[Preparation and identification of the polyclonal antibody against GAPDH protein from Shigella flexneri M90T].
OBJECTIVE
To prepare mouse polyclonal antibodies against Shigella flexneri M90T glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and verify its titer and specificity.


METHODS
The gapA gene of Shigella flexneri M90T was amplified by PCR from extracted genomic DNA and inserted into expression vector pET24a. The plasmid pET24a-gapA was transformed into E.coli BL21(DE3) and induced to express the tagged protein GAPDH-His. After condition optimization, the protein was purified with Ni Sepharose(TM); 6 Fast Flow. The polyclonal antibodies were prepared by immunizing the mice with the purified recombinant protein and then harvesting mouse sera. The specificity and efficiency of antibodies in sera were analyzed by Western blotting and ELISA against whole cell proteins of Shigella flexneri M90T.


RESULTS
The recombinant plasmid pET24a-gapA was successfully constructed. The soluble expression of the tagged protein GAPDH-His was achieved with 2-hour induction of 1 mmol/L IPTG at 30°C. Western blotting and ELISA demonstrated that the polyclonal antibody harvested from the mice immunized with GAPDH-His was specific and efficient against Shigella flexneri M90T GAPDH. Conclusion The mouse polyclonal antibodies against the Shigella flexneri M90T GAPDH protein were successfully prepared.
College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018; Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
Yu
Y
Wang
Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
Xiang
X
Liao
Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
Junjie
J
Yue
Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
Wei
W
Zhou
Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
Long
L
Liang
College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China.
Bateer
B
Huhe
D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D005818:Genetic Engineering / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction; D012763:Shigella flexneri / Q000201:enzymology*
D000906:Antibodies; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases