口蹄疫病毒基因重组鸡痘病毒活载体疫苗系统鉴定研究
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摘要
本研究以共表达FMDV衣壳蛋白前体P1-2A和蛋白酶3C基因的重组鸡痘病毒vUTAL3CP1为研究对象,较系统地进行遗传稳定性、理化学特性、生物学特性、病毒毒种系统鉴定等方面的研究,探索实验室的疫苗制备工艺;将重组鸡痘活载体疫苗分别接种小鼠、豚鼠、猪、牛,以及妊娠小鼠、豚鼠、猪、牛及其新生子代动物,利用临床观察、病理组织学、PCR、RT-PCR、ELISA、IFA、中和抗体检测、免疫组织化学等方法检测其病毒在体内的分布、病毒基因的残留、抗体消长规律、细胞毒性、组织毒性、生态环境影响;将接种动物与非接种动物同居,通过抗体水平和病毒基因检测,证明重组鸡痘活载体疫苗不会造成同居感染,接种动物不会造成垂直感染。通过临床观察、病理组织学检测等方法证明该疫苗在接种动物体内有无毒性;利用PCR、RT-PCR方法检测在免疫动物体内残留时间短,正常剂量和超大剂量对妊娠动物和子代动物无安全威胁,对生态环境无影响,从而进一步证明所构建的重组鸡痘病毒活载体疫苗的生物安全性。
With the development of molecular biology and biotechnology, viruses were used to construction virus vectors .At present, recombinant virus vector is one of the most hot spots in virus gene engineering. There are two type vectors, the replaceable recombinant virus vector and the other is nonreplaceable vector, such as recombinant viral plasmid vector.
The use of virus vectors for heterologous antigen delivery is currently being explored in a variety of fields. The possible development and use of vaccinia virus as recombinant vaccines for protective immunization against infectious diseases have stimulated research into the development of other poxviruses as potential recombinant vectors. Consequently, avipoxviruses were considered first. Reasons for choosing them include: avipoxviruses have large DNA genomes, replicate in the cytoplasm of permissive cell, and may be genetically modified (GM) by insertion of at least 25–30 kbp of foreign DNA, i.e. a number of cDNAs may be inserted into the same genome. It has been shown that recombinant versions of avipoxviruses virus may express polypeptides coded by foreign cDNAs in immunologically attractive ways. Indeed, inoculation of avipoxvirus-based recombinants into mammalian resulted in the expression of the foreign gene and the induction of protective immunity. This showed that favorable immune responses are inducible in the absence of productive replication, while eliminating the potential for dissemination of the vector within the vaccinator and the spread of the vector to non-vaccinated contacts, or to the general environment. The bio- safety should be most important when considering constructing effective, nonreplicating vaccine vectors. Fowlpox virus, which should meet these stringent safety criteria, needs to be tested properly for ability to establish productive infection in mammalian origin.
The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease affecting cloven-hoofed animals. From last 10 years, FMD were broken out in many countries around the world, such as in Korea, Great British, French, Dutch and several cities of China. In the countries where the disease has not yet been eradicated, vaccination plays a crucial role in FMDV control. Although inactivated virus vaccines can prevent FMD effectively, their use is accompanied by dangerous residual potency problems, inclding incomplete viral inactivation or virus escape from vaccine-producing facilities. Likewise, the new type genetically engineering vaccine of security and high-performance would be advanced stringent.
The recombinant fowl poxvirus vUTAL3CP1 containing a FMDV capsid polypeptide and 3C coding regions of O/NY00 (Gene Bank AY333431) had been constructed. In this study, the bio-safety and immunogenicity of the recombinant fowlpox virus vUTAL3CP1 were evaluated by clinical observation, histological, viral culture, PCR, immunohistochemistry, ELISA, immunochemistry, neutralizing antibody detection.
The physical chemistry characteristics of the recombinant vaccine strain vUTAL3CP1were researched by observing the change of TCID50 after being dealed with heat, HCl(pH3), NaOH (pH9), ether, chloroform and parenzyme separately. The results indicated that it was not sensitive to the change of pH. The TCID50 of vUTAL3CP1 was not change from pH3.0 to pH9.0. While it was sensitive to heat, chloroform, aether and parenzyme. It was inactivated by 50℃for 60 min, 55℃for 30min or 60℃for 15 min. Dealed with par enzyme in 37℃for an hour and ether for 24 hour , it depressed 2.44 log 10 TCID50 and 1.0 log10 TCID50.
