Preparation of porcine LIF polyclonal antibody and its immunolocalization in pig
- VernacularTitle:猪白血病抑制因子多克隆抗体的制备及其免疫定位
- Author:
Mingtang LI
;
Yong JIANG
;
Yu FU
- Publication Type:Journal Article
- Keywords:
leukemia inhibitory factor;
polyclonal antibody;
immunolocalization
- From:
Journal of Jilin University(Medicine Edition)
2006;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the expression plasmid of porcine leukemia inhibitory factor(LIF) in the prokaryotic system,and purify the expressed recombinant porcine LIF protein as antigen to immune rabbit for preparation of rabbit anti-pig LIF polyclonal antibody,and immunolocalize of LIF in pigs.Methods The cDNA fragment encoding mature porcine LIF protein was amplified by PCR,then cloned into the prokaryotic expression vector PET-31b at restriction sites NdeⅠand XhoⅠ.After transformed into E.coli BL21 for expression,the recombinant porcine LIF protein was purifed for preparation of anti-pig LIF polyclonal antibody as immunogen.Western blotting and ELISA were used to test the specificity and titer of the purifed IgG.Immunostaining was used to assay the expression of LIF in pigs.Results The constructed expression plasmid was identified by DNA sequencing and restriction enzyme digestion.The expression of the recombinant porcine LIF protein was performed exactly in E.coli BL21.By immuning the rabbit,the polyclonal antibody was successfully prepared.The results of ELISA and Western blotting showed that the antibody had high titer(1∶10000) and specificity.By immunostaining,LIF protein was faintly expressed in porcine myocardial cell,splenic cord and monolayer covering epithelium cell and pulmonary fibroblast.Conclusion The prepared polyclonal antibody has high titer and specificity;LIF has faint positive immunoreactivity in the normal porcine,myocardial cell,splenic cord and monolayer covering epithelium cell and pulmonary fibroblast.
