CN106539789A - Application of the biliverdin on anti-bovine viral diarrhea virus medicine is prepared - Google Patents
Application of the biliverdin on anti-bovine viral diarrhea virus medicine is prepared Download PDFInfo
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- CN106539789A CN106539789A CN201610886115.4A CN201610886115A CN106539789A CN 106539789 A CN106539789 A CN 106539789A CN 201610886115 A CN201610886115 A CN 201610886115A CN 106539789 A CN106539789 A CN 106539789A
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
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Abstract
The present invention relates to application of the biliverdin on anti-bovine viral diarrhea virus medicine is prepared, belong to antiviral study technical field, the medicine includes biliverdin and one or more pharmaceutically acceptable adjuvant, and the valid density of the anti-bovine viral diarrhea virus of its mesobiliverdin is 50 200 μm of ol/L.The present invention is initially with qPCR, TCID50Impact bovine viral diarrhea virus replicated in MDBK cells with the method detection biliverdin of Western Blot, further clear and definite its impact to viruses adsorption and internalization, then detect that the biliverdin of variable concentrations processes the impact to MDBK cell-proliferation activities, effect of the biliverdin on anti-bovine viral diarrhea virus is specify that, foundation is provided to prepare anti-bovine viral diarrhea virus medicine.
Description
Technical field
The invention belongs to antiviral study technical field, is preparing anti-bovine viral diarrhea virus more particularly to biliverdin
Application on medicine.
Background technology
Bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) are flaviviridae pestivirus
Representative species, main infection cattle is simultaneously diseases induced, and the bovine viral diarrhea caused by bovine viral diarrhea virus is more to occur
Symptom, persistent infection and immunosuppressant are principal character, are the important virus of the cattle that high risks are caused to global cattle-raising
Property epidemic disease.
Vaccine and antibiotic are mainly adopted to the anti-system of BVDV at present.BVDV vaccines primarily now have conventional vaccine and new
Type vaccine.Conventional vaccine mainly uses cell culture and obtains BVDV strains, and then obtains weak poison or the inactivation of these strains
Seedling, these vaccines are identical to antigen or protection of the less strain of difference is effective, or not synantigen larger to antigenic difference
The animal that infected of BVDV strains can not play the effect of being effectively protected.With the development and application of technique for gene engineering,
BVDV new generation vaccines are born, including subunit vaccine, DNA vaccination and recombinant live-vector vaccine etc., but as BVDV is in natural bar
Part lower variation is high, and the preventing and treating to BVDV causes very big difficulty, from the point of view of the use of vaccine, whether inactivated vaccine, weak toadstool
Or new generation vaccine, all can not play conclusive effect to final control BVDV at aspects such as safety, therapeutic effect.In addition
Based on the concern to healthy and safe food and demand, the use of antibiotics of animal has caused the anti-of increasing consumer
It is right.Therefore, it is badly in need of finding a kind of new antiviral drugs or formulating novel antiviral strategy infecting with effective prevention and control virus.
The structural formula of biliverdin is, the product of its hemoglobin homergy.Category
In a kind of cholochrome of bile pigments, it is bottle-green chromatoplast, for processing the waste in body.Biliverdin is most strong interior
Source property antioxidant, especially has very strong antioxidation in the process for eliminating free radical and anti-lipid peroxidation, its antioxygen
Change ability has been even more than Vitamin E and vitamin C.
The content of the invention
For adopting at present vaccine or the antibiotic therapy bovine viral diarrhea can not effectively and sustainable protection and control
Present situation, it is an object of the invention to study application of the biliverdin on anti-bovine viral diarrhea virus medicine is prepared, be treatment
New thinking and approach are provided by the disease that bovine viral diarrhea virus cause with prevention.
The present invention relates to application of the biliverdin on anti-bovine viral diarrhea virus medicine is prepared, it is characterised in that：Gallbladder is green
Plain Concentraton gradient dependency suppresses the expression of bovine viral diarrhea virus mRNA, the especially expression of non-structural protein NS5B.
The present invention relates to a kind of pharmaceutical preparation of anti-bovine viral diarrhea virus, the pharmaceutical preparation is using biliverdin as having
Effect composition, and form with reference to auxiliaries.
Used as further preferably, the anti-bovine viral diarrhea virus pharmaceutical preparation is injection or oral agents.
As it is further preferably, the injection be biliverdin is dissolved in into DMSO or normal saline in be formulated.
Used as further preferably, the injection is lyophilized injectable powder.
As it is further preferably, the oral agents are to mix any one in biliverdin and starch, sucrose or dextrin
Close uniform, obtain mixture；Plus sterilized water make mixture mass fraction be 50-70%, stir, mediate, crush, cross sieve series
It is standby to form.
Used as further preferably, the oral agents are discrete piece agent, capsule or granule.
The present invention is also related to biliverdin as a kind of bovine viral diarrhea virus inhibitor, it is characterised in that：The suppression
Agent suppresses bovine viral diarrhea virus infection MDBK cells.
Used as further preferably, the valid density of biliverdin is 50-200 μm of ol/L.
Compared with prior art, the invention has the advantages that：
（1）The present invention prepares anti-bovine viral diarrhea virus medicine with reference to relevant auxiliary materials, for controlling with biliverdin as effective ingredient
The disease that treatment is caused by bovine viral diarrhea virus, the medicine are different from antibiotics, will not be to the immune system of body
Work the mischief；Also vaccine type medication is different from, medicine of the present invention can be prepared into ejection preparation or oral agents, be not limited to make
With type, the needs of different levels can be met；The mature production technology of biliverdin, can be anti-bovine viral diarrhea virus in addition
The preparation of medicine provides sufficient raw material, can greatly save production cost.
（2）The present invention is had found with the increasing of biliverdin concentration by relative quantification is carried out to intracellular BVDV viral genes
Plus, the expression of bovine viral diarrhea virus gene is suppressed, and within a certain period of time, biliverdin acts on the time of cell
It is longer, it is more obvious to the inhibition of virus.For further impact of the clear and definite biliverdin to viruses adsorption and internalization, to virus
The expression of copy number, virus titer and virus NS 5 B albumen is detected, is found with the increase of biliverdin concentration, virus
Copy number, virus titer and virus NS 5 B protein expression are gradually reduced.Process right finally by the biliverdin of detection variable concentrations
The impact of MDBK cell-proliferation activities, in the concentration range of 10 μM～200 μM of discovery, biliverdin does not affect the activity of MDBK cells,
But Jing after 300 μM, 400 μM and 500 μM of biliverdin are processed, the vigor of MDBK cells reduces 43%, 53% and 58% respectively.Institute
It is not, by direct killing BVDV particle, but to be realized by intracellular physiological process with, anti-BVDV infection effects of biliverdin
's；There is penetrating in virus replicative cycle and absorption phase in its depression effect to BVDV infection.
Description of the drawings
Fig. 1 is the schematic diagram that variable concentrations biliverdin processes the impact to BVDV mRNA expression；
Fig. 2 is the schematic diagram that variable concentrations biliverdin processes the impact to BVDV virus NS 5 B protein expressions；
Fig. 3 is that variable concentrations biliverdin processes the schematic diagram affected on BVDV virus titers；
Fig. 4 is the schematic diagram that variable concentrations biliverdin processes the impact to BVDV progeny viruss RNA copy numbers；
Fig. 5 is the schematic diagram of the impact that biliverdin changes to the growth kineticses of BVDV；
Fig. 6 is the schematic diagram of impact of the biliverdin to MDBK cell-proliferation activities.
Specific embodiment
The present invention is further explained below by specific test example.