In this study, one avian and twelve mammalian cell lines were evaluated for permissive to the recombinant fowlpox virus strains vUTAL3CP1. As expected, chicken embryo fibroblast (CEF) was permissive for recombinant fowlpox virus strains. However, it was surprised that Syrian baby hamster kidney (BHK),pig kidney (PK), bovine testicular (BT), Vero cell were equally permissive to recombinant strains by multiplication assays. Results from direct negative stain electron microscopy and ultrathin section transmission electron microscopy of infected BHK, PK, BT, Vero cell revealed viral morphogenesis proceeding to various forms of infectious viruses. A number of virus-containing vesicles and plasma membrane-associated mature viruses at an early stage in the budding process were observed. By PCR, other results were certificated by the demonstration of fowlpox virus specific gene expression and foot-mouth diseases (FMD) insert gene expression. Recombinant fowlpox virus strains could stable passage in the chicken embryo fibroblast (CEF) over thirty generations, and could passage in BHK, PK, BT, Vero only two generations.
In this study, in order to detect the bio-safety of the recombinant fowlpox virus, the mice, guinea pig, chicken and pig were innoculated with recombinant fowlpox virus, respectively. Clinical, histological, PCR, neutralizing antibody were used to assess the bio-safety of the recombinant fowl pox virus. During the experiment, the mental status, hydroposia and foraging behaviors of the immune animal were assessed. The pathohistology detection was not pathological change in animal. By PCR, the viral DNA of recombinant fowlpox virus was not detected in the cohabitatio animal and progeny of inoculated animal. The neutralizing antibody of recombinant fowlpox virus was not detected in the cohabitatio animal. So the bio-safety of recombinant fowlpox virus was validated, neither horizontal transmission nor vertical transmission.
During the experiment, the mental status, hydroposia and foraging behaviors of the mice were normal. No detectable pathological change was found in the organs of inoculated mice, cavia cobaya, swine and cattal. Using PCR, viral nucleic acids could be detected at 3 days post inoculation (DPI), viral nucleic acids could be found in all organs; none viral nucleic acids could be detected from any organs at 7 (DPI). In addition, well immunogenicity had been ensured by its ability to induce humoral responses before the bio-safety of this vaccine was study.
Analyze the bio-safety of the recombinant fowlpox virus by clinical, histological and PCR and influence of progeny weight. During the experiment, the mental status, hydroposia and foraging behaviors of the swine were normal. No detectable pathological change was found in the organs of inoculated swine. In addition, no detriment was found in cyesis and progeny with hyper-flushing dose. None viral nucleic acids could be detected from any organs of progeny. No immature labor, abortion, fetal death, ambly-fetus was found in cyesis swine.
In order to essese enviroment release experiment, urina,ejecta,water, soil were collected after the animals were immuned with recombinant folwpox virus at 1、3、5、7、14DPI. By PCR, viral nucleic acids could not be detected and virus could not isolated at above time.To detect survial ability of recombinant fowlpox virus, recombinant fowlpox virus were combined with urina, ejecta, water, soil set up in 37℃、4℃、-20℃. Recombinant fowlpox virus could survival for 21d in 37℃, over half year in 4℃and over two years in -20℃mixed with urina, and so on mixed with ejecta and water. Recombinant fowlpox virus could survival for 21d in 37℃, over half year in 4℃and over one year in -20℃in soil.
With the development of molecular biology and biotechnology, viruses were used to construction virus vectors .At present, recombinant virus vector is one of the most hot spots in virus gene engineering. There are two type vectors, the replaceable recombinant virus vector and the other is nonreplaceable vector, such as recombinant viral plasmid vector.
The use of virus vectors for heterologous antigen delivery is currently being explored in a variety of fields. The possible development and use of vaccinia virus as recombinant vaccines for protective immunization against infectious diseases have stimulated research into the development of other poxviruses as potential recombinant vectors. Consequently, avipoxviruses were considered first. Reasons for choosing them include: avipoxviruses have large DNA genomes, replicate in the cytoplasm of permissive cell, and may be genetically modified (GM) by insertion of at least 25–30 kbp of foreign DNA, i.e. a number of cDNAs may be inserted into the same genome. It has been shown that recombinant versions of avipoxviruses virus may express polypeptides coded by foreign cDNAs in immunologically attractive ways. Indeed, inoculation of avipoxvirus-based recombinants into mammalian resulted in the expression of the foreign gene and the induction of protective immunity. This showed that favorable immune responses are inducible in the absence of productive replication, while eliminating the potential for dissemination of the vector within the vaccinator and the spread of the vector to non-vaccinated contacts, or to the general environment. The bio- safety should be most important when considering constructing effective, nonreplicating vaccine vectors. Fowlpox virus, which should meet these stringent safety criteria, needs to be tested properly for ability to establish productive infection in mammalian origin.
