口蹄疫病毒反向遗传技术体系及分子标记疫苗株的构建
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摘要
构建口蹄疫病毒(FMDV)的反向遗传技术体系,对分子标记疫苗的研究具有重要的理论意义和广阔的应用前景。本研究用RT-PCR法及RACE法构建了口蹄疫病毒WFL株全长cDNA克隆,经体外转录后进行细胞转染,结果证明可以拯救出口蹄疫病毒;为了提高拯救病毒的数量,又分别构建了口蹄疫病毒全基因组真核表达质粒和口蹄疫病毒ORF真核表达质粒,并将两种真核表达质粒与体外转录RNA共转染细胞,结果揭示,两种真核表达质粒与体外转录本共转染后均可拯救出口蹄疫病毒;在全长cDNA克隆的基础上,又将GFP基因插入到口蹄疫病毒P3基因内,构建了口蹄疫病毒P3基因插入突变的分子标记毒株。本研究成功地建立了口蹄疫病毒反向遗传技术体系,并获得了口蹄疫病毒分子标记疫苗株,从而为分子标记疫苗的开发应用奠定了基础。
FMD is one of the most highly contagious diseases of animals or humans.The disease affects domestic cloven-hoofed animals, including cattle, swine, sheep, and goats,as well as more than 70 species of wild animals, including deer, and is characterized by fever, lameness, and vesicular lesions on the tongue, feet, snout, and teats.In sheep and goats the disease is generally mild and can be difficult to distinguish from other common conditions.Although FMD does not result in high mortality in adult animals, the disease has debilitating effects, including weight loss, decrease in milk production, and loss of draught power,resulting in a loss in productivity for a considerable time.Mortality,however, can be high in young animals, where the virus can affect the heart. In addition, cattle, sheep, and goats can become carriers, and cattle can harbor virus for up to 2 to 3 years.
FMDV rapidly replicates and spreads within the infected animal, among in- contact susceptible animals,and by aerosol. Disease signs can appear within 2 to 3 days after exposure and can last for 7 to 10 days. So,FMD is on the A list of infectious diseases of animals of the Office International desépizooties (OIE) and has been recognized as the most important constraint to international trade in animals and animal products.The recent outbreaks of FMD in a number of FMD-free countries, in particular Taiwan in 1997 and the United Kingdom in 2001, have significantly increased public awareness of this highly infectious disease of clovenhoofed livestock.Furthermore,worldwide concern following the terrorist attacks in the United States has raised the possibility that terrorist organizations or rogue states might target livestock industry by employing the etiologic agent of FMD.
Vaccination is the only way to prevent FMD, but we cannot differentiate the diseased from the vaccinated animals after the current FMD vaccine used,so it recommended the development of modern diagnostic methods to rapidly detect an outbreak, as well as the investment of additional funds in animal disease research to develop marker vaccines that would allow easy diagnostic distinction of vaccinated from infected or convalescent animals by OIE.
Reverse genetic manipulation give a new live to develop new vaccine for FMDV and differentiate the diseased from the vaccinated.Reverse genetic manipulation is a new technique for virus rescue,which is reverse to classical genetic manipulation.The technique make it possible to manipulate virus genome on the level of cDNA,for example point mutantion,deletion,insertion,translocation and so on,so that to study the mechanism of replication,the mechanism of expression and regulation, the strategy for antivirus,gene therapy,and the most important is to develop new vaccine,especially molecular marker vaccine.
Molecular marker vaccine is a newly vaccine which is to insert a marker gene into full length cDNA of virus genome,when the vaccine is used the marker gene or its product can be detected to differentiate the diseased from the vaccinated animal.All of the current vaccine of FMDV is inactivated vaccine,its effectiveness depent on the serotype of FMDV.On the contrary, molecular marker vaccine based on the reverse genetics is a more dependable live vaccine with wider prospective for vaccine development.Furthermore,development of molecular marker vaccine of FMDV based on the full length infectious molecular clone may give a nice pave for attenuated vaccine in our country and Europe.
