CN103740755A - Application of IFIT1 gene of pig in resisting PRRS (porcine reproductive and respiratory syndrome) virus - Google Patents
Application of IFIT1 gene of pig in resisting PRRS (porcine reproductive and respiratory syndrome) virus Download PDFInfo
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- CN103740755A CN103740755A CN201310718015.7A CN201310718015A CN103740755A CN 103740755 A CN103740755 A CN 103740755A CN 201310718015 A CN201310718015 A CN 201310718015A CN 103740755 A CN103740755 A CN 103740755A
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Abstract
The invention relates to a new function of an IFIT1 gene of a pig in resisting PRRS (porcine reproductive and respiratory syndrome) virus, and specifically provides an application of an IFIT1 gene of a pig in resisting PRRS (porcine reproductive and respiratory syndrome) virus. The PRRS virus is inhibited from replicating in cells through overexpression of the IFIT1 gene in cells. The invention also provides a method for screening pigs resisting PRRS virus through IFIT1 gene expression quantity and a method for preparing clone embryo over expressing IFIT1 gene.
Description
Technical field
The present invention relates to genetically engineered field, specifically, relate to the new function of pig IFIT1 gene in anti-PRRS virus.
Background technology
Porcine reproductive and respiratory syndrome (PRRS), being commonly called as " blue otopathy " is caused by porcine reproductive and respiratory syndrome virus (PRRSV), this virus is a member of nido virales Arteriviridae, the virus belonging to together also has equine arteritis virus, mouse serum lactic dehydrogenase enzymophathy poison and SHF virus.This disease mainly causes breeding and the respiratory symptom of pig, shows as interstitial pneumonia and sow miscarriage mummy tire etc., every year global pig industry has been caused to huge financial loss.Within 2007, in China, broken out a hyperpyrexia disease, this disease in the whole country diffusion, surpasses 2,000,000 pigs infected rapidly, 400,000 pig death, and last confirmation of this disease is to be caused by a kind of highly pathogenic blue otopathy poison.The pulmonary alveolar macrophage of PRRSV main infection pig, research now shows after infecting PRRSV, in congenital immunity, PRRSV infects and can not cause effective I type interferon response, humoral immunization can not produce effective neutralizing antibody in time, and can form the viral reinforcing effect that antibody relies on, finally cause immunosuppression and persistent infection.Because this viral mutation rate is high, traditional vaccine method can not effectively be controlled this disease, and a lot of research is all devoted to find RNAi, and the method that Interferon, rabbit etc. are new is come for this virus, for making up the deficiency of traditional method.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide the new gene IFIT1 that a kind of and anti-blue otopathy is relevant, and by IFIT1 gene was carried out to expression, thereby suppress PRRS virus copying in cell.
Technical scheme of the present invention is as follows:
First the present invention provides the application of pig IFIT1 gene in anti-PRRS virus, and it is that the expression of crossing in cell suppresses PRRS virus copying in cell by IFIT1 gene.
The present invention also provides a kind of mistake to express the carrier of IFIT1 gene.
As preferably, described carrier is for importing the pCMV-Myc-N carrier of IFIT1 gene C DS sequence.
The present invention also provides the transgenic cell that contains described carrier.
Further, described transgenic cell is the porcine somatic cell except embryonic cell.
The present invention also provides a kind of method of preparing clone embryos, and it take transgenic cell described in claim 5 is nuclear transplantation donorcells, and in vitro ovocyte is nuclear transplantation recipient cell, by nuclear transfer technology, obtains clone embryos.
The present invention also provides the method for utilizing IFIT1 gene expression amount to screen anti-PRRS virus pig, described method is for to measure by the expression amount of the IFIT1 gene to pig, and the pig pig lower than IFIT1 expression amount that IFIT1 expression amount is high has higher anti-PRRS virus infection ability.
Beneficial effect of the present invention is:
The new gene IFIT1 relevant to anti-blue otopathy that the present invention identifies (interferon-induced protein with tetratricopeptide repeat1) is a kind of interferon inducible protein, there are some researches show that it has antiviral effect.The inventor utilizes information biology in conjunction with the method for Cell Biology Experiment first, has proved that IFIT1 has the effect that suppresses PRRS virus replication.
The gene IFIT1 that the new inhibition PRRSV that the present invention finds copies can have following two purposes:
1. analyze and show, with respect to the cell that there is no to express IFIT1 gene, cross the cell of expressing IFIT1 gene and can significantly suppress copying of PRRS virus, therefore, can measure by the expression amount of the IFIT1 gene to pig, the pig that IFIT1 expression amount is high may be lower than IFIT1 expression amount the infection of the more anti-PRRSV of pig, thereby reach the pig of just screening anti-PRRSV according to the expression amount of IFIT1 gene.
