid; With 1 × PBS (NaCl:8g, KCl:0.2g, the Na of pH7.4 ₂hPO ₄: 1.44g, KH ₂pO ₄: 0.24g, H ₂o:800ml; Hydrochloric acid is adjusted pH7.4, mends H ₂o is to 1000ml) wash attached cell 2-3 time.

2.2 press 2-4MOI (multiplicity of infection) inoculation reovirus (Reovirus) in cultured cell; 37 DEG C of viral adsorptions, after 1～2 hour, are abandoned virus liquid, add the DMEM culture medium containing 2% hyclone, and 37 DEG C, 5%CO ₂(v/v) maintain under condition.

Once, there is obvious pathological changes effect to cell in 2.3 every 12 hours subsequently light Microscopic observations, gathers in the crops the attached cell bottle of viral infection when CPE reaches 90%, puts-20 DEG C and save backup.

Embodiment 3.Reovirus toxicity test

3.1 take out-20 DEG C saves backup Reovirus infection cell, multigelation virus 3 times, and releasing virus becomes Reovirus suspension.

3.2 use 1 × PBS (pH7.4) make 10 × gradient dilution to Reovirus suspension, and gradient is from 10 ⁰-10 ^-8, when dilution, note replacing rifle head after each gradient dilution completes.

After 3.3 trypsinization Reovirus, add 15ml cell culture fluid with cell dispersion.

3.4 blood counting chamber cell countings (1ml cell number=every lattice average cell number × 4 × 10 ⁶× extension rate) must cell concentration be 3.86 × 10 ⁸/ ml, every hole adds 100 μ l cell suspension.

3.5 96 orifice plates are placed in 37 DEG C, 5%CO ₂(v/v) under condition, hatch after 12 hours, suck culture fluid, add the virus liquid of gradient dilution, each gradient 6 holes, every hole 100 μ l, need change rifle head while noting each gradient liquid feeding.

3.5 use sealed membranes seal 96 orifice plate junctions, are placed in 37 DEG C, 5%CO ₂(v/v) under condition, hatch, within every 12 hours, observe once, record cytopathy hole count, use Reed-Muench method to calculate TCID ₅₀(TCID ₅₀logarithm poor of highly diluted logarithm+distance proportion x coefficient of dilution of positive percentage ratio of logarithm=be greater than 50%) be 4.86 × 10 ⁶/ ml.

Embodiment 4.Reovirus just extracts

4.1 discard culture fluid in the cytopathogenic culture bottle of Reovirus, and valve rubber broom scrapes band poison cell, and centralized collection is in sterile test tube;

4.2 add 10 times of volume pyrocarbonic acid diethyl esters (DEPC) water, make to be with poison cell fully to dissolve;

4.3-20 DEG C of multigelation 3 times, 12000-15000rpm, 4 DEG C of centrifugal 10-15min, abandon precipitation, collect supernatant;

4.4 supernatant adds saturated ammonium sulfate, making ammonium sulfate final concentration is 70% (by resetting and adding 3 parts of saturated ammonium sulfates in 1 part of virus), precipitates 2 hours, the then centrifugal 30min of 4000rpm in 25 DEG C (or room temperature), get containing virus precipitation, for being used as viral dsRNA purification.

Embodiment 5.TRIzol method is extracted preparation Reovirus RNA

5.1 equal-volume 1 × PBS (pH7.4) dissolve above-mentioned containing virus precipitation, dress bag filter;

5.2 use DEPC water, in 4 DEG C of dialysis 24-48 hour, changed DEPC water once every 3-6 hour during this time, after thoroughly removing ammonium sulphate, proceeded to EP pipe;

5.3 add 9.5 times of volume Trizol Reagent whirlpool concussions 30 seconds, lytic virus, and after solution is limpid, ice bath 8-12 minute;

5.4 add 0.2 times of volume chloroform to lytic virus liquid, agitating solution, and ice is educated 10-15min;

5.5 use 12000g, 4 DEG C, centrifugal 12-15min, gets upper strata water and collects Reovirus RNA in EP pipe;

