将分别来自禽流感病毒A/Goose/Guangdong/3/96(H5N1)毒株的HA基因和A/Goose/Guangdong/1/96(H5N1)NP基因重组到禽痘病毒基因组中,获得了能同时高效表达这两种蛋白的重组禽痘病毒(rFPV HA NP)。将rFPV HA NP经翅膀刺种途径接种8周龄SPF鸡...将分别来自禽流感病毒A/Goose/Guangdong/3/96(H5N1)毒株的HA基因和A/Goose/Guangdong/1/96(H5N1)NP基因重组到禽痘病毒基因组中,获得了能同时高效表达这两种蛋白的重组禽痘病毒(rFPV HA NP)。将rFPV HA NP经翅膀刺种途径接种8周龄SPF鸡,并设亲本禽痘病毒免疫和非免疫对照组。免疫后4周分别用10LD50的高致病力禽流感病毒(HPAIV)A/Goose/Guangdong/1/96(H5N1)和A/FPV/Rostock/34(H7N1)毒株进行攻击。结果重组禽痘病毒rFPV HA NP免疫鸡群后能够诱导产生高水平的特异性抗体,可完全抵抗H5N1毒株的致死性攻击,并可有效阻止H7N1病毒攻击后病毒在泄殖腔的排出。而禽痘病毒免疫组和非免疫对照组在攻毒后全部发病并死亡。结果表明NP在与HA共同表达时,在诱导交叉保护性方面有一定的免疫增强功能。展开更多
Objective:To discuss the possibility of expressing the haemagglutinin-neuraminidase（HN） protein in prokaryotic system such as Escherichia coli（E.coli） cells by cloning the full length HN gene.Methods:The full leng...Objective:To discuss the possibility of expressing the haemagglutinin-neuraminidase（HN） protein in prokaryotic system such as Escherichia coli（E.coli） cells by cloning the full length HN gene.Methods:The full length HN gene of Newcastle Disease Virus（NDV） of size 1 734 bp was preciously isolated by RT-PCR.The sequence was assessed and submitted to Nucleic Acid Databank（NCBI） and the gene ID was EU215390.1 after cloning and sequencing.Now the assessed HN gene was subcloned into pET 32 a+ expression vector for production the HN protein in E.coli, BL21（DE3） P<sup>LYS</sup>S cells following standard protocols.The crude lysate protein from the induced positive clone was size assessed by sodium dodecyl sulfale-polyacrylamide gel electrophoresis （SDS-PAGE） and their haemagglutination（HA） property against chicken RBC was assessed by standard micro HA test.Results:The molecular size of the full HN gene of NDV as assessed by cloning and digesting the positive clone to release the insert was 1.7 kb.The expressed protein in both crude and pure form was assessed to be 63 kDa and 81 kDa,respectively.The HA activity of the crude protein of the positive clone was 1 in 40.Conclusions:This finding indicates that the fusion protein retains the biological activity of native protein in the crude form and therefore could be used as a diagnostic reagent for antibody detection and for routine assessment of immune status in commercial layer forms.展开更多
A recombinant fowlpox virus co-expressing Haemagglutinin (HA) and Neuraminidase (NA)named as rFPV-HA-NA was produced by HA and NA gene of A/Goose/Guangdong/3/96(H5N1)isolate of avian influenza virus recombined into th...A recombinant fowlpox virus co-expressing Haemagglutinin (HA) and Neuraminidase (NA)named as rFPV-HA-NA was produced by HA and NA gene of A/Goose/Guangdong/3/96(H5N1)isolate of avian influenza virus recombined into the genome of fowlpox virus. In thisstudy, to evaluate its ability of protecting chickens against challenge with a lethaldose of highly pathogenic isolates of avian influenza virus, eight-week-old specific-pathogenic-free (SPF) chickens were vaccinated with recombinant virus or the wildtypefowlpox virus by wing-web puncture. After challenge 4 weeks with 10 LD50 highly pathogenicavian influenza virus H5N1 and H7N1 isolate, all chickens vaccinated with recombinantvirus were protected, while the chickens vaccinated with the wildtype fowlpox virus orunvaccinated controls experienced 100% mortality respectively following challenge. Thiscomplete protection was accompanied by the high levels of specific antibody response tothe respective components of the recombinant virus.展开更多
In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus （A/PR/8/ 34） and studied their expressio...In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus （A/PR/8/ 34） and studied their expression in HEK293 cells. HA and HA1 genes were amplified by RT-PCR and cloned into pcDNA3. 1 （ ＋ ） to generate pcDNA3. 1 （ ＋ ）/HA and pcDNA3. 1 （ ＋ ）/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3. 1（＋）/HA and pcDNA3. 1（＋）/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3. 1 （＋）/HA or pcDNA3. 1 （＋）/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HAl , which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.展开更多