1 biliverdin of test example infects the impact of MDBK cells to BVDV
Step one, biliverdin process the MDBK cells of BVDV infection
S1：6 orifice plates are inoculated in after MDBK cells with the dilution exponential phase of the DMEM culture medium containing 10% hyclone, per hole
2mL；When cultivating to 70%～80% degree of converging, the BVDV strains diluted with DMEM culture medium are inoculated with, adsorb 1h；Abandon virus liquid,
Per hole add 1mL PBS wash 1 time, then be separately added into containing 3% hyclone DMEM culture medium prepare biliverdin concentration be 50
μM, the solution of 100 μM and 150 μM, 2mL/ holes are placed in 37 DEG C, 5% CO2In incubator continue culture, 24hpi collect cell and
Supernatant.
S2：6 orifice plates are inoculated in after MDBK cells with the dilution exponential phase of the DMEM culture medium containing 10% hyclone,
Per hole 2mL；When cultivating to 70%～80% degree of converging, 0.05MOI is inoculated with（Infection multiplicity）BVDV strains, adsorb 1h；Abandon virus
Liquid, adds 1mL PBS to wash 1 time per hole, then in every hole, add 2mL green with the gallbladder that the DMEM culture medium containing 3% hyclone is prepared
Plain concentration is 150 μM of solution, is subsequently placed at 37 DEG C, 5% CO2In incubator continue culture, respectively at 6hpi, 12hpi,
24hpi, 36hpi, 48hpi, 60hpi and 72hpi collect cell and supernatant.
Step 2, qPCR detect the relative expression quantity of intracellular BVDV genes
The cell total rna collected is prepared in extraction step one S1 and S2 respectively.Extraction step is：It is thin for adhere-wall culture MDBK
Born of the same parents, remove culture fluid, and appropriate RNAiso Plus cell lysis are added after being washed with PBS, and it is total that reference explanation book extracts cell
RNA, be stored in after the completion of extraction -80 DEG C it is standby.With extract cell total rna as template, with table 1 according in GeneBank
BVDV Oregon C24V strain gene orders（Serial number： AF091605.1）The BVDV-q F and BVDV-q R of design is to draw
Thing, carries out reverse transcription using reverse transcription reagent box, and the cell total rna of extraction is transcribed into cDNA, reaction system and reaction condition
Reference inversion records kit specification.To reverse transcription product（That is cDNA）Carry out 1:10 dilution, with dilute cDNA as template, with
BB2M-q F and bB2M-q R in table 1 according to the cattle B2M reference gene sequential designs selected is primer, using SYBR GREEN
Fluorescence quantitative kit carries out the detection of gene expression amount, and reaction condition is 95 DEG C, 10 min；95 DEG C, 15 s；60 DEG C, 60s；
95 DEG C, 15 s；Reaction totally 40 circulations, gather first order fluorescence signal at the end of 60 DEG C of extensions in each cycle.Wherein, primer
It is described as follows shown in table 1.
As a result show：As shown in Figure 1A, as the increase of biliverdin concentration, viral gene expression are gradually lowered, illustrate gallbladder
Verdazulene suppresses the expression of 5 ' UTR gene mRNAs of BVDV in Concentraton gradient dependency；As shown in Figure 5A, with process without biliverdin
Compared with control cells is compared, and in the cell of 0.05MOI BVDV infection, during virus infection 6hpi, in cell, BVDV gene expression amounts are notable
Decline, in virus infection 12hpi, the expression of viral gene drops to peak value, then gradually rises, until virus infection
During 72hpi, the expression of viral gene is not significantly different from untreated compared with control cells.
Wherein, the RNAiso Plus reagents are purchased from Takara, model D9108A；
Reverse transcription reagent box is purchased from Takara, model RR037A；
PCR instrument and quantitative real time PCR Instrument are purchased from the silent winged generation that of match；
Roche company of the SYBR GREEN fluorescence quantitative kits purchased from Switzerland.
