Molecular Cloning Of The CDNA Encoding Adult Bullfrog Growth Hormone And Its Transgenetic Expression Effects On Experimental Bullfrog

Animal growth hormone (GH) is a very important protein hormone which is secreted by the anterior pituitary cells and plays many important roles on animals, such as regulating somatic growth and stimulating appetite, affecting various metabolic activities and improving the food transformation rate, etc. Although GH has so many functions, it is limited in the application and functional researches because the natural growth hormone content is very low and difficult to purify. Along with the development of biotechnology, we can obtain large amount of recombinant growth hormone protein which shows it have the same biological activities of natural GH, and its widely reported application studies are showed that the recombinant GH proteins can significantly stimulate the experimental animals growth and appetite when it is continuously and large dosages used on the experimental animals every day.Although DNA vaccine research shows stable effect of enhancing the immune responses and the abilities against diseases on the experimental animals that are injected with antigen genes' recombinant plasmids, such study of growth hormone gene are seldom reported. Applications of animal's growth hormone are mostly studied by means of protein type, up to now, no other reports about GH gene directly used in animals have been found except one paper about the transfection of human GH gene into mice.In this research, we studied the changes in the bullfrogs after they are separately injected with the recombinant bullfrog GH protein (re-BfGH), bullfrog GH plasmid (VB/GH), grass carp GH plasmid (VgcGH) and expression vector VR1020 while the 0.85% salt-water as the control, for the purpose to determine the possibility of that the eukaryotic expression plasmid VBfGH and VgcGH are expressed in adult bullfrogs and affect their growth rate and plasma GH. We hope the results will help developing a new approach to promote the animal growth. Main works and their important results areshowed as following.Firstly, the cDNA encoding bullfrog GH is amplified by use of the total RNA, extracted from adult bullfrog's pituitary, as the template, PI (5'-CGGATCC ATG GCT TCA GGG TTA GGC) and P2 (5'-CGAATTC TTA AAA GGT GCA GTT GCT) as the primer pair and one particular cDNA product, 660bp, was obtained after the RT-PCR-The product was purified by using DNA agarose gel extraction method. After the purified cDNA and pMD18-T vector were ligated at 16℃ over night, the ligation products were transformed into E.coli strain DH5a and then the transformants were screened by clone PCR method. The recombinant plasmids were extracted and the positive plasmid, named pBjGH was determined by enzymatic digestion method. After sequencing of the positive clone and analyzed by BLAST on line, it is proved that the positive one is the full-length cDNA encoding the pre-GH of bullfrog and it encodes a deduced peptide of 215 amino acids (aa), with molecular weight 24.9KDa, isoelectric point at 7.77, and hydrophobic amino acids 37.7%, hydrophilic amino acids 35.8%, basic amino acids 14.9% and acidic amino acids 11.6%. It consists of 25aa making up the signal peptide and the signal peptide is same as other bullfrog GHgene, other 190 aa constituting the mature-peptide. In the latter, some residues differ from other reports on bullfrog GH, in detail, BfGH in this study shares 97%, 95% and 94% aa sequence similarity with the known sequences from other reports that their identification accession are respectively AAB24792 (Takahashi N, et al., 1991), AAB19428 (Kobayashi T, et al., 1992), CAA31038 ( Pan FM, et al., 1988). New cloned BjGHgene and its deduced amino acids sequence were deposited to GenBank under the accession number AY251538 (protein accession number AAP04356).Secondly, the expression of BjGH in E.coli and purification of the recombinant BjGH protein (re-BfGH) have been done after the prokaryotic expression plasmid pGBfGH has been constructed. A predominant band corresponding to the expected size of BfGH fusion protein (50.9KDa) was observed in total cell extract...