In vitro expression and antibody preparation of black carp (Mylopharyngodon piceus) GH.
The cDNA fragment encoding the mature polypeptide of growth hormone (GH) for the black carp (Mylopharyngodon piceus) was PCR amplified and subcloned into pET-28a. The recombinant expression plasmid pET-bcGH was transformed into E.coli BL21(DE3) and fusion polypeptide containing a 6xHis-tag at the N-terminus was expressed after IPTG induction. The fusion protein band of 23 kDa or so showed immunoreactivity to the polyclonal antibody aganist grass carp GH. The recombinant GH for black carp was purified by affinity chromatography and dialysis. Using the fusion protein as an antigen, through the modified immunization method, the polyclonal antiserum to black carp GH was obtained. Immunochemistry results showed that the antiserum could detect the antigen as low as 4 ng. The protein of 21 kDa in black carp pituitary protein extracts and blood serum could be detected by western blot analysis in which polyclonal antiserum to black carp GH was used as the primary antibody. All these results showed that the polyclonal antiserum against black carp GH was not only effective but also highly specific.
Feng
Hao
H
Key Lab of Protein Biochemistry and Developmental Biology, Education Department of China, College of Life Science, Hunan Normal University, Changsha 410081, China. fenghao_99@yahoo.com
Cheng
Jia
J
Liu
Yan
Y
Luo
Jian
J
Li
Jian-Zhong
JZ
Liu
Shao-Jun
SJ
Liu
Jun
J
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D002347:Carps / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D013006:Growth Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007106:Immune Sera / Q000096:biosynthesis / Q000276:immunology; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D014170:Transformation, Genetic
D000906:Antibodies; D018076:DNA, Complementary; D007106:Immune Sera; D011994:Recombinant Proteins; D013006:Growth Hormone