Cloning and expression of P. gonionotus growth hormone CDNA in Escherichia coli by the polymerase chain reaction (PCR) method

Abstract

Puntius gonionotus or Thai silver carp is an economically important fish in our country, and there is a need to improve the quantity and quality of this fish. Growth Hormone (GH) is a 22 kDa polypeptide synthesized in the anterior part of the pituitary gland. This hormone is widely distributed in all vertebrates and regulates the essence function of growth. Because of its importance and its potential in agriculture, the GH gene has been extensively studied in many species. The general strategy used to clone this GH gene involves in the construction of genomic or cDNA library, however nowadays there is a more powerful technique to clone this gene namely the Polymerase Chain Reaction (PCR). This method can amplify DNA fragment of interest by using two specific primers located at the boundaries of the desired sequence. Since P. gonionotus belongs to the same family of Cyprinidae and as the GH cDNA of Cyprinus carpio or common carp that had been cloned and sequenced, the nucleotide sequence of that carp was used to provide an information in the design of two specific oligonucleotide primers located at the 3-end and 5-end of the GH gene used to clone the PgGH cDNA by PCR method. PgGH cDNA reverse-transcribed from total RNA was amplified by PCR and cloned in E. coli using the BS- vector. The recombinant clones were selected and characterized using two restriction enzymes, EcoRI and RsaI, and their inserts were subsequently sequenced. The PgGH cDNA was found to encode a polypeptide of 188 amino acids with two points of polymorphism and had a 97% amino acid homology with carpGH cDNA. When compared with other GHs, the PgGH cDNA structural feature contained the 5 common conserved domains. In order to express the PgGH cDNA in E. coli, the PgGH cDNA was cloned into pUC19 vector under the control of the lac Z promoter of the vector and using the SD sequence and the starting codon (ATG) designed in primer sequence. The GH protein of P. gonionotus produced in E. coli comprised about 8% of the total cellular protein with a molecular weight of about 21 kDa. Moreover, the Southern blot hybridization of P. gonionotus genome with PgGH cDNA showed that the fish contained only one gene coding for GH.