Prokaryotic Expression Of Transmissible Gastroenteritis Virus Spike Protein C And D Antigenic Sites

Transmissible gastroenteritis(TGE) caused by transmissible gastroenteritis virus(TGEV) is an acute and highly contagious disease. All ages and breeds of pigs are susceptible to TGEV. The mortality rates of two-week-old piglets are up to 100%. TGEV is a widespread infectious virus. The infections of TGEV are an imminent threat to the commercial porcine industry.The nucleotides coding TGEV spike protein linear antigenic sites C and D were synthesized. After reannealing, the according fragment was cloned into prokaryotic expression vector pET32 a, respectively. The recombant plasmids were named respectively pET32a-C1C2 and pET32a-D. Furthermore, the D fragment was cloned into recombant plasmid pET32a-C1C2. The new recombant plasmid was named pET32a-C1C2 D.The three recombant plasmids above-mentioned were transformed respectively into E. coli. Rosetta(DE3). The recombant Rosetta(DE3) were induced respectively by 1.0 mmol/L IPTG for 4 h. The approximate molecular weights of recombant protein were 21 kDa, 22 kDa and 25 kDa. The three recombant proteins were purified by elution from sodium dodecyl sulphate(SDS) polyacrylamide gels. The antigenicity of the three recombant proteins was analyzed by Western blot and Dot-ELISA. All the three recombant proteins were recognizated by TGEV-positive sera. After optimizing the induce time and IPTG concentration, the expression quantity of Rosetta(DE3)(pET32a-C1C2) was elevated from 38.0% to 41.4%, the expression quantity of Rosetta(DE3)(pET32a-D) was elevated from 30.9% to 33.8%, the expression quantity of Rosetta(DE3)(pET32a-C1C2D) was elevated from 15.7% to 26.5%. This research lays a necessary material foundation of serological diagnosis for TGEV.