文摘将分别来自禽流感病毒A/Goose/Guangdong/3/96(H5N1)毒株的HA基因和A/Goose/Guangdong/1/96(H5N1)NP基因重组到禽痘病毒基因组中,获得了能同时高效表达这两种蛋白的重组禽痘病毒(rFPV HA NP)。将rFPV HA NP经翅膀刺种途径接种8周龄SPF鸡,并设亲本禽痘病毒免疫和非免疫对照组。免疫后4周分别用10LD50的高致病力禽流感病毒(HPAIV)A/Goose/Guangdong/1/96(H5N1)和A/FPV/Rostock/34(H7N1)毒株进行攻击。结果重组禽痘病毒rFPV HA NP免疫鸡群后能够诱导产生高水平的特异性抗体,可完全抵抗H5N1毒株的致死性攻击,并可有效阻止H7N1病毒攻击后病毒在泄殖腔的排出。而禽痘病毒免疫组和非免疫对照组在攻毒后全部发病并死亡。结果表明NP在与HA共同表达时,在诱导交叉保护性方面有一定的免疫增强功能。
文摘Objective:To discuss the possibility of expressing the haemagglutinin-neuraminidase（HN） protein in prokaryotic system such as Escherichia coli（E.coli） cells by cloning the full length HN gene.Methods:The full length HN gene of Newcastle Disease Virus（NDV） of size 1 734 bp was preciously isolated by RT-PCR.The sequence was assessed and submitted to Nucleic Acid Databank（NCBI） and the gene ID was EU215390.1 after cloning and sequencing.Now the assessed HN gene was subcloned into pET 32 a+ expression vector for production the HN protein in E.coli, BL21（DE3） P<sup>LYS</sup>S cells following standard protocols.The crude lysate protein from the induced positive clone was size assessed by sodium dodecyl sulfale-polyacrylamide gel electrophoresis （SDS-PAGE） and their haemagglutination（HA） property against chicken RBC was assessed by standard micro HA test.Results:The molecular size of the full HN gene of NDV as assessed by cloning and digesting the positive clone to release the insert was 1.7 kb.The expressed protein in both crude and pure form was assessed to be 63 kDa and 81 kDa,respectively.The HA activity of the crude protein of the positive clone was 1 in 40.Conclusions:This finding indicates that the fusion protein retains the biological activity of native protein in the crude form and therefore could be used as a diagnostic reagent for antibody detection and for routine assessment of immune status in commercial layer forms.
文摘A recombinant fowlpox virus co-expressing Haemagglutinin (HA) and Neuraminidase (NA)named as rFPV-HA-NA was produced by HA and NA gene of A/Goose/Guangdong/3/96(H5N1)isolate of avian influenza virus recombined into the genome of fowlpox virus. In thisstudy, to evaluate its ability of protecting chickens against challenge with a lethaldose of highly pathogenic isolates of avian influenza virus, eight-week-old specific-pathogenic-free (SPF) chickens were vaccinated with recombinant virus or the wildtypefowlpox virus by wing-web puncture. After challenge 4 weeks with 10 LD50 highly pathogenicavian influenza virus H5N1 and H7N1 isolate, all chickens vaccinated with recombinantvirus were protected, while the chickens vaccinated with the wildtype fowlpox virus orunvaccinated controls experienced 100% mortality respectively following challenge. Thiscomplete protection was accompanied by the high levels of specific antibody response tothe respective components of the recombinant virus.
基金This project was supported by a grant from the NationalNatural Sciences Foundation of China (No .39970830)
文摘In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus （A/PR/8/ 34） and studied their expression in HEK293 cells. HA and HA1 genes were amplified by RT-PCR and cloned into pcDNA3. 1 （ ＋ ） to generate pcDNA3. 1 （ ＋ ）/HA and pcDNA3. 1 （ ＋ ）/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3. 1（＋）/HA and pcDNA3. 1（＋）/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3. 1 （＋）/HA or pcDNA3. 1 （＋）/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HAl , which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.
