抗猪瘟病毒转基因猪的构建及初步研究
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摘要
猪瘟是一种传染性极强的急性、热性、高致死性传染病,发病率和死亡率很高,是威胁养猪业最重要的传染病,建立新的抗病毒方法,加快抗猪瘟动物育种研究,控制猪瘟的流行具有重要的现实意义。RNA干扰是广泛存在于生物体内,由双链RNA引发的转录后基因沉默机制,随着RNAi机制的逐步阐明及其应用技术的不断发展,现已成为最有潜力和应用价值的抗病毒感染方法之一。为了获得抗猪瘟病毒的新品种,本研究通过将RNAi技术和转基因猪制备技术相结合,培育出了稳定表达针对猪瘟病毒的shRNA的转基因克隆猪。
首先根据RNAi的技术原理,基于载体PGKneotpAlox2,设计并构建了针对猪瘟病毒N~(pro)基因的shRNA表达载体PGKneotlaAlox2-shRNA-N1N2和阴性干扰shRNA表达载体PGKneotpAlox2-shRNA-N1N2-Ctrl,转染原代猪胚胎成纤维细胞,经G-418筛选,获得猪瘟病毒特异性siRNA稳定表达细胞株28株,阴性对照siRNA克隆稳定表达细胞株30株,感染猪瘟病毒72h的间接免疫荧光和病毒滴度测定结果显示,所得克隆中PEF-N1N2-6,PEF-N1N2-11,PEF-N1N2-15,PEF-N1N2-21,PEF-N1N2-25对猪瘟病毒复制的抑制率超过90%。
分别以获得的上述抑制率超过90%的细胞株为核供体细胞,通过细胞核移植技术制备转基因克隆猪,共获得到15头克隆猪,分离培养转基因仔猪细胞和同品种仔猪细胞,扩大培养后感染猪瘟病毒Shimen株,间接免疫荧光实验和病毒滴度测定实验表明,与同日出生的同品种非转基因仔猪相比,所构建的转基因克隆猪细胞上猪瘟病毒的增殖受到明显抑制。
上述研究成果为研制抗猪瘟病毒的转基因克隆猪奠定了基础,并为建立抗其它病毒的转基因动物育种提供了参考数据。
Classical swine fever(CSF,hog cholera) is a highly contagious disease in domestic pigs.Pigs at any age,of any species,can be infected in any season.The morbidity and mortality of CSF caused by virulent CSFV can be 100%.It is the most important infectious diseases of pig-raising industry.The World Organization for Animal Health(OIE) has classified CSF as one of sixteen kinds of class a legal communicable diseases,our country has set CSF as a class of serious infectious diseases.Some countries in Europe announced the elimination of swine fever after killing all animals on infected farms.But in recent years,these countries have also emerged with the outbreak of classical swine fever epidemic;there is the danger of spread of the epidemic.Our country has controlled the disease pandemic with large-scale vaccine inoculation or slaughter the infected pigs.Although this measure prevented the disease efficiently,failure of immunization and persistent infection of CSFV occur occasionally.It is still a serious threat to large-scale pig-raising industry.thus,the development of new antiviral approaches and research of new resistance breeding in swine to control swine fever epidemic effectively has important practical significance.
RNA interference(RNAi) is a naturally occurring,sequence-specific mechanism for gene silencing.RNAi-related events were discovered in almost all eukaryotic organisms,including protozoa,flies,nematodes,insects,parasites,plants,and mouse and human cell lines.It refers to the phenomenon of post-translational silencing of gene expression that occurs in response to the introduction of double-stranded RNA into a cell.It is a process through which endogenous or exogenous double-stranded RNA(dsRNA) induces the sequence-specific post-transcriptional silencing of gene.Its related processes seem to be a cellular defense mechanism against gene invaders such as transposons and viruses.Researchers have developed post-transcriptional gene silencing technology to degrade the homologous single-stranded mRNAs through the introduction of the functional double-stranded RNA(siRNA) which is homologous to the target gene in cells. They think highly of this technology because a large number of studies have shown that siRNA may inhibit the proliferation of a variety of viruses efficiently in vivo study or in vitro study;It is has been used as a new powerful tool for gene-specific therapeutics for viral disease and showed a good application prospects.