5.6 add equal-volume isopropyl alcohol to manage in EP, fully mix, and ice is hatched 10min, 10000-12000g, 4 DEG C, centrifugal 8-12min, the pipe visible water white transparency Reovirus in end RNA precipitation

5.7 abandon supernatant, add 0.8-1.2ml75% ethanol, put upside down washing precipitation for several times;

5.8 12000g, 4 DEG C, centrifugal 4-6min, abandons supernatant, stays precipitation Reovirus RNA, air-dry;

5.9 add appropriate DEPC water dissolution Reovirus RNA, and-20 DEG C store for future use.

Embodiment 6. prepares the characterization and evaluation of Reovirus dsRNA

6.1 adopt conventional 10-12% polyacrylate hydrogel electrophoretic techniques (PAGE) to resolve Reovirus Genome Atlas, to identify Reovirus dsRNAs.With tbe buffer liquid (10mmol/L Tris-Cl, pH7.4,1mmol/L EDTA) preparation 10-12% polyacrylate hydrogel, the thick 1.5mm of glue; Make electrode buffer with electrophoretic buffer (89mmol/L Tris-boric acid, 2mmol/L EDTA, pH8.0); Reovirus dsRNA testing sample adds the 6 × load buffer (Loading buffer:0.25% bromophenol blue, 0.25% diformazan cyanophenyl indigo plant, 40w/v% sucrose) of 1/6 volume, after mixing by every hole 3 μ g dsRNA loadings.Enter glue with 7V/cm voltage, 4V/cm voltage separates.Electrophoresis 4～5 hours, 0.5 μ g/ml bromination second indigo (EB) dyeing 30～60min, after rinsed with deionized water, analysis result under uviol lamp.

The purity of Reovirus dsRNA is prepared in 6.2 ultraviolet spectrophotometry evaluations.Surveying object Reovirus dsRNA due to this patent is natural macromole dsRNA, unartificial synthetic oligonucleotide or primer, and its CG content is more even, so UV spectrophotography can be ideally for purity testing.

6.2.1 the Reovirus dsRNA that gets-20 DEG C of storages prepared by 2 μ l, is diluted to 50 μ l through blank solution, as testing sample, measures respectively OD by UV spectrophotometer ₂₃₀, OD ₂₆₀, OD ₂₈₀, OD ₃₂₀deng the value of four points, in order to conjoint analysis testing sample purity.

6.2.2 calculate OD ₂₆₀/ OD ₂₈₀ratio, to assess the purity of extracting Reovirus dsRNA, ratio is more than or equal to 2.0 for qualified.Pure RNA sample, ratio is greater than 2.0.If ratio, lower than 1.8 or 2.0, represents to exist the impact of protein or aldehydes matter.If OD ₂₆₀/ OD ₂₈₀ratio is between 1.8-2.0, and interpret sample purity is up to standard, OD ₂₆₀numerical value effective; Otherwise, OD ₂₆₀value be judged to invalid.Take out 2 μ l from the Reovirus of-20 DEG C of storages dsRNA, then do to use UV spectrophotometer measuring OD after 25 times of dilutions with DEPC water ₂₆₀and OD ₂₈₀value, is respectively: OD ₂₆₀=0.428; OD ₂₈₀=0.207.The Reovirus dsRNA purity of extracting is A ₂₆₀/ A ₂₈₀value=2.0676, illustrates that the Reovirus dsRNA purity of extracting is higher.

6.2.3OD ₂₃₀represent to exist in sample the pollution of polypeptide, phenol, carbohydrate etc.Pure sample, OD ₃₂₀generally 0.We preparation Reovirus dsRNA requires OD ₃₂₀be 0.

The further purity evaluation of 6.3 preparation Reovirus dsRNA.

6.3.1 the Reovirus dsRNA purity of extracting in conjunction with the PAGE collection of illustrative plates context analyzer of the Reovirus dsRNA of aforementioned extraction, assesses the existence of other nucleic acid.

