2.5 SDS-PAGE and western blot analysis
The total protein extracted from transgene positive wheat seeds tissues was then separated by SDS-PAGE on their molecular masses. The expression of vaccine antigen in seeds was verified by western blot analysis. The band were separated using 8% gel and were incubated with PBST containing 5% skimmed milk for one hour. The blot was then incubated with primary with 1:10000 ration with polyclonal rabbit anti-CTB antibody (Sigma-Aldrich, Inc,) over night at 4°C with continuous shaking. The blot was then washed three times with PBST and then was incubated with secondary goat anti‐rabbit IgG‐HRP antibody (Southern Biotechnology, Birmingham, AL) with ratio 1:5000 in PBST containing 5% skimmed milk. The expected bands corresponding to TM-1 protein was detected by substrate NBT/ BCIP and developed with X-Ray for 5 seconds exposure. The quantitation of specific band was performed using software ImageJ.
2.6 ELISA quantification Enzyme-linked immunosorbent assay (ELISA) was performed using Pierce™ Nickel Coated Plates (Thermo Fisher Inc.) to detect the expression of TM-1 recombinant vaccine in wheat seeds by following manufacture’s instruction. All the plant samples were analyzed in duplicate. For detection of antibody titer in immunized chickens, chickens were bled, and the serum was analyzed by the ELISA against GM antibody (Idexx Laboratories) following manufacture’s instruction.