CN106420812A

CN106420812A - Carbon monoxide release molecule-2 preparation, application of carbon monoxide release molecule-2 preparation in preparing medicines for resisting porcine reproductive and respiratory syndrome viruses and detection method of carbon monoxide release molecule-2 preparation

Info

Publication number: CN106420812A
Application number: CN201610864322.XA
Authority: CN
Inventors: 肖书奇; 周恩民; 张昂克
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2016-09-29
Filing date: 2016-09-29
Publication date: 2017-02-22

Abstract

The invention discloses a carbon monoxide release molecule-2 preparation, an application of the carbon monoxide release molecule-2 preparation in preparing medicines for resisting porcine reproductive and respiratory syndrome viruses and a detection method of the carbon monoxide release molecule-2 preparation. The carbon monoxide release molecule-2 preparation is Chinese name of CORM-2 (tricarbonyldichlororuthenium (II) dimer). In-vitro experiments prove that the carbon monoxide release molecule-2 has a significant effect of obviously inhibiting replication of the porcine reproductive and respiratory syndrome viruses, therefore a strong support is provided for enhancing prevention and control of the porcine reproductive and respiratory syndrome viruses through drug therapy.

Description

Carbon monoxide-releasing molecules -2 preparation and its preparing anti-porcine reproductive and respiratory syndrome Application on virus drugs and detection method

Technical field

The invention belongs to antiviral study technical field and in particular to carbon monoxide-releasing molecules -2 preparation and its preparation Application on anti-porcine reproductive and respiratory syndrome virus medicine and detection method.

Background technology

Porcine reproductive and respiratory syndrome (Porcine repsoductive and respiratory syndrome, PRRS), also known as pig blue-ear disease, it is a kind of by porcine reproductive and respiratory syndrome virus (Porcine repsoductive and Respiratory syndrome virus, PRRSV) cause, can cause in-pig breeding difficulty (later stage occur miscarriage, Weak tire, stillborn foetus, mummy tire etc.) and each age group pig particularly piglet respiratory symptom (interstitial pneumonia) and high mortality be The pig Important Infectious Diseases of principal character, therefore very harmful to pig industry.

Porcine reproductive and respiratory syndrome constitutes a serious threat to whole pig industry, because people are to this sick immunologic escape Molecular mechanism is short in understanding, current vaccine using nor effective and continuable disease control is provided, special standby be to those RNA viroid is even more helpless；It is simultaneously based on the concern to healthy and safe food and demand, the use of antibiotics of animal is drawn Play the opposition of more and more consumers.Therefore, develop effective antiviral drugs and formulate novel antiviral strategy still extremely Important and urgent.

CORM-2 (Tricarbonyldichlororuthenium (II) dimer), Chinese is carbon monoxide release Molecule -2, its structural formula is shown in formula I：

Content of the invention

It is an object of the invention to, carbon monoxide-releasing molecules -2 preparation is provided, upper existing for prior art to overcome State shortcoming and defect.

It is a further object to provide carbon monoxide-releasing molecules -2 preparation is to prepare the breeding of anti-pig comprehensive with breathing Application on simulator sickness virus drugs.

It is a further object to provide the detection method of carbon monoxide-releasing molecules -2 preparation.

By experiment in vitro, the present invention finds that CORM-2 has what obvious suppression porcine reproductive and respiratory syndrome virus replicated Effect, is to strengthen the prevention and control offer to porcine reproductive and respiratory syndrome virus by drug therapy to provide powerful support for.Therefore, the present invention There is provided a kind of application on preparing anti-porcine reproductive and respiratory syndrome virus medicine for CORM-2, the present invention provides one kind simultaneously CORM-2 is preparing anti-porcine reproductive and respiratory syndrome virus replica preparations.

The technical problem solving required for the present invention, can be achieved through the following technical solutions：

By experiment in vitro, the present invention finds that CORM-2 has what obvious suppression porcine reproductive and respiratory syndrome virus replicated Effect, is to strengthen the prevention and control offer to porcine reproductive and respiratory syndrome virus by drug therapy to provide powerful support for.Therefore, the present invention There is provided a kind of application on preparing anti-porcine reproductive and respiratory syndrome virus medicine for CORM-2, the present invention provides one kind simultaneously Application in preparing anti-porcine reproductive and respiratory syndrome virus replica preparations for the CORM-2.

