Production And Characterization Of Monoclonal Antibodies To VP4 Of Group A Rotavirus LLR

Rotavirus, one genera belongs to the family Reovoridae, known as the main cause of severe, dehydrating diarrhea in infants and young children, is estimated to cause about 125 million cases of diarrhea annually, and to cause 900 thousands deaths in infants under 5 years old. Inprovement of hydgiene condition cannot decrease morbitity of rotavirus gastroenteristis, development and universal use of a safe and effective vaccine is considered as the first strategy for prevention of RV disease. Our team is devoted to developping rotavirus vaccine by construction of attenuated reassortants of GARV backboned with rotavirus LLR using reverse genetic method. The rotavirus reverse genetic method Komoto estanblished in 2006 need a helper rotavirus for offering replication and other gene, at the same time, a neutralzing antibody to suppress helper rotavirus is also needed to select the RV reassortant as selecting pressure. In order to provide neutralzing antibodies to rotavirus LLR for selecting ressortant backboned with rotavirus LLR, production and characterization of monoclonal antibodies to VP4 of rotavirus LLR are described in this paper.Chapter One: Rotavirus virion structure, physicochemical properties, genome and its coding assignments, molecular epidemiology and vaccine development, especially the approach in reverse genetics method of rotavirus are reviewed. And our strategy for reverse genetics of rotavirus is also introduced.Chapter Two: Multiplication and purification of rotavirus LLR (G10P[12]) were described. Rotavirus LLR was isolated from live rotavirus vaccine: VICMIC produced by Lanzhou institute of biological products and was cultured in MA104 cells. The Rotavirus LLR cultured was detected by PAGE and Rotavirus Group A Colloidal Gold immunochromagraphy Diagnostic Kit, and the titer was determined by TCID50 method. The virus was purified using sucrose density gradient centrifugation combined with gel chromatography method, and purified virus was negative stained and observed by transmitted electronic microscope.Chapter Three: Monoclonal antibodies to VP4 of rotavirus LLR (G10P[12]) were produced and characterized. 6-8 week-old female Balb/c mouse was immunized with purified rotavirus LLR at dose of 175μg with complete Freund's adjuvant per mouse, and then boosted injected with equal quantitity antigen with incomplete Freund's adjuvant twice, and equal quantitity antigen were intravenously injected before 3 days of cell fusion at last. The spleen cells from immunized mice were fused with mouse sp2/0 myeloma cells, hybridoma cells stably secreting monoclonal antibody against LLR was screened by indirect ELISA and then were cloned by limited dilution method. Four stable hybridoma cell lines, named 1B1,1B8,1F11 and 1G10 were established and characterized. The chromosome number of hybridomas is 82,99,87 and 99 respectively, the isotypes of the MAbs secreted by these hybridomas are IgM and IgG1 respectively, the ascetic fluids antibody titers of four hybridomas are all above 1.0×10~4. All four monoclonal antibodies recognized the same antigen VP4 of rotavirus LLR identified by western-blot, and can neutralized rotavirus LLR.