6.3.2 by aforementioned PAGE, adopt protein polypeptide dyeing, to show that PAGE collection of illustrative plates background has or not protein polypeptide to pollute or pollution level.

Embodiment 7. prepares mensuration and the cubage thereof of Reovirus dsRNA concentration

To its OD of UV spectrophotometric determination for Reovirus dsRNA sterling ₂₆₀value, to calculate the concentration of purification of samples, and calculates the content of prepared Reovirus dsRNA.

Take out 2 μ l from the Reovirus of-20 DEG C of storages dsRNA, then do to use UV spectrophotometer measuring OD after 25 times of dilutions with DEPC water ₂₆₀=0.428, so, the Reovirus dsRNA solution concentration=OD of low temperature storage ₂₆₀× extension rate × 50=0.428 × 25 × 50=535.0 μ g/mL=0.535mg/mL; Reovirus dsRNA content=0.535mg/mL × 8650.48mL=4627mg=4.628g in 8.65L (8650.48mL) solution of-20 DEG C of storages.

Embodiment 8. herpes simplex virus type 1s (HSV-1) propagation

Can serve as its proliferative cell by responsive any cell of breeding herpes simplex virus type 1 (HSV-1).This embodiment 7 is taking Vero cell as example.

8.1 choose cultured cell in exponential phase (as Vero cell, Hep-2 cell ... .. etc.), converge rawly when reaching 80%-90% and converging until cell, inhale and abandon culture fluid; 1 × PBS (pH7.4) washing attached cell 2-3 time.

8.2 inoculation HSV-1 virus liquid 1.0ml, in culture bottle, rock culture bottle gently, allow virus liquid cover cell.

8.3 make viruses adsorption 1 hour in 37 DEG C, inhale and abandon residue virus liquid; Add containing the maintain liquid of 2% calf serum and cultivate.

8.4 put 37 DEG C, 5%CO ₂cultivate 24 hours.Treat that cell occurs, justify, come off as cell swells, becomes etc., when obvious cytopathic effect (CPE), collect virus-culturing fluid.

8.5 by virus-culturing fluid in-20 DEG C of multigelations 3 times, fully to disengage virus, become HSV-1 suspension, 800-1000rpm, centrifugal 8-15min, aseptic results virus supernatant, TCID ₅₀technology for detection virus titer is about 10 ⁷tCID ₅₀/ ml ,-20 DEG C save backup.

Embodiment 9. herpes simplex virus type 1s (HSV-1) toxicity test

Any cell of the responsive propagation of energy HSV-1 can be served as the cell that this virus titer is measured.This embodiment 8 is also taking Vero cell as example.

The 9.1 Vero cells that go down to posterity, trypsinization becomes individual cells suspension, and piping and druming is evenly.

9.2 draw a small amount of cell suspension, and blood counting chamber counting, adjusts to the suitable order of magnitude by cell quantity.

9.3 draw cell suspension, add 96 orifice plates by every hole 100 μ l, and 37 DEG C, 5%CO ₂under condition, cultivate.

9.4 use maintenance mediums are to HSV-1 stock solution (10 ⁰) make log10 dilution with 1 × PBS (pH7.4), to become 10 ⁰, 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8, 10 ^-9deng, dilution factor of every work is changed a suction pipe, in case prolong after virus titer.

9.5 grow up to after monolayer in 96 orifice plate bottoms until cell, inhale and abandon culture fluid, and every hole adds the virus liquid 100 μ l of dilution; 96 orifice plates all add 1 × PBS (pH7.4) in hole around, and every dilution factor adds 6 multiple holes.

9.6 culture plates were put 37 DEG C of absorption after 1.5-2.0 hour, inhaled and abandoned virus liquid, added the not cell maintenance medium containing serum; Put 37 DEG C, 5%CO ₂under condition, cultivate, every 12 hours observation of cell pathological changes, record each gradient cytopathy hole count.