A kind of pharmaceutical preparation of anti-porcine reproductive and respiratory syndrome virus, the CORM-2 including effective dose and pharmaceutically can connecing The auxiliary material being subject to.Preferably, described pharmaceutical preparation is ejection preparation or oral formulations.It is highly preferred that described ejection preparation is lyophilized Powder-injection；Described oral formulations are discrete piece agent, capsule or granule.

The present invention is initially with qPCR, western blot, TCID₅₀This with IiT detection CORM-2 The impact that medicine replicates in Marc-145 cell to porcine reproductive and respiratory syndrome virus.Then qPCR, western are passed through again Blot and TCID₅₀Detection CORM-2 to porcine reproductive and respiratory syndrome virus PAM cellular replication impact, in the above results On the basis of the antiviral effect of COMR-2 is evaluated.

Beneficial effects of the present invention：

1st, the CORM-2 mature production technology of the present invention, can be the medicine preparation of anti-porcine reproductive and respiratory syndrome virus Furnish ample material.

2nd, substantially, the present invention passes through experiment in vitro to the effect of the anti-porcine reproductive and respiratory syndrome virus of the CORM-2 of the present invention Find, CORM-2 can significantly inhibit PRRSV and replicate in Marc-145 cell and PAM cell and propagation.

3rd, the present invention is easy to use, and medicine is powder formulation, and 2-8 DEG C of sealing preserve can preserve it is easy to transport, and instant Use.

Brief description

Fig. 1 is PRRSV ORF7 mRNA expression after qPCR detection variable concentrations CORM-2 is processed.

Fig. 2 is that western blot detects that variable concentrations CORM-2 processes the impact to PRRSV infection Marc-145 cell.

Fig. 3 is Marc-145 cells and supernatant PRRSV TCID after variable concentrations CORM-2 is processed₅₀Mensure.

Fig. 4 indirect immunofluorescene assay variable concentrations CORM-2 processes the impact to the expression of PRRSV N protein.Fig. 4 is to close And the result of ruddiness and blue light, wherein, middle blue portion is nuclear fractions, is dyeed by DAPI；The red fluorescence in left side For marked the viral N proteins of TRITC.

Fig. 5 is PRRSV ORF7mRNA expression in PAM cell after qPCR detection variable concentrations CORM-2 is processed.

Fig. 6 is that western blot detects that variable concentrations CORM-2 processes the impact to PRRSV infection PAM cell.

Fig. 7 is PAM cells and supernatant PRRSV TCID after variable concentrations CORM-2 is processed₅₀Mensure.

Specific embodiment

Below in conjunction with specific embodiment, the present invention is made with progressive explanation.It should be understood that following examples are merely to illustrate this Bright not for limit the scope of the present invention.

In embodiment if no special instructions, it is this area conventional laboratory techniques.

Marc-145 cell (the derived cell system of RhMK MA-104) conduct is selected in the embodiment of the present invention The nutrient matrix of the high strain GD-HD that causes a disease of PRRSV, breeding for PRRSV provides nutrition, therefore selects Marc-145 cell not to this The scope of invention does any restriction.

Material therefor source in embodiment：

CORM-2 is purchased from Sigma company；DMEM, RPMI1640, hyclone (FBS) are purchased from Gibco；GD-HD-PRRSV Separated by veterinary vaccination biology seminar of animal medicine institute of Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology；FastStart Universal SYBR Green Master is purchased from Roche company；It is precious raw that Primescript RT reagent Kit is purchased from Dalian；Inactivation CORM-2 (iCORM-2) prepare is as described below：After CORM-2 is dissolved in DMSO, at 37 DEG C, 5%CO₂Wet condition under be incubated 24h, with Fully release carbon monoxide.Marc-145 cell (the derived cell system of RhMK MA-104) publishes in Chinese Typical Representative training In the catalogue of foster thing collection (deposit number GDC041), it is in open state, scientific worker can be in this culture presevation The heart is asked for.Porcine alveolar macrophage (PAM) is originated as detailed below described.Monoclonal antibody specific for PRRSV N protein Standby by animal medicine institute of Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology veterinary vaccination biology project team system.

