ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2012, Vol. 43 ›› Issue (8): 1273-1280.

• 预防兽医 • Previous Articles     Next Articles

Construction and Identification of Attenuated Salmonella typhimurium Harbouring S/N Double Fusion Genes of Porcine Transmissible Gastroenteritis Virus

HUANG Xiaobo 1, LI Chunsong 1， YANG Heng 1,2， CAO Sanjie 1,

WEN Xintian 1*， LIAO Xiaodan 1， ZHANG Xinmiao 1

  

  1. 1. Laboratory of Animal Infectious Disease and Microarray / Key Laboratory of Animal Disease and Human Health of Sichuan Province， College of Veterinary Medicine， Sichuan Agricultural University, Ya’an 625014, China； 2. College of Life Science and Technology, Kunming University of Science and Technology， Kunming 650224， China
  • Received:2011-12-07 Revised:1900-01-01 Online:2012-08-24 Published:2012-08-24
  • Contact: WEN Xintian

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Abstract: To provide a new vaccine for oral immunization of transmissible gastroenteritis virus (TGEV), attenuated Salmonella typhimurium harbouring S/N double fusion genes of (TGEV) was constructed and identified. The S gene fragment (2.1 kb) and the N gene fragment (1.2 kb) were respectively amplified from the recombinant plasmid 19TS and 19TN of TGEV by RTPCR, and then the two gene fragments were successively inserted into the eukaryotic expression vector pVAX1 to construction the recombinant eukaryotic expression plasmid pVAXS/N that expressing the SN double fusion gene. The plasmid pVAXS/N was identified by PCR and restrictive digestion, and then the pVAXS/N was transfected into COS7 cells through liposome transfection to identify the expressions of the two target genes by indirect immunofluorscence assay. The plasmid pVAXS/N was transformed by electroporation into attenuate S. typhimurium SL7207, the stability of pVAXS/N in SL7207 (pVAXS/N) cultured in vitro was detected. The transcription of pVAXS/N in mouse ileal tissue cells orally immunized with SL7207 (pVAXS/N), BALB/c mice were inoculated orally with SL7207 (pVAXS/N) at different dosages to detect the safety and stability in vivo. The results showed that the recombinant plasmid pVAXS/N was constructed correctly and the expression of S gene and N gene were detected in COS7 cells. The transcription and expression of S/N double fusion gene were detected in mouse ileal tissue cells after 3 days infected with SL7207 (pVAXS/N). The recombinant bacteria SL7207 (pVAXS/N) was highly stable cultured with Kanamycin Resistance medium in vitro. The recombinant SL7207 (pVAXS/N) were all safe to mouse at dosage of 0.5×109, 1×109 and 2×109 CFU. The SL7207 (pVAXS/N) were eventually eliminated from the spleen and liver at about four weeks postimmunization. These results indicated that the recombinant bacteria SL7207 (pVAXS/N) had good safety and stability, which lay a foundation for oral immunization of TGEV.

Key words: attenuated Salmonella typhimurium, transmissible gastroenteritis virus, double fusion gene, construction, biological characteristics