In order to develop new ways of prevention and control of classical swine fever and explore the feasibility of breeding for disease resistance to classical swine fever virus with the production technique of transgenic pigs and RNAi,we designed and constructed shRNA expression vector PGKneotpAlox2-shRNA-N1N2,which contains two separate shRNA expression cassette according to the principle of DNA-vector-based RNAi technology.The shRNA expression cassette is under the control of respective H1 promoter.It will be recognized by polymeraseⅢof the cells and will be transcribed into shRNAs;it will be further processed into a classical swine fever virus-specific siRNAs whose target is the N~(pro) of CSFV When the vector is introduced into the cells.The negative control shRNA expressing plasmid was constructed in the same way and was named PG KneotpAlox2-shRNA-N1N2-Ctrl.
Large-scale Endotoxin-free DNA were prepared and purified by using the Endotoxin-free plasmid DNA extraction kit and were digested with restriction enzyme ApaI and SacI,leaving only the DNA sequence of G-418 expression cassette flanked by loxP sequences and two shRNA expression cassettes as the transgenic construct.For transduction of fetal porcine fibroblasts, 1×10~5 cells porcine fetal fibroblasts that were obtained from 35-day-old fetuses were transferred to 35-mm culture dish(Corning).Gene transfer was carried out according to the manufacturer's instructions of transfection reagent FuGENE HD.According to the conventional methods,the cells recovered from the dish were seeded in 6-well plates(2×10~4cells/well).At 48 h after gene transfer,the cells were incubated in standard culture medium including 350μg/ml of G-418. Colonies resistant to G-418 appeared after cells were cultured in selection medium for an additional 7 days.The surviving cell colonies resistant to G-418 were obtained and subcultured in 24-well plates and propagated and the cells were frozen.In order to examine if the gene have integrated into genome of the cells,the genomic DNA of cell clones were used for PCR amplification.Twenty-eight cell strains expressing the CSFV-specific-shRNAs were obtained and thirty cell strains expressing the short hairpin RNAs of negative shRNA were obtained too.When the cells have grown to confluence in a well of 24-well plates,split them into 96-well plates and assay individual clones for antivirus effect.These cell strains in a 96-well tissue culture cluster were infected by 100TCID_(50) classical swine fever virus shimen strain respectively,and 72h later, the virus replication were detected and evaluated by undirected immunofluorescence assay(IFA). The results showed the infected cell were above 90%in all of the cell strains expressing the short hairpin RNAs of negative shRNA.But the inhibition ratio of the virus replication in cell strains PEF-N1N2-6,PEF-N1N2-11,PEF-N1N2-15,PEF-N1N2-21,PEF-N1N2-25 were above 90%. These results showed that siRNAs expressed in these cell strains specifically suppress CSFV gene expression.Further more,the TCID_(50) of CSFV of these cell strains were significantly reduced,in contrast.
Based on the aforementioned studies,cell stains exhibiting noticeable inhibition effect were selected and were used as the nuclear donor cells.The reconstructed embryos were produced by somatic cell nuclear transfer technology and were surgically transferred into the oviducts of the recipients after making a mid-ventral incision under general anesthesia.Following transfer of cloned embryos;two recipients became pregnant and then delivered fiveteen piglets.Samples obtained from the transgenic-clone pigs,donor cells and recipients were analyzed by using STR-PCR method and the result confirmed that the piglets were clones of the donor cells. Detection of exogenous gene integration was conducted by using PCR and hiTail-PCR analysis. The PCR results together with the hiTail-PCR analysis of the piglets indicated that the transfected gene had integrated into genome of one pig.In order to make a preliminary assessment of antivirus ability of the transgenic piglet,cells were isolated from transgenic piglet and non transgenic piglet and were infected with 100TCID_(50) classic swine fever virus Shimen strain respectively,the results of indirected immunofluorescence assay(IFA) and titration determination test at 48h and 72h post-infection of CSFV showed that the amount of virus multiplication were reduced in cells of the transgenic-clone pig significantly.It indicates that the expression of siRNA in the cell of transgenic-clone pig can effectively inhibit the proliferation of classical swine fever virus.