Embodiment 1 qPCR detects the collection of intracellular PRRSV ORF7 gene mRNA relative expression quantity sample

PRRSV inoculates the Marc-145 cell of 80% degree of converging, 37 DEG C of incubation 1h, inhales and abandons virus liquid, cell is with 1 × PBS Wash 3 times, be separately added into the CORM-2 containing variable concentrations (50 μM, 100 μM, 150 μM) and the DMEM of 3% hyclone and maintain Liquid culture medium in 37 DEG C, 5%CO₂Humidified incubator in cultivate；Tieed up with the DMEM of 3% hyclone without CORM-2 simultaneously Hold liquid culture medium and add the maintenance liquid culture medium containing 150 μM of iCORM-2 as comparison, 24hpi collects cell and supernatant.

Cell sample Total RNAs extraction

(1) every 10⁷Individual/cell adds 1ml Trizol, and piping and druming mixes to being visible by naked eyes precipitation, the static 5min of room temperature.

(2) add chloroform (volume of 1/5Trizol) in above-mentioned homogenate lysate, cover tightly centrifugation lid, with being vortexed Shaker vibrates 15s.After solution fully emulsified (no obvious lamination), then room temperature standing 5min.

(3) 12000g, 4 DEG C of centrifugation 5min.

(4) careful from centrifuge take out centrifuge tube, now homogenate is divided into three layers, colourless supernatant, middle white albumen Layer and band coloured lower floor organic phase.Careful absorption supernatant is transferred in another new centrifuge tube (to be made sure to keep in mind not suction out in white Interbed).

(5) add isopyknic isopropanol in supernatant, after the centrifuge tube that turns upside down fully mixes, stand under room temperature 10min.

(6) 4 DEG C of centrifugation 10min of 12000g；After centrifugation, white precipitate in test tube bottom.

(7) cleaning of RNA precipitate：Careful supernatant discarded, lentamente adds 75% ethanol 1ml (to be sure not to touch along centrifugation tube wall And precipitation), gently turn upside down washing centrifuge tube wall, 4 DEG C of centrifugation 5min of 12000g, carefully discards ethanol, should make ethanol as far as possible Volatilization is clean.

(8) RNA dissolving：Drying at room temperature precipitates, and adds appropriate RNase-free water dissolves, treats that RNA precipitate is completely dissolved Stand-by after -80 DEG C of preservations.

Method and step that real-time fluorescence quantitative PCR (qPCR) detects

(1) mensure of total rna concentration：Each draws 2.0 μ l total serum IgE samples, is measured using BioTek micropore quantitative approach The concentration (ng/ μ l) of the sample total serum IgE being extracted；

(2) preparation of reaction mixture：Prepare reaction mixture qPCR reverse transcription reaction system by following component on ice, As shown in table 1.

Table 1

(3) carry out reverse transcription reaction：Reverse transcription condition is as follows, as shown in table 2.

Table 2

QPCR detection primer and detection method

QPCR detection primer sequence, as shown in table 3.

Table 3

CDNA Sample Dilution：The cDNA that reverse transcription is obtained is according to 1:10 (10 μ l cDNA+90 μ l ddH2O) ratio is carried out Dilution fully mixing are placed in wait on ice to do and detect further.

QPCR detects：

QPCR reaction mixture is formulated as follows, as shown in table 4.

Table 4

Above-mentioned each composition is sequentially added in EP pipe and mixes, mixed liquor is transferred to qPCR and reacts in eight connecting legs, 7.5 μ l/ hole, then takes the cDNA having diluted to be separately added in eight connecting legs, and 2.5 μ l/ holes, after covering eight connecting leg lids, are flicked tube wall and filled Divide and carry out qPCR reaction immediately after mixing.

(4) qPCR reaction condition, as shown in table 5.

Table 5

Can be seen that with CORM-2 concentration from intracellular PRRSV ORF7 gene relative quantification (Fig. 1) testing result Increase, the expression of virus O RF7 gene is gradually lowered, and notable with control group comparing difference, and the iCORM-2 inactivateing is to PRRSV ORF7 gene expression does not make significant difference.

Embodiment 2Western blot detects the impact to PRRSV infection Marc-145 cell for the CORM-2

Western blot detects that protein sample is derived from the cell sample collected by embodiment 1.

(1) prepare：Rinse beaker, electrophoresis apparatus and its accessory with running water, then deionized water is rinsed well, dry standby With.Glass plate and comb rinse well respectively after again deionized water rinse, dry standby.