2. can utilize the method for the genetic engineerings such as transgenosis, the expression amount of IFIT1 gene in pig be raise, thereby obtain the pig that IFIT1 expression amount is high, and then cultivate the pig that anti-PRRSV copies.
Accompanying drawing explanation
Fig. 1 is the IFIT1 that obtains of the embodiment of the present invention 1 transcription group data analysis infects PRRSV at Tongcheng pig and landrace the 0th day, the 3rd day, and expression pattern and relative expression quantity in the 5th day and the 7th day lung tissue; X-coordinate represents time point, and ordinate zou represents RPKM(relative expression quantity).
Fig. 2 is the carrier collection of illustrative plates of initial carrier pCMV-Myc-N in the embodiment of the present invention 2.
Fig. 3 is the copy number that in the embodiment of the present invention 2, MARC145 cell is attacked latter 24 hours PRRSV of poison experiment; X-coordinate represents control group and attacks malicious group; Ordinate zou represents the relative expression quantity of PRRSV ORF7.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is all according to normal experiment condition, as Sambrook equimolecular cloning experimentation handbook (New York:Gold Spring Harbor Laboratory Press, 1989), or according to the condition of manufacturer's specification sheets suggestion.
Embodiment 1 utilizes to analyze and transcribes the gene IFIT1 that group data method finds that new anti-PRRSV infects
Due to different pig varieties and individual genetic background difference, also different to the resistance of blue otopathy, study and show on clinical symptom and physiological and biochemical index, the domestic pig varieties such as Tongcheng pig, plum mountain pig have stronger resistance after the infection highly pathogenic PRRSVs such as external landrace, Large White.By the method for high-flux sequence, to infecting the tissue of PRRSV front and back landrace and Tongcheng pig, do differential genes expression analysis, some Ia gene expression amount has significant difference between former and later two kinds of infection.By PRRSV between different varieties being infected to the analysis of the gene expression difference cause, not only can study the infection mechanism of blue otopathy, can also find out the gene that resistance or susceptible are relevant, thereby provide new method for treatment and the prevention of blue otopathy.
Previous experiments is carried out the screening of disease-resistant pig by 6 product boars (landrace, Large White, duroc, peaceful pig, plum mountain pig, Tongcheng pig) are infected to high-pathogenicity blue ear disease poison (PRRSV), by clinical symptom record: the detection of food consumption, body temperature, Physiology and biochemistry, cytokine and survival time etc., has finally obtained two kinds larger to blue otopathy Resistant Difference: landrace (susceptible pig), Tongcheng pig (disease-resistant pig).
Choose each 8 of disease-resistant pig and susceptible pigs, respectively the 0th day and infect the high PRRSV of causing a disease after within the 3rd, 5,7 days, slaughter sampling, lung tissue is extracted total RNA, builds storehouse and carries out high-flux sequence, passes through bioinformatic analysis.Analyze and find: attacking poison, within the 0th day, be control group, the relative expression quantity (RPKM) 78.6 of 0th day of IFIT1 in the pig lung tissue of Tongcheng is greater than 6.49 of landrace; And in the pig lung tissue of Tongcheng the expression of IFIT1 to reach peak value be after infection the 5th day, in landrace lung tissue, to reach peak value be after infection the 3rd day (as shown in Figure 1) in the expression of IFIT1.
Embodiment 2 utilizes the anti-PRRSV of cell experiment method validation IFIT1 gene to infect
1.1. design the primer for IFIT1 gene (GenBank accession number is HQ679904.1), utilize reverse transcription PCR technology reverse transcription mRNA amplification to obtain the CDS sequence of IFIT1 gene, primer sequence is as follows:
Goal gene IFIT1:
Upstream primer IFIT1-F:
5′-GCGTCGACCATGGTTTATTCCAACTGTG-3′；
Downstream primer IFIT1-R:
5′-CCCTCGAGTCAGCCTGGGAACTTG-3′。
The CDS sequence of the IFIT1 gene that amplification is obtained (as shown in SEQ ID No.1) imports initial carrier pCMV-Myc-N(purchased from Clonetech company, and carrier collection of illustrative plates is as shown in Figure 2), build pCMV-IFIT1-Myc carrier.Because having CMV strong promoter, pCMV-Myc-N can make the IFIT1 gene importing wherein carry out expression.
