Cloning and expression of bovine prolactin cDNA in Escherichia coli.
Total mRNA isolated from bovine pituitary was used as a template to synthesize double-stranded cDNA with reverse transcriptase and E. coli DNA polymerase. Recombination was performed using pBR322 as the cloning vector and the oligo dG-tailed and oligo dC-tailed method. The recombinant plasmid was then introduced into E. coli to construct the cDNA library of bovine pituitary mRNA. The labelled synthetic bovine prolactin (bPrl) gene fragment was used as hybridization probe to screen the positive clones, which were then subjected to enzymatic mapping and DNA sequence analysis. The results demonstrate that the positive clones contain a full length bPrl cDNA sequence. The clones obtained were subsequently trimmed, linked to a tac promoter, introduced into E. coli JM103, and expressed under the induction of IPTG. The SDS-PAGE indicates the existence of expression product, and the result of ELISA shows that the product has the same immune activity as native bPrl.
Shu
Y M
YM
Institute of Developmental Biology, Chinese Academy of Sciences, Beijing.
Shi
Y X
YX
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA; D008969:Molecular Sequence Data; D010957:Plasmids; D011388:Prolactin / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D011994:Recombinant Proteins; D011388:Prolactin; D004247:DNA