| A novel reverse co-immunoprecipitation method was used to get the purified BTV-HbC3 particles and gel filtration, electron microscopy(EM), TCID50 and plaque assay were used to analyze the purification results. Sephadex G-200 gel filtration analysis of the purified sample by absorbance values measured at 280nm , only one steep UV absorbance peak can be seen ; background of electron micrograph of the purified virus is clean and the complete structure of virus particles can be clearly seen; the 50% tissue culture infective dose on Vero cell line is 10-6.6/ml before purification and 10-5.7/ml after purification; purified and unpurified virus liters can be attained from plaque assay of BTV-HbC3 on Hep-3B cell line . Purification recovery rate can be gotten by comparing between virus titers of purified and unpurified. On 10-3, 10-4, 10-5 dilution degrees respectively , the recovery rates are 69.6 %, 70.8 %, 50.4 %. From the results described above we can draw the conclusion that the new method introduced can produce large amounts of high purified viral particles with high infectivity so it can be applied to further research on developing a large scale production system.BTV-HbC3 can selectively replicate in 7402, Hep-3B cell lines and induce cytopathic effects (CPE), and eventually cause complete lysis and death of cells. Between the two human liver cell lines, Hep-3B cells are more susceptible to BTV-HbC3 sinfection, the CPE of which occur quickly and can reach 90% at 36h p.i., while 7402 cells are the less sensitive. The inhibition activity of BTV-HbC3 on the two liver cell lines (7402, Hep-3B ) was tested by MTT. Under the same condition, the inhibition ratio of BTV is 77.69% on Hep-3B; 51.45% on 7402.BTV-HbC3 can induce apoptosis in 7402, Hep-3B cell lines. The result of flow cytometry assay shows the apoptosis ratio of 48hr p.i. is 20.1% on 7402; 33.0% on Hep-3B. The result of confocal microscopy also indicates typical apoptosis induced by BTV-HbC3 in two human liver cell lines. |
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