In this study, the full length cDNA of FMDV WFL strain was cloned by RT-PCR or RACE,and sequenced.The results showed that the full length of the genome was 8155nt including 1059nt of the 5′UTR,6969nt of the ORF and 127nt of the 3′UTR.The ORF has a leader protein and a polyprotein with 201aa and 2122aa, respectively.
In order to construct reverse genetic manipulation system of FMDV and rescue FMDV, the full length cDNA plasmid pFMDV-AI was linearized and transcribed in vitro,and RNA was transfected into sensitive cell.The results showed that BHK-21 has CPE(cytopathic effect),and it had FMDV in cell culture fluid proved by ELISA and RT-PCR,which showed we constructed the reverse genetic manipulation system of FMDV and rescued FMDV successfully.
Furthermore,two eukaryotic expression plasmids, pEGFP-C1-FMDV-A2I2 and pEGFP-C1-A3I3,were constructed based on the pEGFP-C1.The former plasmid has the full length genome of FMDV,and the latter has FMDV ORF gene.The results of transient expression showed that two eukaryotic expression plasmids can express in BHK-21,and green fluorescence was seen by fluorescence microscope,but we cannot get any positive result by sandwich ELISA when the plasmids transfected into cell alone.But,when the plasmid cotransfected with RNA transcribed from linearized cDNA clone,FMDV antigen and nucleotide acid was detected in cell culture fluid by sandwich ELISA and RT-PCR,respectively.And the virus particles were also found by electron microscope.Animal inoculation study showed that level of FMDV antibody in baby mouse was increased with inoculation times.The results proved that cotransfection of eukaryotic expression plasmid with RNA can be used to rescue virus.And for the first time we successfully rescued FMDV by cotransfecting eukaryotic expression plasmid with RNA transcribed in vitro.
In order to develop a molecular marker vaccine of FMDV,GFP gene was inserted into P3 gene of the full length cDNA clone of FMDV to construct a recombinated plasmid,named pFMDV-AI-GFP.The result of transfection showed that the recombinated RNA transcribed in vitro can expressed in BHK-21 cell,and green fluorescence can be seen by fluorescence microscope.Results of RT-PCR which amplified GFP gene and AB fragment of FMDV and sandwich ELISA also showed that mutanted FMDV was rescued successfully.We successfully rescued a molecular marker strain of FMDV for the first time,based on which molecular marker vaccine of FMDV may be developed.
FMD is one of the most highly contagious diseases of animals or humans.The disease affects domestic cloven-hoofed animals, including cattle, swine, sheep, and goats,as well as more than 70 species of wild animals, including deer, and is characterized by fever, lameness, and vesicular lesions on the tongue, feet, snout, and teats.In sheep and goats the disease is generally mild and can be difficult to distinguish from other common conditions.Although FMD does not result in high mortality in adult animals, the disease has debilitating effects, including weight loss, decrease in milk production, and loss of draught power,resulting in a loss in productivity for a considerable time.Mortality,however, can be high in young animals, where the virus can affect the heart. In addition, cattle, sheep, and goats can become carriers, and cattle can harbor virus for up to 2 to 3 years.
FMDV rapidly replicates and spreads within the infected animal, among in- contact susceptible animals,and by aerosol. Disease signs can appear within 2 to 3 days after exposure and can last for 7 to 10 days. So,FMD is on the A list of infectious diseases of animals of the Office International desépizooties (OIE) and has been recognized as the most important constraint to international trade in animals and animal products.The recent outbreaks of FMD in a number of FMD-free countries, in particular Taiwan in 1997 and the United Kingdom in 2001, have significantly increased public awareness of this highly infectious disease of clovenhoofed livestock.Furthermore,worldwide concern following the terrorist attacks in the United States has raised the possibility that terrorist organizations or rogue states might target livestock industry by employing the etiologic agent of FMD.