Step 3, Western Blot detection biliverdin process the impact to virus NS 5 B protein expression
By the expression change of virus protein N S5B in the cell for preparing in one S1 of Western Blot detecting steps.Disease
Malicious NS5B protein extraction steps：The cell sample that the Jing variable concentrations biliverdin that step S1 is prepared is processed is collected in respectively
In the centrifuge tube of 1.5 mL, 4 DEG C, 500g centrifugation 10min abandon supernatant, add 1mL PBS solutions resuspended, same 500g centrifugations
10min, discards PBS supernatant solutions, estimates cell total amount according to wet cell weight, according to 1 × 106Individual cell adds 100 μ L NP40
The ratio of lysate, adds appropriate NP40 lysates, is placed in；12000g is centrifuged 10 min, removes cell
Fragment, takes supernatant ,+45 μ L PBS of 5 μ L lysate samples, using the total egg of Pierce BCA protein quantification kit measurements
White concentration, then with each 50 μ g total protein of swimming lane, carries out Western blot detections.
Preparation concentration is 12% SDS-PAGE separation gels and 5% concentration glue；Carry out concentrating glue and separation by voltage stabilizing 200V
The electrophoresis of glue；Half-dried transferring film method constant pressure 15V transferring film 1h；After taking out film, add confining liquid, room temperature after film 5min being washed with 1 × PBS ' T
Slow shake 2h（Or 4 DEG C overnight）；Confining liquid is discarded, and film 3 times, each 5min is washed with PBS ' T；Add the one of proper ratio dilution
Anti- solution, room temperature slowly shake 2h or 4 DEG C of night incubation；An anti-solution is discarded, and film 3 times is washed with PBS ' T, each 5min, it
The two corresponding anti-solution of proper ratio dilution, room temperature is added slowly to shake 1h afterwards；Two corresponding anti-solution is discarded, and film 3 times is washed with PBS ' T, every time
5min；ECL develops the color, and exposes on luminescence imaging instrument.
As a result show：As shown in Fig. 2 biliverdin suppresses the table of BVDV non-structural protein NS5B with Concentraton gradient dependency
Reach.
Wherein, the Pierce BCA protein quantifications test kit is purchased from Thermo Scientific.
Virus titer after step 4, drug treating in cell conditioned medium（TCID50）Determine
The cell conditioned medium that S1 in step one and S2 are prepared is made 10 times with DMEM culture medium and is serially diluted 10-1To 10-8, connect
Plant in 96 porocyte culture plates for covering with MDBK cell monolayers, each dilution factor is inoculated with 8 holes, per 100 μ L of hole, 37 DEG C of absorption 1h
Afterwards, supernatant is abandoned, DMEM culture medium maintaining liquids of the 100 μ L containing 3% hyclone, 37 DEG C, 5% CO is added per hole2It is quiet in incubator
4～7d of culture is put, daily observation of cell pathological changes calculate the TCID of virus according to record result using Karber methods50。
As a result show：As shown in figure 3, biliverdin is in BVDV in the dependent reduction MDBK cells and supernatants of Concentraton gradient
Virus titer；As shown in Figure 5 B, compared with the compared with control cells processed without biliverdin, on the cell of 0.05MOI BVDV infection
Clear virus titer, in virus infection 6hpi, virus titer is remarkably decreased, and in virus infection 12hpi, virus titer is dropped to
Peak value, then gradually rises, and when virus infection 72hpi, the expression of viral gene is not had with untreated compared with control cells
Significant difference, the trend consistent with the expression presentation of viral gene in cell.
