Developing A Method Of Purifying High Active Bluetongue Virus With Agarose Protein A In Reverse Co-immunoprecipitation

It is an indispensable approach of getting high amount, high infectivity and high purity products with high throughput in the purification and preparation of biological products. The traditional methods of purifying are centrifugation, precipitation and chromatography, etc. These methods had many defections and could not meet the need completely. This article will introduce a novel technology of purifying active bluetongue viruses (BTV) isolated and systematically studied as a model in our laboratory, which is named as the technology of purifying viruses by reverse co-immunoprecipitation and affinity with agarose Protein A for getting the purified BTV particles. The antibodies of the bovine serum and Vero cell fragments were made from immuned rabbits. Then use agarose Protein A to adsorb these antibodies under the optimized temperature, pH value and time. Use the antibodies-Protein A complexes to adsorb the antigens of the bovine serum and Vero cell fragments in BTV preparation proliferated on the culture monolayer of Vero cell under the optimized temperature, pH value and time. The immune complexes from these antibodies to antigens of the bovine serum and Vero cell fragments were adsorbed on the agarose Protein A and can be precipitated by centrifuging the reaction test tube at low speed, and the purified viral particles can be retained in the suspension. The immune complexes from these antibodies to antigens of the bovine serum and Vero cell fragments were adsorbed on the agarose Protein A can be washed down with fit buffer, and agarose Protein A can be used again. Agarose double immunodiffusion shows that there are no bovine serum and Vero cell fragments'antigens in the viral suspension. The electron microscopic detection on the negative staining virus showing that the background of the purified virus suspension and pellet are clean and clear(Fig.4). HPLC shows that there is a single peak among suspension of the purified virus(Fig.5). The ratio of virus peak's square among all peaks is 98.9%,which meets the need of Chinese biological product quality detection. The TCID50 of the viral suspension on the Vero cell line is 4×10^-5.25/ml before purification and 4×10^-4.4/ml after purification. The recovery of virus under the titer of 10°, 10 ^-1, 10^-2,10^-3are 79.81%, 77.90%, 78.13%, 55.42% separately. From TCID50 it can be seen that the viral infectivity is maintained.The conclusion is that this method to purify BTV is significant and feasible. And it is a base for producing high purification, high yield and high infectivity bluetongue virus usingas a nature oncolytic virus and using for other aids. Further more, this method holds the character of high throughput. We can purify all kinds of biological product with this principal.