In the present study,we produced transgenic-clone pig which has the ability to express CSFV-specific siRNAs and accumulated essential experimental data of the application of RNAi in the researches of transgenic-clone pig which can resist CSFV infection.Our results provide a reference for the establishment of other anti-virus breeding of transgenic animals.
首先根据RNAi的技术原理,基于载体PGKneotpAlox2,设计并构建了针对猪瘟病毒N~(pro)基因的shRNA表达载体PGKneotlaAlox2-shRNA-N1N2和阴性干扰shRNA表达载体PGKneotpAlox2-shRNA-N1N2-Ctrl,转染原代猪胚胎成纤维细胞,经G-418筛选,获得猪瘟病毒特异性siRNA稳定表达细胞株28株,阴性对照siRNA克隆稳定表达细胞株30株,感染猪瘟病毒72h的间接免疫荧光和病毒滴度测定结果显示,所得克隆中PEF-N1N2-6,PEF-N1N2-11,PEF-N1N2-15,PEF-N1N2-21,PEF-N1N2-25对猪瘟病毒复制的抑制率超过90%。
分别以获得的上述抑制率超过90%的细胞株为核供体细胞,通过细胞核移植技术制备转基因克隆猪,共获得到15头克隆猪,分离培养转基因仔猪细胞和同品种仔猪细胞,扩大培养后感染猪瘟病毒Shimen株,间接免疫荧光实验和病毒滴度测定实验表明,与同日出生的同品种非转基因仔猪相比,所构建的转基因克隆猪细胞上猪瘟病毒的增殖受到明显抑制。
上述研究成果为研制抗猪瘟病毒的转基因克隆猪奠定了基础,并为建立抗其它病毒的转基因动物育种提供了参考数据。
Classical swine fever(CSF,hog cholera) is a highly contagious disease in domestic pigs.Pigs at any age,of any species,can be infected in any season.The morbidity and mortality of CSF caused by virulent CSFV can be 100%.It is the most important infectious diseases of pig-raising industry.The World Organization for Animal Health(OIE) has classified CSF as one of sixteen kinds of class a legal communicable diseases,our country has set CSF as a class of serious infectious diseases.Some countries in Europe announced the elimination of swine fever after killing all animals on infected farms.But in recent years,these countries have also emerged with the outbreak of classical swine fever epidemic;there is the danger of spread of the epidemic.Our country has controlled the disease pandemic with large-scale vaccine inoculation or slaughter the infected pigs.Although this measure prevented the disease efficiently,failure of immunization and persistent infection of CSFV occur occasionally.It is still a serious threat to large-scale pig-raising industry.thus,the development of new antiviral approaches and research of new resistance breeding in swine to control swine fever epidemic effectively has important practical significance.
RNA interference(RNAi) is a naturally occurring,sequence-specific mechanism for gene silencing.RNAi-related events were discovered in almost all eukaryotic organisms,including protozoa,flies,nematodes,insects,parasites,plants,and mouse and human cell lines.It refers to the phenomenon of post-translational silencing of gene expression that occurs in response to the introduction of double-stranded RNA into a cell.It is a process through which endogenous or exogenous double-stranded RNA(dsRNA) induces the sequence-specific post-transcriptional silencing of gene.Its related processes seem to be a cellular defense mechanism against gene invaders such as transposons and viruses.Researchers have developed post-transcriptional gene silencing technology to degrade the homologous single-stranded mRNAs through the introduction of the functional double-stranded RNA(siRNA) which is homologous to the target gene in cells. They think highly of this technology because a large number of studies have shown that siRNA may inhibit the proliferation of a variety of viruses efficiently in vivo study or in vitro study;It is has been used as a new powerful tool for gene-specific therapeutics for viral disease and showed a good application prospects.
