Expression Of Pesudorabies Virus GE Gene And Preliminary Apply

Pseudorabies is a intense infectious disease endangering the world porcineculture and make great economic losses in the world. Thinking of the great harm of the disease, many countries in Europe have succeeded to use "marker vaccine" and start up "eradicating project". At present, the disease is also very populous in our country and the same method is adopted. To use TK/gE double gene deleting vaccine and set up corresponding differentiating diagnose method is a request of the project. Before setting up the method, producing a diagnose antigen with the gene engineering method is required.Cut out the whole gE gene from pgE with BamHI and Hindlll , linked .with the expression vector pET-32c. With the method of double restriction enzyme digest and PCR, some masculine plasmids are checked out, named with pET-32wgE. Transform the BL21 strain with pET-32wgE, choose several single .bacterium drops at random and contaminate 2X YT, vibrate at 37癈 over night. Contaminate another tube with the above culture to the final concentration of 2%, continue to culture to OD6ou0.6, add IPTG to the final concentration of Immol/L, inducing 6 hours. Analyse the expression with SDS-PAGE and Western-blot, no distinctive strip is observed.Devise a pair of primer to put out the section of pgE which code the dense antigen episode, clone into pET-32a, name the masculine plasmid with pET-32pgE. Transform the BL21 strain with pET-32pgE, choose several single bacterium drops at random and contaminate 2X YT. vibrate at 37癈 over night. Contaminate another tube with the above culture to the final concentration of 2%, continue to culture to OD6oo0.6, add IPTG to the final concentration of Immol/L. inducing 6 hours. Analyse the expression with SDS-PAGE and Western-blot, the result is that the expression has actually happened and the product has immunoreactivity. Purify the product with organic solvent and histidine column, link the purified protein to polystyrene board and latex to check the serum, the former result is preferable and the latter has a deeper background, vacancy control and negative control coagulate in some degree but the coagulation of positive is obvious.Cut out the whole gE gene from pET-32wgE, clone into pSecTagHygroC, a mammal secret expression vector, the masculine plasmid is named with pSecTaggE. Transinfect vero cell with pSecTaggE, continue to culture 60 more hours, Analyse the expression with SDS-PAGE and Western-blot, no distinctive strip is observed, but the secret expression product is checked out with mediate ELISA in the supernatant. The cell expression preserve the whole episode and reduce the complexed purified step, if a stable cell expression strain is made out and the background reaction is reduced, it will be a good method to produce the check antigen.