To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rab...To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as immunogens to immunize 6-8 week old female BALB/c mice. Spleen cells and SP2/0 myeloma cells were fused according to conventional methods: the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally, systematic identification of subclass, specificity and sensitivity was carried out. Two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3B12 and 4A12, with ascitic fluid titers of 1:8000 and 1:10000, respectively. Both belonged to the IgG2a subclass. These strains secrete potent, stable and specific anti-rabies virus monoclonal antibodies, which makes them well suited for the development of rabies diagnosis reagents.展开更多
The global burden caused by dengue has increased dramatically in recent decades. Dengue Fever and its severe clinical manifestation, Dengue Hemorrhagic Fever and Dengue Shock Syndrome, have become international public...The global burden caused by dengue has increased dramatically in recent decades. Dengue Fever and its severe clinical manifestation, Dengue Hemorrhagic Fever and Dengue Shock Syndrome, have become international public health problem endemic in more than 100 countries, risking 2.5 to 3 billion world populations in tropical and subtropical region. Envelope （E） protein of dengue virus has been proposed as the most important antigen that enables it as vaccine candidate or diagnostic materials. Recombinant protein E production is desirable for dengue vaccine and diagnostic development, especially in Indonesia, where dengue is epidemic. Cloning E gene in an expression vector is essential as an initial method to produce dengue E antigens. The purpose of this research was to clone E gene of dengue virus type 3 （Indonesia D3-1703 strain） into Saccharomyces cerevisiae expression vector pYES2/CT. The cloning method used was the in vitro ligation protocol. First, the cDNA from dengue virus type 3 strain （D3-1703） was generated. Then the polymerase chain reaction （PCR） amplification product of E gene cassette from this cDNA was obtained. The E gene cassette was ligated into linearized pYES2/CT resulting a recombinant vector named pYES2/CT-E. The cloned E gene was verified by restriction enzyme digestion and PCR. Sequencing analysis at its 5＇ end showed that the E gene was inserted at the right open reading frame. In conclusion, the results showed that for the first time the E gene originated from an Indonesian dengue virus type 3 strain was successfully cloned within the yeast expression vector pYES2/CT. In the future, this clone could be expressed and provided as materials for dengue vaccine and diagnostic kit, specific for Indonesian dengue virus strain.展开更多
基金National Department Public Benefit Research Foundation (201103032)Pathogens Network Monitoring Technology Research (2008ZX10004-008)
文摘To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as immunogens to immunize 6-8 week old female BALB/c mice. Spleen cells and SP2/0 myeloma cells were fused according to conventional methods: the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally, systematic identification of subclass, specificity and sensitivity was carried out. Two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3B12 and 4A12, with ascitic fluid titers of 1:8000 and 1:10000, respectively. Both belonged to the IgG2a subclass. These strains secrete potent, stable and specific anti-rabies virus monoclonal antibodies, which makes them well suited for the development of rabies diagnosis reagents.
文摘The global burden caused by dengue has increased dramatically in recent decades. Dengue Fever and its severe clinical manifestation, Dengue Hemorrhagic Fever and Dengue Shock Syndrome, have become international public health problem endemic in more than 100 countries, risking 2.5 to 3 billion world populations in tropical and subtropical region. Envelope （E） protein of dengue virus has been proposed as the most important antigen that enables it as vaccine candidate or diagnostic materials. Recombinant protein E production is desirable for dengue vaccine and diagnostic development, especially in Indonesia, where dengue is epidemic. Cloning E gene in an expression vector is essential as an initial method to produce dengue E antigens. The purpose of this research was to clone E gene of dengue virus type 3 （Indonesia D3-1703 strain） into Saccharomyces cerevisiae expression vector pYES2/CT. The cloning method used was the in vitro ligation protocol. First, the cDNA from dengue virus type 3 strain （D3-1703） was generated. Then the polymerase chain reaction （PCR） amplification product of E gene cassette from this cDNA was obtained. The E gene cassette was ligated into linearized pYES2/CT resulting a recombinant vector named pYES2/CT-E. The cloned E gene was verified by restriction enzyme digestion and PCR. Sequencing analysis at its 5＇ end showed that the E gene was inserted at the right open reading frame. In conclusion, the results showed that for the first time the E gene originated from an Indonesian dengue virus type 3 strain was successfully cloned within the yeast expression vector pYES2/CT. In the future, this clone could be expressed and provided as materials for dengue vaccine and diagnostic kit, specific for Indonesian dengue virus strain.