Vaccination is the only way to prevent FMD, but we cannot differentiate the diseased from the vaccinated animals after the current FMD vaccine used,so it recommended the development of modern diagnostic methods to rapidly detect an outbreak, as well as the investment of additional funds in animal disease research to develop marker vaccines that would allow easy diagnostic distinction of vaccinated from infected or convalescent animals by OIE.
Reverse genetic manipulation give a new live to develop new vaccine for FMDV and differentiate the diseased from the vaccinated.Reverse genetic manipulation is a new technique for virus rescue,which is reverse to classical genetic manipulation.The technique make it possible to manipulate virus genome on the level of cDNA,for example point mutantion,deletion,insertion,translocation and so on,so that to study the mechanism of replication,the mechanism of expression and regulation, the strategy for antivirus,gene therapy,and the most important is to develop new vaccine,especially molecular marker vaccine.
Molecular marker vaccine is a newly vaccine which is to insert a marker gene into full length cDNA of virus genome,when the vaccine is used the marker gene or its product can be detected to differentiate the diseased from the vaccinated animal.All of the current vaccine of FMDV is inactivated vaccine,its effectiveness depent on the serotype of FMDV.On the contrary, molecular marker vaccine based on the reverse genetics is a more dependable live vaccine with wider prospective for vaccine development.Furthermore,development of molecular marker vaccine of FMDV based on the full length infectious molecular clone may give a nice pave for attenuated vaccine in our country and Europe.
In this study, the full length cDNA of FMDV WFL strain was cloned by RT-PCR or RACE,and sequenced.The results showed that the full length of the genome was 8155nt including 1059nt of the 5′UTR,6969nt of the ORF and 127nt of the 3′UTR.The ORF has a leader protein and a polyprotein with 201aa and 2122aa, respectively.
In order to construct reverse genetic manipulation system of FMDV and rescue FMDV, the full length cDNA plasmid pFMDV-AI was linearized and transcribed in vitro,and RNA was transfected into sensitive cell.The results showed that BHK-21 has CPE(cytopathic effect),and it had FMDV in cell culture fluid proved by ELISA and RT-PCR,which showed we constructed the reverse genetic manipulation system of FMDV and rescued FMDV successfully.
Furthermore,two eukaryotic expression plasmids, pEGFP-C1-FMDV-A2I2 and pEGFP-C1-A3I3,were constructed based on the pEGFP-C1.The former plasmid has the full length genome of FMDV,and the latter has FMDV ORF gene.The results of transient expression showed that two eukaryotic expression plasmids can express in BHK-21,and green fluorescence was seen by fluorescence microscope,but we cannot get any positive result by sandwich ELISA when the plasmids transfected into cell alone.But,when the plasmid cotransfected with RNA transcribed from linearized cDNA clone,FMDV antigen and nucleotide acid was detected in cell culture fluid by sandwich ELISA and RT-PCR,respectively.And the virus particles were also found by electron microscope.Animal inoculation study showed that level of FMDV antibody in baby mouse was increased with inoculation times.The results proved that cotransfection of eukaryotic expression plasmid with RNA can be used to rescue virus.And for the first time we successfully rescued FMDV by cotransfecting eukaryotic expression plasmid with RNA transcribed in vitro.
In order to develop a molecular marker vaccine of FMDV,GFP gene was inserted into P3 gene of the full length cDNA clone of FMDV to construct a recombinated plasmid,named pFMDV-AI-GFP.The result of transfection showed that the recombinated RNA transcribed in vitro can expressed in BHK-21 cell,and green fluorescence can be seen by fluorescence microscope.Results of RT-PCR which amplified GFP gene and AB fragment of FMDV and sandwich ELISA also showed that mutanted FMDV was rescued successfully.We successfully rescued a molecular marker strain of FMDV for the first time,based on which molecular marker vaccine of FMDV may be developed.
引文
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