Construction and Identification of a Dural-promoter Eukaryotic Vector pVAXD-N/S of Transmissible gastroenteritis virus (TGEV)
Porcine transmissible gastroenteritis (TGE) is one important causative enteric infection caused by Transmissible gastroenteritis virus (TGEV). The S and N genes of TGEV have important immunological function respectively, a DNA vaccine co-expressing S and N gene will provide better immune efficacy for controlling. In this research, the S gene fragment (2.1 kb, including A, B, C and D antigenic sites of the S protein) and the full-length TV gene (1.2 kb) of TGEV were amplified by RT-PCR, respectively. The /V and S genes were respectively inserted into the multiple cloning sites (MCS) of the dural-promoter eukaryotic vector pVAXD to construct the recombinant plasmid pVAXD-TV and pVAXD-5 firstly. The TV gene was subsequently inserted into the recombinant plasmid pVAXD-S to construct the dural-promoter eukaryotic plasmid pVAXD-N/S. The plasmid pVAXD-N/S and the control plasmids (pVAX-5, p VAX-TV and pVAXD) were transfected into COS-7 cells to express. The transcription of the 5 and TV genes could be detected by RT-PCR from transfected COS-7 cells. The expression of pVAXD-TV/S in COS-7 cells could react with the rabbit anti-S protein serurruthe rabbit anti-TV protein serum and the rabbit anti-TGEV serum, respectively, by indirect immunofluorescence assay (IFA), the fluorescence intensity detected from the pVAXD-N/S transfected COS-7 cells was consistent with that detected from the pVAXD-TV group and pVAXD-S group. The results indicated that the dural-promoter eukaryotic vector pVAXD-TV/S could express the S protein and N protein at the same time. Six-weeks-old BALB/c mice {Mus musculus) were immunized with the plasmid pVAXD-TV/S and the control groups(pVAXD-/V, pVAXD-S and pVAXD). The serum IgG antibody was measured by indirect ELISA. The result showed that specific anti-TGEV serum IgG antibody could be detected at day 14 post-immunization, and the antibody peak was detected at day 35 post-immunization. The IgG antibody level induced by pVAXD-TV/S was significantly higher than that induced by the pVAXD-TV group or pVAXD-S group (P<0.5), and was consistent with that induced by the group(pVAXD-N+pVAXD-5). This study provides a basic materials for developing a bivalent DNA vaccine of TGEV.
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