9.7 calculate than distance, with Reed-Muench method calculating TCID ₅₀(TCID ₅₀logarithm poor of highly diluted logarithm+distance proportion × coefficient of dilution of positive percentage ratio of logarithm=be greater than 50%), obtaining virus concentration is 10 ⁶-10 ⁸tCID ₅₀/ ml, physiological saline solution is diluted to 10 ⁵tCID ₅₀/ ml ,-20 DEG C save backup.

The foundation of embodiment 10.HSV-1 infected animal model

10.1 60 kunming mices (4 week age), male and female half and half, are divided into 6 groups at random, and 10 every group, isolated rearing.

The HSV-1 (10 of 10.2 propagation ⁵tCID ₅₀/ ml) become 10 with physiological saline solution (0.9%) logarithm gradient dilution ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5five gradients.

The corresponding gradients of 10.3 every groups of mices, after cotton ball soaked in alcohol sterilization every animal along canthus, right side to ear after root line mid point inserting needle, intracranial injection 30 μ l; Matched group injection physiological saline solution 30 μ l.

10.4 sustainable supply food and water, observe the variation such as feed, body weight, hair color of mice, and whether enlargement variable color of abdominal part, whether have the appearance of the tic phenomenon of trembling; Every day, record was observed, and statistics mortality rate, observes 14 days, calculates median lethal dose(LD 50) LD by Reed-Muench method ₅₀.This experiment shows, 3 × 10 ²tCID ₅₀hSV-1 can make experiment mice half death (LD in 14 days ₅₀).

Embodiment 11. laboratory animal grouping and processing

11.180 mices are divided into 8 groups at random, do respectively different processing.

Group 1 (blank group), compares with normal saline, numbering A0;

Group 2 (HSV-1 matched groups), only infect and compare with HSV-1, numbering A1;

Group 3 (Reov-dsRNA matched groups), only compare with Reov-dsRNA EnII, numbering A2;

Group 4 (Poly[I:C] positive drug matched group), only use positive drug Poly[I:C] and compare numbering A3;

Group 5 (Reov-dsRNA+HSV-1 groups), Reov-dsRNA EnII prevention HSV-1 infected group, first uses Reov-dsRNA EnII, the rear HSV-1 counteracting toxic substances of using, numbering B1;

Group 6 (HSV+Reo-dsRNA groups), Reov-dsRNA EnII treatment HSV infected group, first infects with HSV-1, rear with Reov-dsRNA EnII antiviral therapy, numbering B2;

Group 7 (Poly[I:C]+HSV group), positive drug Poly[I:C] and prevention HSV-1 infection matched group, first prevent to use positive drug Poly[I:C], the rear HSV-1 counteracting toxic substances of using, numbering C1;

Group 8 (HSV-1+Poly[I:C] group), positive drug Poly[I:C] and treatment HSV-1 infection matched group, first infect the rear positive drug Poly[I:C that uses with HSV-1] and treatment, numbering C2.

11.2 process and observe.

11.2.1 for Prevention Research group (B1, C1), every group at first 12 hours of viral infection and 24 hours intraperitoneal injections 2 times, each every mice 100 μ g Reov-dsRNA EnII or Poly[I:C].

11.2.2 for treating seminar (B2, C2) and HSV-1 matched group (A1) at intracranial injection 50LD ₅₀after/ml HSV-1 suspension 30 μ l, respectively at every injected in mice 100 μ g Reov-dsRNA EnII or Poly[I:C of 24 hours, 48 hours each groups].

11.2.3 for matched group (A0, A1, A2, A3) except experiment is processed, in all the other processing times of each experimental group, each control animals is all used the normal saline injection of equivalent, identical to ensure that laboratory animal used stimulates.

11.2.4 observe 7 days, get cerebral tissue and make histopathological analysis, and the mensuration of other indexs of correlation.

