Cloning And Expression Of Prolactin Gene Of Cestode In Prokaryotic Cell

Cloning and Expression of Prolactin gene of Taenia Hydatigena.Using antibody of prolactin(PRL) of ovine.we analysized the expression of PRL of cestode by western blot.The SDS-PAGE result showed that the coarse proteins extracted from scolex and organs concerning reproduction has a brighter band at the 23kDa position than what from other part and antibody of PRL from ovine can react with the protein.This result is in accordence with result ealier. Acording to conservative domain of the published sequence of prolactin(PRL) gene of vertebrate.a pair of primers that can amplify cestode PRL gene was desighned and synthesized.By RT-PCR ,a single DNA fragment of about 800bp was obtained.After recovery from agarose gel the target DNA fragment cloned into pMD-18T vector and sequenced.The complete nucleotide sequence is 893bp,encoding 222aa.OF the complete amino acid sequence,the first 22aa is signal peptide.and mature protein has 200aa,By comparing with published sequence in genebank with BLAST.the sequence showed hightest identity with PRL gene.The amino sequence translated from the nucleotide sequence is compared with published amino sequence of database by BLASTP.the result showed that the amino sequence has the highest identity with PRL.RPS-BLAST found a hormone conserve dormain in the amino sequence.By interproscan program two motif which belongs to prolactin gene was found. These proved that the sequence we cloned is PRL gene of cestode. Analysis of the amino sequnce of cestode PRL gene demonstrated that the molecular weight of the mature protein is 23kDa and isoelectric point is 6.67.Cestode PRL gene has the highest homogeneity with huamn and ovine PRL gene,the homogeneity is 74.3% and 68.0% respectively.The homogeneity between cestode and fish is the lowest,which is no more than 30%.From the pHylogenetic tree of PRL gene,fish forms one of the branch of tree because of the low homogeneity with other animals and the others form the other.Cestode is located between human and rat of rodent.The distribution of various animals on the pHylogenetic tree is not strictly in accordence to the evolution level.Cestode is one of invertebrate animals.and its evolution level among invertebrate animals is low.but its PRL gene on pHylogenetic tree is close to animals with higher evolution level.PRL gene has two motif domains.one of them has 34 amino acids and the other has 28 aminoacids.The number of the amino acids of motif domains is constant.The homogeneity of motif domain is much higher than that of other domains.Some of the amino acid in these two domains is indnetical among all animals.which is important for the conservative function.The recombinent vector pMD-18T-PRL is cut by ECORI and HindIII,the PRL gene is recovered from gel.The expression vector(pET-28c) is cut by similar enzymes and recovered.PRL and expression vector is linked by T4 DNA ligase and then transformed into DH5a competent cell.Recombinent vectors is indentified by PCR and enzyme-cut.The positive recombinent vector pET-28c-PRL is transformed into BL21 and induced by IPTG.The Cestode PRL gene is successfully expressed and can react with antibody of PRL from ovine.Because of the absence of modifycation mechanism of prokaryotic expression systems.the protein expressed by these systems can not form the native conformation and so has lower activity.This is why the PRL protein expressed has lower reactivity with PRL antibody than that extracted from cestode.