CDNA Cloning And Prokaryotic Expression Of Buffalo Pituitary Prolactin Gene

This thesis studied cDNA cloning and prokaryotic expression of buffalo pituitary prolactin gene. It is arranged in two parts: Part I includes chapter 1, which is a review of literatures; Part II includes chapter 2 and chapter 3, which focuses on the experimental works.Buffalo is one of the main economical animal species in southern China, which not only can produce good quality of milk , but also can be rough feeding and resisting disease. However, the shortage of buffalo is the poor reproductivity and lower milk yield while compares to dairy cow. Pituitary prolactin (PRL) is a polypeptide hormone which is synthesized and secreted by acidophil in the anterior pituitary gland. It can improve reproductivity and promote milk yeild. The objective of this study is to investigate the construction and function of buffalo pituitary prolactin gene by cDNA cloning and prokaryotic expression thereby provides basic information for a further research in improvement of buffalo reproductivity and lactating performance.The second chapter of this thesis is focused on the cloning and sequence analysis of buffalo PRL gene. According to the conservative domain of mammalian PRL cDNA, a pair of primers that can amplify buffalo PRL gene was designed and synthesized. Total RNA was extracted from pituitary of buffalo. The cDNA of buffalo PRL gene was synthesized and amplified by reverse transcriptase polymerase chain reaction (RT-PCR). A single DNA fragment about 860bp was obtained. After recovery from agarose gel, the target DNA fragment was inserted into pMD18-T vector. The positive clone identified by PCR and double digestion restricting enzyme was sequenced. The results of Sequencing revealed that buffalo prolactin cDNA contains an open read frame (ORF) of 690bp which encoded 229 amino acids, 19 of which are signal peptide. By blasting with the published prolactin genes sequence in NCBI, the sequence showed high identity with PRL gene. The multiple sequence homology of buffalo and cattle is 98. 4%, which proved that the sequence is buffalo PRL gene. The sequence had been submitted to GenBank (GenBank accession No. EF054878). The above studies first amplified the cDNA sequence of buffalo pituitary prolactin, which provides basic information for further study of prokaryotic expression of buffalo prolactin pituitary.The third chapter of this thesis was focused on the prolactin prokaryotic expression. On the base of buffalo pituitary prolactin cDNA, a pair of primers which had been added two restriction endonuclease sites was redesigned to amplify the objective fragment. The singe peptide was cut off from PRL cDNA. After obtaining the objiectives, the target fragment together with its pQE-30 vector were digested by restriction endonucleases BamH I and HindIII, then ligated by T4 DNA ligation enzyme and transfected into E. coli DH5α. The results showed that the prokaryotic expression vector pQE-30-PRL system was successfully established. The positive recombinant clones we're identified by restriction endonuclease digestion, PCR and sequencing. Then positive recombinant plasmid was transfected into E. coli M15. After induction by IPTG, the SDS-PAGE analysis showed that a specific expression band was detected. Result analysis indicated the expression product existed mainly in inclusion body. The molecular weight of recombinant protein was approximately 23kDa, which was consistent with its theory molecular weight. The present studies on PRL provided a basic work for furher purification of PRL protein and further investigation of its biology of gene structure and function.