(2) prepare 12% separation gel and first assemble offset plate, every kind of according to lower table search according to required resolving gel concentration The usage amount of composition and ratio, as shown in table 6.

Table 6 12% separation gel formula

Sequentially add ultra-pure water, 30% acrylamide, 1.5M Tris-HCl buffer solution, 10%APS in pumping in beaker 10%SDS is added on machine, finally plus TEMED after fully draining the air in mixed liquor.Fully mix after adding TEMED, It is added in offset plate with 1ml pipettor immediately.Then, glue gently add deionized water to close, with vaporization prevention.Separation gel room Warm 30min about i.e. solidifiable.Then according to following table prepares 5% spacer gel, as shown in table 7.

The spacer gel formula of table 7 5%

Sequentially add in beaker ultra-pure water, 30% acrylamide, 1.0M Tris-HCl buffer solution, 10%SDS, 10% APS, now, outwells the ultra-pure water on separation gel, and carefully blots residual moisture with filter paper, then TEMED is added in beaker, mixes It is added on separation gel with pipettor immediately after even, insert suitable comb.Spacer gel 20min about can solidify completely.

(3) it is loaded：Carefully pull out comb, assemble electrophoresis apparatus, be subsequently poured into 1 × Running Buffer, by albumen Marker and sample sequentially add in glue hole, and make a record.

(4) run glue：Bio-Rad electrophoresis apparatus is fixed on 200V and runs glue, stops running glue when bromophenol blue dye band just will be run out of.

(5) transferring film：The process of pvdf membrane：Cutting out in advance and adhesive tape pvdf membrane of a size are immersed in methyl alcohol, lives Change 15s, then film is directly immersed in transferring film liquid, filter paper is then directly immersed in transferring film liquid so as to balance.Transferring film：Using Bio- Rad transferring film instrument, is followed successively by sponge, 3 metafiltration paper, adhesive tape, pvdf membrane, 3 metafiltration paper, sponge from negative pole to positive pole.With scoop gently Drive filter paper, adhesive tape out of, after the bubble between film, fix clip and start to be started in mixture of ice and water kind with 150mA constant current Transferring film.

(6) immune response：

A. after transferring film finishes, pvdf membrane is carefully taken out, be placed in room temperature jog incubation 1h in confining liquid.

B. discard confining liquid, acutely rocked on shaking table with PBS ' T and wash film, 5min × 4 time.

What c. addition confining liquid had diluted resists for the one of destination protein, 4 DEG C of shaking table jog overnight incubation.

D. discard one to resist, PBS ' T acutely rocks on shaking table and washes film, 5min × 4 time.

E. add being resisted with confining liquid has diluted two of HRP- mark, room temperature jog is incubated 1h.

F. discard two to resist, PBS ' T washes film, 5min × 4 time.

(7) ECL luminescence reagent box colour developing.

Figure it is seen that when CORM-2 suppresses PRRSV N protein to express in concentration gradient dependence mode, and ICORM-2 does not make significant difference to N protein expression.

Marc-145 cells and supernatant PRRSV TCID after embodiment 3 variable concentrations CORM-2 process₅₀Mensure

(1) bed board, it is 1 × 10 that the Marc145 cell that pancreatin has been digested adjusts density⁵Individual/ml, is added to 96 ghost trainings In foster plate, 175 μ l/well about 24h grow up to individual layer.

(2) in 96 porocyte culture plates, with the DMEM culture medium of serum-free, by virus liquid, (virus liquid is derived from embodiment 1 institute Collect supernatant) make continuous 10 times of gradient dilution, from 10^-1～10^-10.

(3) by the virus inoculation having diluted to Marc145 cell monolayer, each dilution factor inoculates a tandem totally 8 hole, often Hole inoculation 100 μ L, normal cell controls work two tandems, 37 DEG C, 5%CO₂3%FBS+DMEM maintaining liquid juxtaposition is changed into after incubation 1h In 37 DEG C, 5%CO₂Humidified incubator in cultivate.

(4) observe day by day and record result, generally require observation 5～7 days.

(5)TCID₅₀Calculating：Karber method is calculated as follows：lgTCID₅₀=L-D (s-0.5)；L：Highest dilution right Number；D：Difference between dilution factor logarithm；S：Positive boring ratio rate summation.