The copy number of virus in step 5, qPCR detection supernatant cells
The supernatant cell total rna collected is prepared in extraction step one S1 and S2 respectively.Extraction step is：Collect 400 μ L of supernatant
Liquid is in the 1.5mL EP pipes of sterilizing, plus 400 μ L RNAiso Plus vibrations are mixed, and reference explanation book extracts cell total rna, carries
Be stored in after the completion of taking -80 DEG C it is standby.With extract cell total rna as template, with table 1 according to BVDV in GeneBank
Oregon C24V strain gene orders（Serial number：AF091605.1）The BVDV-q F and BVDV-q R of design is primer, sharp
Reverse transcription is carried out with reverse transcription reagent box, the cell total rna of extraction is transcribed into into cDNA, reaction system is with reaction condition with reference to anti-
Transcript reagent box description.To reverse transcription product（That is cDNA）Carry out 1:10 dilution, with dilute cDNA as template, with table 1
It is primer according to the bB2M-q F and bB2M-q R of selected cattle B2M reference gene sequential designs, using SYBR GREEN fluorescence
Quantification kit carries out the detection of gene expression amount, and reaction condition is 95 DEG C, 10 min；95 DEG C, 15 s；60 DEG C, 60s；95 DEG C,
15 s；Reaction totally 40 circulations, gather first order fluorescence signal at the end of 60 DEG C of extensions in each cycle.
As a result show：As shown in figure 4, in supernatant copy number and the corresponding intracellular BVDV viral genes of BVDV viruses and
The expression trend of albumen is consistent；As shown in Figure 5 C, compared with the compared with control cells processed without biliverdin, 0.05MOI BVDV senses
Virus titer in the copy number of the progeny viruss in the cell conditioned medium of dye, with the expression and supernatant of viral gene in cell is in
Now consistent trend.
Impact of 2 biliverdin of test example to MDBK cell-proliferation activities
96 orifice plates are inoculated in after MDBK cells with the dilution exponential phase of the DMEM culture medium containing 10% hyclone, per hole 100
μ L, to prevent evaporation, 96 porocyte culture plates surrounding marginal pores from filling the aseptic PBS of same volume, being placed in 37 DEG C, 5%CO2Training
24h is cultivated in foster case, trial drug biliverdin is added.The treatment group of biliverdin variable concentrations is set（Biliverdin be diluted to 0 μM,
10 μM, 25 μM, 50 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, 500 μM of ten kinds of variable concentrations）.With reference to CCK-8 reagents
Box description carries out MDBK cytoactive detections.Inhaled with 100 μ L pipettors successively and abandon old culture medium, plus after PBS washs 1 time, plus
Enter the biliverdin solution of preprepared Concentraton gradient, per 100 μ L of hole, each Concentraton gradient experimental port is provided with 6 repeating holes,
Control wells are provided with 4 repeating holes.Then 96 porocyte culture plates are placed in into 37 DEG C, 5%CO2Culture 48h in incubator.Ultra-clean
In workbench, under the conditions of lucifuge, 10 μ L CCK-8 solution are added to every hole with 10 μ L liquid-transfering guns, make sure to keep in mind to wipe off, stab thin
Born of the same parents get bubble, in 37 DEG C, 5% CO2Continue measure OD of culture to setting in incubator450Time point, and in microplate reader
Upper reading 450nm absorbances, finally according to absorbance statistical analysiss.
As a result show, as shown in fig. 6,10 μM～200 μM of biliverdin processes the activity to MDBK cells without notable shadow
Ring；Compared with normal cell, after 300 μM, 400 μM and 500 μM of biliverdin is processed, then the vigor of MDBK cells is reduced respectively
43%th, 53% and 58%, illustrate that biliverdin in 10 μM～200 μM of concentration range does not affect the activity of MDBK cells.
Wherein, the green skies biotechnology research institute in CCK-8 test kits source, article No. is C0038.