The checking of 2.IFIT1 gene overexpression
By the pCMV-IFIT1 carrier that is connected with IFIT1 gene C DS sequence building and the pCMV-Myc-N carrier that is not connected with the CDS sequence of IFIT1 gene, transfection MARC145 cell respectively.
3. by the pCMV-IFIT1 carrier that is connected with IFIT1 gene C DS sequence building and the pCMV-Myc-N carrier that is not connected with the CDS sequence of IFIT1 gene, difference transfection MARC145 cell, after cultivating 24 hours, extract total RNA of cell, to the 7th coding reading frame of the ORF7(of PRRS virus) carry out quantitative PCR detection.
PRRS virus O RF7 primer:
Upstream primer PRRS-F:AATAACAACGGCAAGCAGCA;
Downstream primer PRRS-R:GCACAGTATGATGCGTCGGC.
In order to calculate the relative expression quantity of the ORF7 of PRRS virus, when the ORF7 of PRRS virus is carried out to quantitative PCR detection, introduce reference gene GAPDH(GAPDH more constant in cell inner expression amount), carry out pcr amplification simultaneously.
Reference gene GAPDH(GenBank accession number is NM_001206359.1):
Reference gene GAPDH primer:
Upstream primer GAPDH-F:CCTTCCGTGTCCCTACTGCCAAC;
Downstream primer GAPDH-R:GACGCCTGCTTCACCACCTTCT.
4. analyze transfection with the copy number of the PRRSORF7 in the cell of the plasmid of IFIT1 gene C DS sequence and transfection without the copy number of the ORF7 in the cell of the plasmid of IFIT1 gene C DS sequence, whether there were significant differences.Our result shows, complete attacking poison, and cultivate after 24 hours, transfection cross the relative expression quantity of the ORF7 of the PRRS virus in the cell of expressing IFIT1 gene C DS sequence will be significantly lower than control group (as shown in Figure 3).
By relatively crossing the copy number express the MARC145 cell of IFIT1 gene and not turn the PRRS in the MARC145 cell of any gene, analyze the impact that IFIT1 gene copies in MARC145 cell for PRRS virus.We study and found after expression IFIT1 gene, PRRS virus copying obviously in MARC145 cell is suppressed, this explanation IFIT1 gene is inhibited for copying of PRRS virus, and we can express IFIT1 gene by mistake and reach the object that suppresses PRRS virus replication.
In the present embodiment experimental implementation, comprise:
1) extraction of total RNA
The TRIzol reagent that the extraction Shi Yong Invitrogen company of total RNA produces, and require to operate in strict accordance with product description.
2) reverse transcription PCR
Reverse transcription adopts is MMLV reverse transcription archaeal dna polymerase and requires to operate in strict accordance with the specification sheets of Promega company.
3) utilize the expression of fluorescence real-time quantitative PCR technology for detection PRRSV ORF7
Fluorescence real-time quantitative PCR:
Instrument model: the Applied BiosystemsViiA7 that ABI company produces
Test kit: the FastSYBR mixture that Kang Wei ShiJi Co., Ltd produces (CoWin Biotech Co., Ltd.[CWBIO]), and operate according to product description.
PRRSV ORF7 relative expression analysis method:
Δ Δ Ct method: Δ Ct experimental group=(experimental group goal gene Ct-experimental group reference gene Ct)
Δ Ct control group=(control group goal gene Ct-control group reference gene Ct)
2
-Δ Δ Ct=2
-(Δ Ct experimental group-Δ Ct control group)
Adopt GAPDH as reference gene.
1, the recovery of donorcells.From liquid nitrogen, take out cryopreservation tube, drop into fast in 37 degree water-baths, and constantly Quick shaking moves cryopreservation tube.After refrigerating fulid thoroughly thaws, be transferred in centrifuge tube, with 3 times of fresh medium dilutions, 1000rpm, centrifugal 5 minutes, go out supernatant, with fresh nutrient solution suspension cell, precipitate, cell dilution, to desired concn, is inoculated in culture dish and is cultivated.
2, utilize the method for liposome transfection, object plasmid is proceeded in donorcells.Ordinary method peptic cell, dilutes 0.8ug with 100ul serum free medium DMEM and treats Pignus pignoris grain and 2ul liposome (lipofectamineTM2000).After incubated at room 20 minutes, in each hole of 24 well culture plates, add 100ul aforesaid liquid.By before and after culture plate, rock and shake up gently, cultivating and changing nutrient solution after 4-6 hour is complete cell culture medium.