From figure 3, it can be seen that with the increase of CORM-2 concentration, in supernatant, the titre of PRRS virus declining, and is in agent Amount dependence.And iCORM-2 no significantly inhibits effect to viral supernatant titre.

Embodiment 4 IIF detects that variable concentrations CORM-2 processes the impact to viral N proteins expression

(1) bed board：By Marc-145 cell with 1 × 10⁵The density of individual cell/ml is added in 24 porocyte culture plates, often Hole 500 μ l, about 24h reach 80% degree of converging.

(2) connect poison：PRRSV connects poison and measures as 0.1MOI, 37 DEG C of incubation 1h, discards virus liquid, 1 × PBS washes 3 times, and addition contains Continue culture after the 3%FBS+DMEM maintaining liquid of variable concentrations CORM-2.

(3) fixing：Slightly CPE phenomenon in the cells such as culture about 24h, and venom is discarded, and add 75% second in 400 μ l/ holes Alcohol (need to be placed on -20 DEG C of precoolings) in advance, 4 DEG C of fixing 30min.

(4) wash plate：Discard fixer, plate is washed in concussion on washing trigger, and 400 μ l/ hole PBS wash 5min/ time × 4 times, are giving birth to In thing safety cabinet, 24 orifice plates are dried up.

(5) incubation one resists：The monoclonal antibody 6D10 (1 of the anti-PRRSV N protein that every hole addition 150 μ l PBS have diluted:300 is dilute Release), 37 DEG C of incubation 1h.

(6) wash plate：On washing trigger, plate is washed in concussion, and 400 μ l/ hole PBS wash 5min/ time × 4 times.

(7) incubation two resists：Every hole adds the sheep anti-mouse igg fluorescence two that the TRITC that 150 μ l PBS have diluted marks to resist (1: 300 dilutions), 37 DEG C of lucifuges are incubated 1h.

(8) contaminate nucleus：The last 5min that will terminate in the anti-incubation of fluorescence two, add 100 μ l/ holes 4 ', 6 '- (DAPI presses 1 with PBS to diamidino-2-phenylindole (DAPI) staining cell core:10000 dilution proportion).

(9) wash plate：On washing trigger, plate is washed in concussion, and 400 μ l/ hole PBS wash 5min/ time × 4 times.

(10) observe fluorescence：Remove water, microscopy after lucifuge is dry.Or add direct microscopy after 100 μ l PBS.

From fig. 4, it can be seen that with the increase of CORM-2, marked N protein expression (red fluorescence in left side) of TRITC In minimizing, and it is in dose dependent.And it is not added with the control group of CORM-2 and the expression of iCORM-2 treatment group N protein has no notable change Change.Thus more confirming that CORM-2 can play antivirus action in PRRSV infection Marc-145 cell processes.

The detection of PRRSV ORF7 mRNA expression in PAM cell after embodiment 5 variable concentrations CORM-2 process

The separation of PAM cell

(1) pallet, waste cylinder are placed in Biohazard Safety Equipment and are sterilized through ultra violet lamp.

(2) by the complete lungs collecting in pig body with rinsing well containing dual anti-PBS.

(3) lungs are placed in pallet, clamp oesophagus with haemostatic clamp, and lungs are gently above carried, with scissors from tracheae knotting point Slightly descend and at 2cm, cut off tying, drawn with 10ml suction pipe and pour into lungs from there containing dual anti-PBS, volume about 150ml-250ml, Gently lungs are patted in kneading, then insert in tracheae by suction pipe, constantly suction out irrigating solution and extremely sterilize in blue mouth bottle；So repeatedly Lavation lungs 2～4 times, lavation as far as possible goes out pulmonary alveolar macrophage.

(4) collect the irrigating solution stainless steel mesh obtaining to filter to new aseptic indigo plant mouth bottle.

(5) irrigating solution after filtering is dispensed in 50ml centrifuge tube, and 500r/min is centrifuged 10min.

(6) supernatant discarded, adds 20ml to contain dual anti-PBS re-suspended cell, 500r/min is centrifuged 10min, washed cell 1 time.

(7) supernatant discarded, with RPMI 1640 culture medium of 10ml 10%FBS, will be resuspended for the PAM in all pipes and be incorporated into In one pipe, prepare cell count.

(8) count to after the PAM cell dilution obtaining.