Embodiment 12. animal subject HSV-1 infect clinical manifestation

After HSV-1 infecting mouse, second day mice starts to occur the early symptom infecting, and feed reduces, body weight sharply declines, become thin, movablely reduce, eyes micro-ly close, health is rolled up and trembled; Indivedual mices have the stage casing of the continuous excitement nose of forelimb and eyes; The 3rd day starts there is dead mouse, mice tremble aggravation and with twitch symptom.

The mensuration (the results are shown in Table 1) of the tested mice serum interferon of embodiment 13. (IFN-α/β/γ) level

13.1 tested mices are got blood and serum preparation

13.1.1 left hand thumb forefinger is caught mouse ear and skin of neck, little finger of toe and nameless fixing mousetail and hind leg; Middle finger is pushed down the left fore of mice, cuts off beard, by mouse head down;

13.1.2 pin eyes both sides with tweezers, make mice one's eyes became bloodshot and exophthalmos, then remove eyeball with tweezers folder;

13.1.3 collect blood applying light cardia, many collection blood, probably can collect 0.6～1ml for every as far as possible;

13.1.4 in the time that blood no longer flows out, cervical spinal is from disconnected execution mice;

13.1.5 the blood of having collected is placed in to room temperature two hours, 4 DEG C are spent the night;

13.1.6 treat blood coagulation, after serum is separated out, 2500-3200rpm, centrifugal 8-12min, collects serum to another EP pipe, and-20 DEG C are frozen, for subsequent use.

13.2 animal subject serum interferon level determinations (by specification operation)

This test kit adopts enzyme linked immunosorbent assay (ELISA) to measure the level of the interferon such as IFN-α, IFN-β in mice serum, IFN-γ.Operation sequence is as follows:

13.2.1 take out ELISA test kit, place 15～30 minutes in room temperature (20～25 DEG C).Experimentation should carry out in room temperature (20～25 DEG C).

13.2.2 establish respectively blank well (blank hole does not add sample and enzyme marking reagent, and all the other each step operations are identical), standard substance hole, testing sample hole.On the coated plate of enzyme mark, in standard substance hole, add standard substance 50 μ l; On the coated plate of enzyme mark, in testing sample hole, first add sample diluting liquid 40 μ l, and then add testing sample 10 μ l (the final dilution factor of sample is 5 times).Except blank well, every hole adds enzyme marking reagent 50 μ l.Rock and mix gently, 37 DEG C of incubations 60 minutes.

13.2.3 discard liquid, dry, cleaning mixture (beforehand dilution) is filled it up with in every hole, vibrates 30 seconds, discards cleaning mixture, pats gently and blots liquid with absorbent paper, so repeats 3 times, pats dry.

13.2.4 every hole first adds developer A50 μ l, then adds developer B50 μ l, rocks and mixes at experiment table top, develops the color 15 minutes in 37 DEG C of darkrooms.

13.2.5 take out ELISA Plate, every hole adds stop buffer (30%-40% concentration hydrogen sodium oxide) 50 μ l, and cessation reaction (now visible blue is vertical turns yellow), notes not bubbling.

13.2.6 survey OD value: taking blank well as contrast zeroing, add after stop buffer in 15 minutes, survey the absorbance (OD value) in each hole at 450nm wavelength place.

13.2.7 calculate the linear regression equation of standard curve according to the OD value of the concentration of standard substance and correspondence; OD value per sample calculates corresponding sample concentration on regression equation again.Because sample has diluted 5 times before test, so the sample concentration finally obtaining should be multiplied by the dilution factor (the contrast dilution that practical operation adopts) of 5 times.

The mice serum IFN-α of the each seminar of table 1., IFN-β, IFN-γ concentration determination result statistical analysis table

[note] ^*represent each seminar and the comparison of blank group, P < 0.05; ^*represent each seminar and the comparison of HSV-1 infection model group, P < 0.05; △ represents Reov-dsRNA+HSV-1 group and Poly[I:C]+HSV-1 organizes relatively, P < 0.05; △ △ represents HSV-1+Reov-dsRNA group and HSV-1+Poly[I:C] organize relatively P < 0.05.