The result of compbined test example 1 and test example 2, biliverdin can be by suppressing bovine viral diarrhoea in effective dose scope
The expression of virus mRNA, especially suppresses the virus titer expressed to reduce bovine viral diarrhea virus of non-structural protein NS5B
With the copy number of progeny viruss, so as to affect the infection to MDBK cells, it is that biliverdin is used to develop anti-bovine viral diarrhea
Cytotoxic drug is laid a good foundation and provides theoretical condition.Can suppress on the premise of MDBK cytoactives are not affected as far as possible again
The optium concentration of the biliverdin that BVDV is replicated is 50-200 μM.
Above content be interpreted as it is illustrative, elaborate the present invention ultimate principle and advantages of the present invention, rather than limit
Protection scope of the present invention processed.Protection scope of the present invention is defined by the content of claims, to those skilled in the art
Speech, on the premise of without departing substantially from spirit and scope of the present invention, some the nonessential modifications and adaptations made to the present invention still belong to
In protection scope of the present invention.
Claims (10)
1. application of the biliverdin on anti-bovine viral diarrhea virus medicine is prepared, it is characterised in that：Biliverdin Concentraton gradient according to
Bad property suppresses the expression of bovine viral diarrhea virus mRNA.
2. application of the biliverdin as claimed in claim 1 on anti-bovine viral diarrhea virus medicine is prepared, it is characterised in that：
Biliverdin Concentraton gradient dependency suppresses the expression of bovine viral diarrhea virus non-structural protein NS5B.
3. a kind of pharmaceutical preparation of anti-bovine viral diarrhea virus, it is characterised in that：Using biliverdin as effective ingredient, and combine
Auxiliaries are formed.
4. the pharmaceutical preparation of a kind of anti-bovine viral diarrhea virus according to claim 3, it is characterised in that：The medicine
Preparation is injection or oral agents.
5. a kind of pharmaceutical preparation of anti-bovine viral diarrhea virus as claimed in claim 4, it is characterised in that：The injection
Be biliverdin is dissolved in into DMSO or normal saline in be formulated.
6. the pharmaceutical preparation of a kind of anti-bovine viral diarrhea virus as described in claim 4 or 5, it is characterised in that：The note
Agent is penetrated for lyophilized injectable powder.
7. a kind of pharmaceutical preparation of anti-bovine viral diarrhea virus as claimed in claim 4, it is characterised in that：The oral agents
It is that biliverdin is mixed homogeneously with any one in starch, sucrose or dextrin, obtains mixture；Plus sterilized water makes the matter of mixture
Amount fraction is 50-70%, is stirred, and mediates, crushes, sieving is prepared from.
8. the pharmaceutical preparation of a kind of anti-bovine viral diarrhea virus as described in claim 4 or 7, it is characterised in that：The mouth
Agent is taken for discrete piece agent, capsule or granule.
9. biliverdin is used as a kind of bovine viral diarrhea virus inhibitor, it is characterised in that：The inhibitor suppresses bovine viral
Diarrhea virus infection MDBK cells.
10. biliverdin as claimed in claim 9 is used as a kind of bovine viral diarrhea virus inhibitor, it is characterised in that：Gallbladder is green
The valid density of element is 50-200 μm of ol/L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111172249A (en) * | 2020-02-25 | 2020-05-19 | 芜湖天明生物技术有限公司 | rhTSG-6 fluorescent quantitative RT-qPCR detection kit and application thereof |
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| US20090203762A1 (en) * | 2005-03-14 | 2009-08-13 | Pendrak Michael L | Methods for the production of biliverdin |
| CN103463626A (en) * | 2013-03-03 | 2013-12-25 | 西北农林科技大学 | HO-1 and HO-1 inducer for inhibiting PRRS virus (PRRSV) infection as novel blocker |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172249A (en) * | 2020-02-25 | 2020-05-19 | 芜湖天明生物技术有限公司 | rhTSG-6 fluorescent quantitative RT-qPCR detection kit and application thereof |
| CN111172249B (en) * | 2020-02-25 | 2021-03-02 | 安徽医科大学 | rhTSG-6 fluorescent quantitative RT-qPCR detection kit and application thereof |
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