(9) according to count results, according to different tests purpose, the RPMI 1640 of the 10%FBS of respective volume is added to cultivate Keynote whole cell density bed board, unnecessary PAM cell carries out frozen.

(10) according to cell quantity, by often pipe 1ml, every milliliter 2 × 10⁷Individual cell density, calculates required frozen liquid Long-pending, according to serum：DMSO=9:1 proportional arrangement frozen stock solution.

(11) 500r/min centrifugation 10min, supernatant discarded, will be resuspended for cell and dispense to cryopreservation tube with frozen stock solution, press Requirement marks in the cooling box that loads program after understanding in -80 DEG C overnight, and next day proceeds to and carries out freezen protective in liquid nitrogen.

Sample collection

Primary PAM cell is with 5 × 10⁵The density of individual cells/well is laid in 24 porocyte culture plates.After cell attachment, Culture supernatant is abandoned in suction, adds containing PRRSV but does not contain the RPMI1640 culture medium of hyclone, and 37 DEG C of incubation 1h inhale and abandon virus liquid, It is separately added into the RPMI1640 maintaining liquid culture of 3% hyclone containing variable concentrations CORM-2 (50 μM, 100 μM, 150 μM) Based on 37 DEG C, 5%CO₂Humidified incubator in cultivate；Trained with the RPMI 1640 of 3% hyclone without CORM-2 simultaneously The maintenance liquid culture medium of foster base and 150 μM of iCORM-2 of addition collects cell and supernatant as comparison, 24hpi.

QPCR detection method is with the method described in embodiment 1.

Can be seen that the increasing with CORM-2 concentration from PAM intracellular PRRSV ORF7 gene qPCR testing result (Fig. 5) Plus, the expression of virus O RF7 gene is gradually lowered, and notable with control group comparing difference, and the iCORM-2 inactivateing is to PRRSV ORF7 gene expression does not make significant difference.

Embodiment 6Western blot detects the impact to PRRSV infection PAM cell for the variable concentrations CORM-2

Western blot detection sample is derived from the cell sample collected by embodiment 5.

Western blot detection method is with the method described in embodiment 2.

Western blot testing result is as shown in fig. 6, compared with processing control group without CORM-2, CORM-2 is with concentration Dependence mode suppresses PRRSV N protein to express, and iCORM-2 does not make significant difference to N protein expression.

PAM cells and supernatant PRRSV TCID after embodiment 7 variable concentrations CORM-2 process₅₀Mensure

TCID₅₀Measure Supernatant samples and be derived from the cells and supernatant collected by embodiment 5.

TCID₅₀Assay method is with the method described in embodiment 3.

TCID₅₀Measurement result is as shown in fig. 7, compared with processing control group without CORM-2, CORM-2 concentration gradient relies on Property reduce PAM cells and supernatant generation of neutrons virus titer, and iCORM-2 is to supernatant TCID₅₀Do not make significant difference.

Above the specific embodiment of the present invention is illustrated, but the present invention is not limited thereto, without departing from Spirit of the invention, the present invention can also have various change.

Claims

1. carbon monoxide-releasing molecules -2 preparation is it is characterised in that include carbon monoxide-releasing molecules -2 and auxiliary material, a described oxygen Changing carbon emissions molecule -2 English name is CORM-2 (Tricarbonyldichlororuthenium (II) dimer), its structure Shown in formula I, the dosage of described carbon monoxide-releasing molecules -2 is 50-150 μM/L to formula, and described auxiliary material is DMSO.

2. carbon monoxide-releasing molecules -2 preparation according to claim 1 it is characterised in that：Described pharmaceutical preparation is injection Agent or oral formulations.

3. carbon monoxide-releasing molecules -2 preparation according to claim 2 it is characterised in that：Described ejection preparation is lyophilized Powder-injection.

4. carbon monoxide-releasing molecules -2 preparation according to claim 2 it is characterised in that：Described oral formulations are discrete piece Agent, capsule or granule.

5. anti-porcine reproductive and respiratory syndrome virus prepared by carbon monoxide-releasing molecules -2 preparation described in a kind of claim 1 Application on medicine.

6. the carbon monoxide-releasing molecules -2 described in a kind of claim 1 are preparing anti-porcine reproductive and respiratory syndrome virus duplication Application in preparation.