The tested mouse brain tissue sampling of embodiment 14. and histopathology section and observation analysis

13.1 draw materials with fixing: after laboratory animal blood sampling (seeing 12.1.4), cervical spinal is from disconnected execution mice, for making tested mouse brain tissue sampling; Surgical scissors " V " font is cut off the skin of neck of putting to death mice, cuts off scalp along mice cephalocaudal axis direction, and shears is pried open skull, complete taking-up cerebral tissue.

13.2 perusals are also recorded the cardinal principle pathological changes of each group of each laboratory animal cerebral tissue.

13.3 cerebral tissue are immersed in 10% paraformaldehyde solution, and room temperature spends the night.Attention: 1. fix liquid measure and should be 40 times of piece of tissue volume; 2. the fixative kind of set time and use and piece of tissue size, temperature etc. are relevant, are generally 3～24h; 3. fixed temperature can be fixed in room temperature mostly, should extend in low temperature (4 DEG C) time; 4. fixed container should be more greatly.

13.4 dehydrations are transparent: have sequentially through 70%, 80%, the each 2～4h of 90%, 100% ethanol, sloughs the moisture content in piece of tissue gradually.Again piece of tissue is put in clarifier dimethylbenzene transparent, general half an hour.

13.5 waxdip embeddings: the transparent piece of tissue of dewatering is placed in to the paraffin that heating has been dissolved in advance and is incubated 2～4h.After piece of tissue is fully immersed by paraffin, can carry out embedding, paraffin be poured into the container of appointment, the piece of tissue that paraffin has been soaked in rapid gripping is put into wherein, cooled and solidified in bulk.

13.6 section and pasters: embedded wax stone is placed on microtome, is cut into the thick thin slice of 5～8 μ m.Be put in hot water and plate with brush pen picking thin slice, then be attached on the microscope slide that coats in advance Oleum Pini, in 45 DEG C of calorstats, dry.

13.7 dewaxing dyeing:

13.7.1 put into hematoxylin solution and dye 5 minutes being attached to section on microscope slide;

13.7.2 sample is passed through respectively to color separation in ammonia and sour water, each several seconds;

13.7.3 flowing water rinses after 60 minutes and immerses distilled water for a moment;

13.7.4 in 70% and 90% ethanol, dewater each ten minutes;

13.7.5 be placed in Yihong dyeing liquor dyeing 2～3min.

13.8 dehydrations are transparent: the section after dyeing is dewatered through absolute alcohol, then makes to cut into slices transparent through dimethylbenzene.

13.9 mountings: transparent section is dripped to upper canada balsam, covered sealing.After natural gum is slightly dry, labelled.

13.10 pathological study are analyzed the laboratory animal pathologyofbraintissue sheet of animal subject cerebral tissue HE dyeing, and under optical microscope, the each laboratory animal cerebral tissue pathology of the each group of observation analysis change.

Research summary

1. Fig. 1 is that Reovirus structural representation, Fig. 2 are the ultra micro section Electronic Speculum figure that Reovirus-3 propagation infects vero cell, show: virus is spherical in shape, and double capsid, Undivided genome is arranged in monolayer inner capsid.Fig. 3 is that 10%PAGE resolves Reoirus dsRNA Genome Atlas.These conditions have been created the most important condition for we research and develop Reov-dsRNA EnII.

2. the herpes simplex virus type 1 (HSV-1) that Fig. 5 and Fig. 7 are Green Fluorescent Protein infects vero cell image, and a large amount of HSV-1, in the internal breeding of vero cell, sends strong green fluorescence; The CPE such as shrinking significantly appears in infected vero cell, change is justified, come off.Fig. 4 is the vero cell matched group that does not infect HSV-1, has no green fluorescence, and without CPE, Growth of Cells is luxuriant, be fusiformis, queueing discipline.The HSV-1 of Green Fluorescent Protein is the foundation of HSV-1 infected animal model in this patent research, and the safety of Reov-dsRNA EnII and study on the efficiency have been created laboratory research material substance basis.