3, the screening of transgenic positive cell.The liposome transfection cell of 48 hours is added to 800ug/mL G418, and every 2-3 days changes liquid once, screens after 14 days, with PBS, washes 2 times, collects positive cell clone.
4, the DNA detection of transgenic positive cell.According to conventional molecular cloning method, extract the performing PCR checking of going forward side by side of transgenic positive cell DNA.
5, take above-mentioned transgenic positive cell as nuclear transplantation donorcells, the puberty father's former wife's porcine oocytes of maturation in vitro of take is nuclear transplantation recipient cell.Donorcells and mature oocyte are proceeded to 39 degree, 5%CO simultaneously
2, fixedly suction pipe sticking ovocyte is then used in 100% moisture equilibrium 10 minutes on the inverted microscope of being furnished with micrurgy instrument and thermostatic platform, with entry needle, will draw oocyte nuclei.Then select ganoid somatocyte, during from stoning, breach is put into ovum week gap, with entry needle, presses zona pellucida, and donorcells is contacted closely with the after birth of acceptor ovum.The reconstruct ovum that donorcells-ooecium matter is formed is transferred in nutrient solution PZM3, at 39 degree, 5%CO
2, in 100% humidity incubator, recover 1 hour.
6, the reconstruct ovum having recovered is transferred in batches and merged in liquid balance 2 minutes, with after fusion/activation solution washing 3 times, put 5-8 for every batch and put into the integration slot that is paved with fusion liquid, make donorcells-recipient oocyte film contact surface parallel with electrode, with CUY-21 fusion instrument (BEX, Japan) apply 1 100us, activation is simultaneously merged in the electric pulse induction of 1.4kv/cm, then with PZM3 nutrient solution washing 3 times, proceeding to immediately mineral oil covers in embryo medium or in assisted activation liquid, 39 degree, 5%CO
2, 100% humidity is cultivated after 4 hours decision fusion under stereoscopic microscope again.
7, clone embryos vitro culture.The reconstructed embryo merging, with after embryo medium washing 5 times, is proceeded in 2 hours above embryo medium PZM3 of pre-equilibration to culture condition 39 degree, 5%CO
2, 5%O
2, 90%N
2, saturated humidity.
8, select in clone embryos that form is good moves into spontaneous estrus multiparity Gilt Uterus with Nonoperative method and carry out gestation.
9, the cloned, transgenic pig of birth is carried out to PCR and southern detection, carry out functional study.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (7)
1. the application of pig IFIT1 gene in anti-PRRS virus, is characterized in that, it is that the expression of crossing in cell suppresses PRRS virus copying in cell by IFIT1 gene.
2. a mistake is expressed the carrier of IFIT1 gene.
3. carrier according to claim 2, is characterized in that, described carrier is for importing the pCMV-Myc-N carrier of IFIT1 gene C DS sequence.
4. the transgenic cell that contains carrier described in claim 2 or 3.
5. transgenic cell according to claim 4, is characterized in that, it is the porcine somatic cell except embryonic cell.
6. a method of preparing clone embryos, is characterized in that, it take transgenic cell described in claim 5 is nuclear transplantation donorcells, and in vitro ovocyte is nuclear transplantation recipient cell, by nuclear transfer technology, obtains clone embryos.
7. utilize IFIT1 gene expression amount to screen the method for anti-PRRS virus pig, it is characterized in that, described method is for to measure by the expression amount of the IFIT1 gene to pig, and the pig pig lower than IFIT1 expression amount that IFIT1 expression amount is high has higher anti-PRRS virus infection ability.
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| CN102016072A (en) * | 2008-04-21 | 2011-04-13 | 诺瓦提斯研究基金会弗里德里克·米谢尔生物医学研究所 | Antiviral therapy |
| WO2012162367A1 (en) * | 2011-05-25 | 2012-11-29 | Medimmune, Llc | Methods of treating systemic lupus erythematosus, scleroderma, and myositis with an antibody against interferon-alpha |
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| CN101317091A (en) * | 2005-12-08 | 2008-12-03 | Irm责任有限公司 | Methods and compositions for inhibiting HIV infection |
| CN101821629A (en) * | 2007-09-10 | 2010-09-01 | 诺瓦提斯研究基金会弗里德里克·米谢尔生物医学研究所 | Method for predicting the response of subject suffering from viral infection of the liver to antiviral therapy |
| CN102016072A (en) * | 2008-04-21 | 2011-04-13 | 诺瓦提斯研究基金会弗里德里克·米谢尔生物医学研究所 | Antiviral therapy |
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Application publication date: 20140423 |