AIM: To achieve a better understanding of the pathogenesis of new type gosling viral enteritis virus (NGVEV) and the relationship between NGVEV and host cells. METHODS: The apoptosis of duck embryo fibroblasts (DEF) i...AIM: To achieve a better understanding of the pathogenesis of new type gosling viral enteritis virus (NGVEV) and the relationship between NGVEV and host cells. METHODS: The apoptosis of duck embryo fibroblasts (DEF) induced by NGVEV was investigated by fluorescence-activated cell sorter (FACS) and fluorescence microscope after the cells were stained with Annexin V-FITC and propidium iodide (PI). RESULTS: By staining cells with a combination of fluorescein annexin V-FITC and PI, it is possible to distinguish and quantitatively analyze non-apoptotic cells (Annexin V-FITC negative/PI negative), early apoptotic cells (Annexin V-FITC positive/PI negative), late apoptotic/necrotic cells (Annexin V-FITC positive/ PI positive) and dead cells (Annexin V-FITC negative/PI positive) through flow cytometry and fluorescence microscope. The percentage of apoptotic cells increased with the incubation time and reached a maximum at 120 h after infection, while the percentage of non- apoptotic cells decreased.展开更多
The capsid (Cap) protein, which is the only structural protein of duck circovirus (DuCV), is the most important antigen for the development of vaccines against DuCV and the virus's serological diagnostic methods....The capsid (Cap) protein, which is the only structural protein of duck circovirus (DuCV), is the most important antigen for the development of vaccines against DuCV and the virus's serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid (Opt-Cap) gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% (v/v) methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What's more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles (VLPs). These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.展开更多
基金The National Natural Science Foundation of China, No. 39970561The Key Projects in the National Science and Technology Pillar Program, 2007Z06-017+2 种基金Program for New Century Excellent Talents from Universities, Chinese Ministry of Education, No. NCET-04-0906/NCET-06-0818Fund of the Discipline Leaders of Sichuan Province, No. SZD0418Culture Fund for Excellent Doctoral Dissertations of Sichuan Agricultural University, 2008scybpy-1
文摘AIM: To achieve a better understanding of the pathogenesis of new type gosling viral enteritis virus (NGVEV) and the relationship between NGVEV and host cells. METHODS: The apoptosis of duck embryo fibroblasts (DEF) induced by NGVEV was investigated by fluorescence-activated cell sorter (FACS) and fluorescence microscope after the cells were stained with Annexin V-FITC and propidium iodide (PI). RESULTS: By staining cells with a combination of fluorescein annexin V-FITC and PI, it is possible to distinguish and quantitatively analyze non-apoptotic cells (Annexin V-FITC negative/PI negative), early apoptotic cells (Annexin V-FITC positive/PI negative), late apoptotic/necrotic cells (Annexin V-FITC positive/ PI positive) and dead cells (Annexin V-FITC negative/PI positive) through flow cytometry and fluorescence microscope. The percentage of apoptotic cells increased with the incubation time and reached a maximum at 120 h after infection, while the percentage of non- apoptotic cells decreased.
基金supported by the National Science and Technology Support Program(2015BAD12B05)the China Agricultural Research System(CARS-43-8)+2 种基金the Integration and Demonstration of Key Technologies for Duck Industrial in Sichuan Province,China(2014NZ0030)the Ministry of Education Program of China(20125103110013)the Sichuan Province Research Programs,China(2013HH0042/2013 TD0015/2014-002)
文摘The capsid (Cap) protein, which is the only structural protein of duck circovirus (DuCV), is the most important antigen for the development of vaccines against DuCV and the virus's serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid (Opt-Cap) gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% (v/v) methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What's more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles (VLPs). These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.