3. Fig. 6 is the inoculation Vero of HSV-1 infecting mouse model brain tissue homogenate cell, and Vero cell sends green fluorescence clearly, illustrates that we are recovered to HSV-1 from animal pattern.In conjunction with Fig. 8-Figure 11, illustrate that HSV-1 infecting mouse model is successfully established.

4. and then study in vivo safety, the effectiveness of Reov-dsRNA EnII by this model system, and in body prevention HSV-1 infect, the effect of anti-HSV-1 treatment, disclosed: Reov-dsRNA EnII is for safety in body; Induction endogenous interferon IFN-α, IFN-β, IFN-γ are effective; Can be used for preventing HSV-1 to infect and anti-HSV-1 treatment.

5. can obtain conclusion from table 1 and Figure 18: Reov-dsRNA EnII can induce safely IFN-α, IFN-β, IFN-γ, P<0.05 in vivo effectively.

6. can obtain conclusion from Figure 12 and Figure 15: Reov-dsRNA EnII and Poly[I:C] can effectively induce in vivo IFN-α, IFN-β, IFN-γ to express; And the level that Reov-dsRNA EnII induction IFN-α, IFN-β, IFN-γ express is significantly higher than Poly[I:C] (P<0.05).

7. can obtain conclusion from Figure 13 and Figure 16: formerly use Reov-dsRNA EnII or Poly[I:C] Pretreatment Test animal, use again HSV-1 infected animal, result proves: Reov-dsRNA EnII and Poly[I:C] expression that can effectively induce in vivo endogenous interferon IFN-α, IFN-β, IFN-γ etc., can effectively prevent HSV-1 to infect; And Reov-dsRNA EnII induces the level of these 3 kinds of interferon to be significantly higher than Poly[I:C in vivo].

8. can obtain conclusion from Figure 14 and Figure 17: formerly infect HSV-1 and then use Reov-dsRNA EnII or Poly[I:C] process condition, Reov-dsRNA EnII and Poly[I:C] expression that can effectively induce in vivo endogenous interferon IFN-α, IFN-β, IFN-γ etc., the treatment of infecting to realize anti-HSV-1; And further prove that Reov-dsRNA EnII induces the level of these 3 kinds of interferon to be also significantly higher than Poly[I:C in vivo].

9. statistic analysis result shows: Reov-dsRNA EnII prevention HSV-1 infected group mortality rate is significantly lower than poly[I:C] prevention HSV-1 infected group (P<0.05), prompting Reov-dsRNA EnII has the effect that good prevention HSV-1 infects.

10. statistic analysis result shows: Reov-dsRNA EnII treatment HSV-1 infected group mortality rate is significantly lower than poly[I:C] treatment HSV-1 infected group (P<0.05), prompting Reov-dsRNA EnII has the effect that good treatment HSV-1 infects.

Conclusion

Endogenous interferon (ENI) is to use endogenous interferon derivant (EnII) in body, the endogenous IFNs that directly induction body produces, can overcome many deficiencies of exogenous interferon (EXI) and bring into play maximum pharmacological activity.

At present, the mankind best business-like EnII used is Poly[I:C], there is a series of defect, market and clinical practice are all extremely restricted, and the mankind are pursuing safe and effective medical EnII always.So, find and develop the medical EnII of the new second filial generation and be the biomedical family in the whole world the field of contending.

The present invention has just been to provide a kind of so brand-new Poly[I:C that is better than] Reov-dsRNA EnII, have significant novelty.

The present invention is by cell in vitro culture systems propagation Reovirus, and then extraction purification Reovirus dsRNA molecule, is prepared into Reov-dsRNA EnII, is directly used in human body; Body is followed self-defense reactive mode and is induced to produce various endogenous interferons (ENIs), give the treatment of human body viral diseases, viral diseases prevention, antineoplastic treatment and therapeutic alliance and diseases prevention widely, cure the disease and constitutional function, also there is the important function for tackling sudden public health event.

Claims (1)

1. the application of reovirus dsRNA in medical endogenous interferon derivant preparation.