Prokaryotic expression of chicken TGF‑β1 and preparation of polyclonal antibody
-
-
Abstract
The polyclonal antibodies against chicken transforming growth factor β1 （TGF‑β1） was prepared. Firstly， the CDS region of the chickenTGF‑β1 gene was amplified by RT‑PCR using the mRNA of Marek's disease virus transformed T lymphocyte line MSB1 cell as the template， and then the fragment was ligated into the prokaryotic expression vector pET‑32a （+） for the construction of the recombinant plasmid pET‑32a‑TGF‑β1. Subsequently， the recombinant expression plasmid was transformed into the Escherichia coli expression strain BL21 and induced for expression. The recombinant expressed protein was purified by affinity chromatography. Then， polyclonal antibodies were prepared by immunizing rabbits， and the antibody titer was detected by indirect ELISA. The specificity of the polyclonal antibodies was evaluated by Western blot and indirect immunofluorescence. The results demonstrated that the successful construction of the recombinant prokaryotic expression plasmid pET‑32a‑TGF‑β1， and a recombinant protein of approximately 65 kDa was obtained after induction with IPTG. Rabbit anti-chicken TGF‑β1 antiserum was obtained by immunizing rabbits， and the antibody titer was 1∶640 000 as detected by ELISA. Western blot results showed that the rabbit anti‑chicken TGF‑β1 antiserum could specifically recognize the target protein， and indirect immunofluorescence results indicated that the polyclonal antibody could well recognize TGF‑β1 positive cells. In conclusion， polyclonal antibodies against chicken TGF‑β1 with good specificity were successfully prepared， and can be used for the detection of chicken TGF‑β1.
-
-
DownLoad: