Construction And Expression Of Vector Encoding The P32 Protein Of Goatpoxviruswith Deletion Of The Transmembrane Domain

The multiplex PCR meothod were developmented for differentiation goatpoxvirus from orf virus. The sensitivity of the PCR assay was evaluated using 10-fold dilutions of a culture .The detection limit of the method was 4pg DNA. 7 field samples were detected by this multiplex PCR, and 6 were goatpoxvirus, 1 was orf virus.The P32 protein genes of 6 strains from Guangxi were amplified by PCR and sequenced, the Genbank number for our nucleotide sequences are EF522176, EF522177, EF522178, EF522179, EF522180, and EF522181,respectively.The results of nucleotide sequences analysis shows,the homologies of nucleotide and animo acid sequence among the strains from Guangxi are 99.5-99.8%,and 98.5-99.4% ,respectively.The homologies of nucleotide and animo acid sequence between the Chinese vaccine strain and strians from Guangxi are 96.6-99.9%,and 99.1-100%. And the homologies between reference strians published in genbank and the strain from Guangxi are 99.4-99.6%, and 98.1-98.8%.It shows deletion of 2 amino acid at the position 34 to 35 in those stains isolated from Guanggxi. And substitution T 651 C, and a additional restriction sites of Bsp1407 I are found in strians from Guangxi, while it doesn't exist in the other reference strains and vaccine strain. Bsp1407 I can digests the PCR products of P32 gene of strains from Guangxi into 3 fragments of 135bp, 226bp and 378bp, however, vaccine strain was digested into 2 fragments of 135bp and 604bp.Furthermore, the protein P32 was expressed in Eco.li using pGEX-6p-lvecor. For P32 protein has transmembrane domain, the expression level of complete P32 protein using the common method is rather low. So a recombine PCR was used to delete the transmembrane domain. Then, P32 protein with the transmembrane domain deletetion or not were analysised by the software DNAstar. Results indicated that the Alpha amphipathic regions, the Beta amphipathic regions, flexible regions were changed, but the antigenicity wasn't change. The DNA of P32 gene without transmembrane domain was inserted into pGEX-6p-l vector to construct, then recombinant vector pGEX-S6 was transformed into BL21(DE3), and induced by IPTG in different time, concentration and temperature, detected using SDS-PAGE and Western blot, and analysised using BandScan software. The results indicated that protein almost expressed in body of fusion protein with GST protein, molecular weight of expression product was 58KDa, and expression product could be identified by stand serum of goatpox.