In order to develop new ways of prevention and control of classical swine fever and explore the feasibility of breeding for disease resistance to classical swine fever virus with the production technique of transgenic pigs and RNAi,we designed and constructed shRNA expression vector PGKneotpAlox2-shRNA-N1N2,which contains two separate shRNA expression cassette according to the principle of DNA-vector-based RNAi technology.The shRNA expression cassette is under the control of respective H1 promoter.It will be recognized by polymeraseⅢof the cells and will be transcribed into shRNAs;it will be further processed into a classical swine fever virus-specific siRNAs whose target is the N~(pro) of CSFV When the vector is introduced into the cells.The negative control shRNA expressing plasmid was constructed in the same way and was named PG KneotpAlox2-shRNA-N1N2-Ctrl.
Large-scale Endotoxin-free DNA were prepared and purified by using the Endotoxin-free plasmid DNA extraction kit and were digested with restriction enzyme ApaI and SacI,leaving only the DNA sequence of G-418 expression cassette flanked by loxP sequences and two shRNA expression cassettes as the transgenic construct.For transduction of fetal porcine fibroblasts, 1×10~5 cells porcine fetal fibroblasts that were obtained from 35-day-old fetuses were transferred to 35-mm culture dish(Corning).Gene transfer was carried out according to the manufacturer's instructions of transfection reagent FuGENE HD.According to the conventional methods,the cells recovered from the dish were seeded in 6-well plates(2×10~4cells/well).At 48 h after gene transfer,the cells were incubated in standard culture medium including 350μg/ml of G-418. Colonies resistant to G-418 appeared after cells were cultured in selection medium for an additional 7 days.The surviving cell colonies resistant to G-418 were obtained and subcultured in 24-well plates and propagated and the cells were frozen.In order to examine if the gene have integrated into genome of the cells,the genomic DNA of cell clones were used for PCR amplification.Twenty-eight cell strains expressing the CSFV-specific-shRNAs were obtained and thirty cell strains expressing the short hairpin RNAs of negative shRNA were obtained too.When the cells have grown to confluence in a well of 24-well plates,split them into 96-well plates and assay individual clones for antivirus effect.These cell strains in a 96-well tissue culture cluster were infected by 100TCID_(50) classical swine fever virus shimen strain respectively,and 72h later, the virus replication were detected and evaluated by undirected immunofluorescence assay(IFA). The results showed the infected cell were above 90%in all of the cell strains expressing the short hairpin RNAs of negative shRNA.But the inhibition ratio of the virus replication in cell strains PEF-N1N2-6,PEF-N1N2-11,PEF-N1N2-15,PEF-N1N2-21,PEF-N1N2-25 were above 90%. These results showed that siRNAs expressed in these cell strains specifically suppress CSFV gene expression.Further more,the TCID_(50) of CSFV of these cell strains were significantly reduced,in contrast.
Based on the aforementioned studies,cell stains exhibiting noticeable inhibition effect were selected and were used as the nuclear donor cells.The reconstructed embryos were produced by somatic cell nuclear transfer technology and were surgically transferred into the oviducts of the recipients after making a mid-ventral incision under general anesthesia.Following transfer of cloned embryos;two recipients became pregnant and then delivered fiveteen piglets.Samples obtained from the transgenic-clone pigs,donor cells and recipients were analyzed by using STR-PCR method and the result confirmed that the piglets were clones of the donor cells. Detection of exogenous gene integration was conducted by using PCR and hiTail-PCR analysis. The PCR results together with the hiTail-PCR analysis of the piglets indicated that the transfected gene had integrated into genome of one pig.In order to make a preliminary assessment of antivirus ability of the transgenic piglet,cells were isolated from transgenic piglet and non transgenic piglet and were infected with 100TCID_(50) classic swine fever virus Shimen strain respectively,the results of indirected immunofluorescence assay(IFA) and titration determination test at 48h and 72h post-infection of CSFV showed that the amount of virus multiplication were reduced in cells of the transgenic-clone pig significantly.It indicates that the expression of siRNA in the cell of transgenic-clone pig can effectively inhibit the proliferation of classical swine fever virus.
In the present study,we produced transgenic-clone pig which has the ability to express CSFV-specific siRNAs and accumulated essential experimental data of the application of RNAi in the researches of transgenic-clone pig which can resist CSFV infection.Our results provide a reference for the establishment of other anti-virus breeding of transgenic animals.
引文
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