RESUMEN
The expressing cassette, LoxP-CMV-gpt-IRES-LoxP( about 2.9kb), was amplified by PCR from a plasmid, pIRES-gpt, by use of the primers , which contained the loxP sites in 5' terminals, respectively. The loxP sites were designed into primers by the software of Primer primer 5.0. Then the cassette was cloned into the site of BalI in pBUS10 to obtain pUS-gptIRES(L). The sequencing analysis for pUS-gptIRES(L) indicated that two loxP sites with the same direction were correctly inserted into pUS-gptIRES(L).The gpt gene in pUS-gptIRES(L) was replaced by a fragment including the full length GFP gene as well as SV40 poly A sequence to get pUS-GFPIRES(L). pUS-GFPIRES(L) was transiently transfected into CHO cell lines, and then the green fluorescence could be seen, the results showed that GFP gene could be expressed correctly. Moreover, pUS-GFPIRES(L) was transfected into the CEF infected MDV CVI988 strain and recombinant virus was selected by the green fluorescence. The growth curve of virus showed the characteristic of recombinant virus was the same as that of CVI988 in vitro. These results give the basis for further studying the characteristic of MDV in vivo and the application of the Cre/LoxP system to MDV genome.
RESUMEN
The partial segment of Marek′s disease virus (MDV) glycoprotein B (gB) gene was amplified by PCR. The segment was cloned into pET-28a vector to obtain the recombinant pET-gB plasmid. The recombinant plasmid was transformed into E.coli BL21,and expressed in very high level as inclusion body after induced with 1.0mmol/L IPTG. The inclusion body was solubilized in urea (8mol/L) . The purified protein was obtained by use of His?Bind affinity chromatography. Mice were immunized i.p. by the purified protein to make the polyclonal antibody. The titer of the antibody by indirect ELISA was 1?10~ -5 . Moreover, the analysis by western blot proved that antibody was specific to the recombinant protein. These works lay a favorable foundation for the study of the immune response by MDV gB.