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
Duck circovirus infection is an immunosuppressive disease that is characterized by growth retardant and odd deaths. Its infection is usually combined with other pathogens, making the clinical diagnosis more difficult....Duck circovirus infection is an immunosuppressive disease that is characterized by growth retardant and odd deaths. Its infection is usually combined with other pathogens, making the clinical diagnosis more difficult. With the rapid development of molecular biological and immunological technologies, the laboratory diagnostic methods for duck circovirus infection also advance greatly. The paper summarizes the research advances in various laboratory detection methods for duck cireovirus infection including PCR, nested PCR, multiple PCR, fluorescence quantitative PCR, LAMP, nucleic acid probe and ELISA, as well as their ad- vantages and shortages, aiming at providing reference for finding novel detection methods and for the diagnosis and comprehensive prevention and control.展开更多
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.
文摘Duck circovirus infection is an immunosuppressive disease that is characterized by growth retardant and odd deaths. Its infection is usually combined with other pathogens, making the clinical diagnosis more difficult. With the rapid development of molecular biological and immunological technologies, the laboratory diagnostic methods for duck circovirus infection also advance greatly. The paper summarizes the research advances in various laboratory detection methods for duck cireovirus infection including PCR, nested PCR, multiple PCR, fluorescence quantitative PCR, LAMP, nucleic acid probe and ELISA, as well as their ad- vantages and shortages, aiming at providing reference for finding novel detection methods and for the diagnosis and comprehensive prevention and control.