The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease affecting cloven-hoofed animals. From last 10 years, FMD were broken out in many countries around the world, such as in Korea, Great British, French, Dutch and several cities of China. In the countries where the disease has not yet been eradicated, vaccination plays a crucial role in FMDV control. Although inactivated virus vaccines can prevent FMD effectively, their use is accompanied by dangerous residual potency problems, inclding incomplete viral inactivation or virus escape from vaccine-producing facilities. Likewise, the new type genetically engineering vaccine of security and high-performance would be advanced stringent.
The recombinant fowl poxvirus vUTAL3CP1 containing a FMDV capsid polypeptide and 3C coding regions of O/NY00 (Gene Bank AY333431) had been constructed. In this study, the bio-safety and immunogenicity of the recombinant fowlpox virus vUTAL3CP1 were evaluated by clinical observation, histological, viral culture, PCR, immunohistochemistry, ELISA, immunochemistry, neutralizing antibody detection.
The physical chemistry characteristics of the recombinant vaccine strain vUTAL3CP1were researched by observing the change of TCID50 after being dealed with heat, HCl(pH3), NaOH (pH9), ether, chloroform and parenzyme separately. The results indicated that it was not sensitive to the change of pH. The TCID50 of vUTAL3CP1 was not change from pH3.0 to pH9.0. While it was sensitive to heat, chloroform, aether and parenzyme. It was inactivated by 50℃for 60 min, 55℃for 30min or 60℃for 15 min. Dealed with par enzyme in 37℃for an hour and ether for 24 hour , it depressed 2.44 log 10 TCID50 and 1.0 log10 TCID50.
In this study, one avian and twelve mammalian cell lines were evaluated for permissive to the recombinant fowlpox virus strains vUTAL3CP1. As expected, chicken embryo fibroblast (CEF) was permissive for recombinant fowlpox virus strains. However, it was surprised that Syrian baby hamster kidney (BHK),pig kidney (PK), bovine testicular (BT), Vero cell were equally permissive to recombinant strains by multiplication assays. Results from direct negative stain electron microscopy and ultrathin section transmission electron microscopy of infected BHK, PK, BT, Vero cell revealed viral morphogenesis proceeding to various forms of infectious viruses. A number of virus-containing vesicles and plasma membrane-associated mature viruses at an early stage in the budding process were observed. By PCR, other results were certificated by the demonstration of fowlpox virus specific gene expression and foot-mouth diseases (FMD) insert gene expression. Recombinant fowlpox virus strains could stable passage in the chicken embryo fibroblast (CEF) over thirty generations, and could passage in BHK, PK, BT, Vero only two generations.
In this study, in order to detect the bio-safety of the recombinant fowlpox virus, the mice, guinea pig, chicken and pig were innoculated with recombinant fowlpox virus, respectively. Clinical, histological, PCR, neutralizing antibody were used to assess the bio-safety of the recombinant fowl pox virus. During the experiment, the mental status, hydroposia and foraging behaviors of the immune animal were assessed. The pathohistology detection was not pathological change in animal. By PCR, the viral DNA of recombinant fowlpox virus was not detected in the cohabitatio animal and progeny of inoculated animal. The neutralizing antibody of recombinant fowlpox virus was not detected in the cohabitatio animal. So the bio-safety of recombinant fowlpox virus was validated, neither horizontal transmission nor vertical transmission.
During the experiment, the mental status, hydroposia and foraging behaviors of the mice were normal. No detectable pathological change was found in the organs of inoculated mice, cavia cobaya, swine and cattal. Using PCR, viral nucleic acids could be detected at 3 days post inoculation (DPI), viral nucleic acids could be found in all organs; none viral nucleic acids could be detected from any organs at 7 (DPI). In addition, well immunogenicity had been ensured by its ability to induce humoral responses before the bio-safety of this vaccine was study.
Analyze the bio-safety of the recombinant fowlpox virus by clinical, histological and PCR and influence of progeny weight. During the experiment, the mental status, hydroposia and foraging behaviors of the swine were normal. No detectable pathological change was found in the organs of inoculated swine. In addition, no detriment was found in cyesis and progeny with hyper-flushing dose. None viral nucleic acids could be detected from any organs of progeny. No immature labor, abortion, fetal death, ambly-fetus was found in cyesis swine.
In order to essese enviroment release experiment, urina,ejecta,water, soil were collected after the animals were immuned with recombinant folwpox virus at 1、3、5、7、14DPI. By PCR, viral nucleic acids could not be detected and virus could not isolated at above time.To detect survial ability of recombinant fowlpox virus, recombinant fowlpox virus were combined with urina, ejecta, water, soil set up in 37℃、4℃、-20℃. Recombinant fowlpox virus could survival for 21d in 37℃, over half year in 4℃and over two years in -20℃mixed with urina, and so on mixed with ejecta and water. Recombinant fowlpox virus could survival for 21d in 37℃, over half year in 4℃and over one year in -20℃in soil.
引文
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