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.