Heng Yang1, Jiawen Liu, Sanjie Cao, Xiaobo Huang, Xintian Wen. 1. Laboratory of Animal Infectious Disease and Gene Chip, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an 625014, China. 2008yanghengyh@163.com
Abstract
OBJECTIVE: To study the feasibility of using attenuated Salmonella typhimurium as carrier for oral immunization of TGEV DNA vaccine. METHODS: The 2.1 Kb fragments of the TGVE SC-H strain S gene that encompasses all the four major antigenic domains were amplified by RT-PCR and cloned into eukaryotic expression vector pVAX1. The recombinant plasmid pVAX-S was transfected into COS7 cellsand the expression of recombinant plasmids was identified by indirect immunofluorscence assay. Then pVAX-S was transformed by electroporation into attenuated Salmonella typhimurium SL7207. The recombinant was screened and designated as SL7207 (pVAX-S). Mouse peritoneal macrophages were infected with SL7207 (pVAX-S), the transcription and expression of S gene were detected by RT-PCR and indirect immunofluorscence. BALB/c mouse were inoculated orally with SL7207(pVAX-S) at dosage of 5 x 10(8), 1 x 10(9) and 2 x 10(9) CFU for safety analysis. In a vaccination test, BALB/c mouse were immunized orally with recombinant bacterium at dosage of 1 x 10(9) CFU, for 3 times and specific serum IgG and intestinal mucosal IgA antibody were detected by indirect ELISA. RESULTS: Recombinant plasmid pVAX-S was constructed correctly and expressed in COS7 cells. The transcription and expression of S gene were detected after mouse peritoneal macrophages were infected with SL7207 (pVAX-S). The recombinant bacterium was safe to mouse at dosage of 2 x 10(9) CFU. Specific serum IgG and intestinal mucosal IgA antibody against TGEV S protein were detected in SL7207 (pVAX-S) immunized group at 2 weeks post-boosting,and there were significant difference (P < 0.05) in serum IgG and most significant difference (P < 0.01) in intestinal mucosal IgA at 2 weeks after the third immunization, compared with SL7207(pVAX) control group. CONCLUSION: The recombinant Salmonella carrying TGEV S gene DNA vaccines had good immunogenicity and safety in mouse.
OBJECTIVE: To study the feasibility of using attenuated Salmonella typhimurium as carrier for oral immunization of TGEV DNA vaccine. METHODS: The 2.1 Kb fragments of the TGVE SC-H strain S gene that encompasses all the four major antigenic domains were amplified by RT-PCR and cloned into eukaryotic expression vector pVAX1. The recombinant plasmid pVAX-S was transfected into COS7 cellsand the expression of recombinant plasmids was identified by indirect immunofluorscence assay. Then pVAX-S was transformed by electroporation into attenuated Salmonella typhimuriumSL7207. The recombinant was screened and designated as SL7207 (pVAX-S). Mouse peritoneal macrophages were infected with SL7207 (pVAX-S), the transcription and expression of S gene were detected by RT-PCR and indirect immunofluorscence. BALB/c mouse were inoculated orally with SL7207(pVAX-S) at dosage of 5 x 10(8), 1 x 10(9) and 2 x 10(9) CFU for safety analysis. In a vaccination test, BALB/c mouse were immunized orally with recombinant bacterium at dosage of 1 x 10(9) CFU, for 3 times and specific serum IgG and intestinal mucosal IgA antibody were detected by indirect ELISA. RESULTS: Recombinant plasmid pVAX-S was constructed correctly and expressed in COS7 cells. The transcription and expression of S gene were detected after mouse peritoneal macrophages were infected with SL7207 (pVAX-S). The recombinant bacterium was safe to mouse at dosage of 2 x 10(9) CFU. Specific serum IgG and intestinal mucosal IgA antibody against TGEV S protein were detected in SL7207 (pVAX-S) immunized group at 2 weeks post-boosting,and there were significant difference (P < 0.05) in serum IgG and most significant difference (P < 0.01) in intestinal mucosal IgA at 2 weeks after the third immunization, compared with SL7207(pVAX) control group. CONCLUSION: The recombinant Salmonella carrying TGEV S gene DNA vaccines had good immunogenicity and safety in mouse.