Cloning And Expression Studies Of FSH/LH And GH/GHR From Giant Panda And Other Endangered Animals

Wild animal is an important component of natural ecosystem, and it becomes increasingly important to protect wild animal, particularly endangered animals with aim to maintain the biodiversity and ecological balance. As one of strategies for ex-situ conservation of endangered animals, captive breeding will contribute a lot to increase their individual amount and improve population. However, in captive, most of animals exhibit reproductive problem with low fertility which hinders the application and development of captive breeding. Particularly for giant panda, due to its high specialization upon reproductive capability and cub-raising behavior, it has a typical low fertility and high infant mortality which become two problems that seriously need to be solved in ex-situ conservation. Previous studies suggested that the endocrine maladjustment of the female giant panda hypothalamic-pituitary-gonadal axis, especially upon insufficient secretion of FSH and LH might be one of the causes of low fertility. While the possible reason resulted in high infant mortality is that new-born pandas was observed to be congenitally immature with underdeveloped immunity system. Up to now, concerning the molecular mechanism underlying low fertility and high infant mortality is still poorly understood. Accordingly, focusing on reproductive problems of giant panda, lesser panda and Panther tigris altaica, this project is aimed at: (1) Cloning, sequence analysis and expression of FSH/LH and GH/GHR which play vital roles in growth and reproductive regulation. (2) Discussing the significance and possibility of a further study towards the production of their recombinant gonadotropins and GH by recombinant DNA technique, and their application in assisted reproduction. The primary results were as follows.1. By RT-PCR gonadotropins common a , FSH P and LH P subuntis from giant panda encoding 120,129 and 141 amino acid respectively were isolated, with predicted molecular mass of 13.6kD,14.6kD and 15kD respectively. Three subunits all showed high degree of homology with other reported sequences at nucleotide and amino acids levels. Three differences in panda amino acids sequences were found from sequence alignment. Whether these differences affect the molecular conformation or functions of these hormones, or these changes in the molecular structure of three subunits may be responsible for the abnormal secretion of reproductive hormone by influencing the function of the gonadotropin subunits have not yet been determined.2. COS-7 cells was co-tranfected with the panda subunits a together with FSHPor LHP cloned into pcDNA3.1(+ ) expression vectors. RT-PCR analysis revealed that a, FSHP and LHp were normally transcribed in COS-7 cells. Bioassay in vitro exhibited the dose-dependent increase estrogen (17 P -estradiol) in a rat Sertoli cell and testosterone in a rat Leydig's cell stimulating by the cell media using ECLIA methods, which apparently suggested that recombinant panda FSH and LH produced from COS-7 cells are efficiently secreted and biologically active comparable to standard gonadotropins. The results will lay a foundation for a further study to produce recombinant gonadotropins in CHO cells and apply them in artificial breeding.3. The DNA fragments encoding mature a and FSHp, precursor LHp from giant panda were subcloned to the pGEX-4T-l expression vector and highly expressed in E. coli host BL21 with IPTG induction. The expressed proteins fused by GST (GST-ct, GST-FSHP and GST-LHp) with their molecular mass of 36kD,38kD and 41kD respectively, were found to be sequestered into inclusion bodies. To improve the yield of purified GST fusion protein by Glutathione Sepharose 4B affinity column, we optimized the solubilized condition with 8M urea and 0.1M NaOH for three fusion proteins. The results revealed that the urea-solubilized fusion protein did not bind to the column and no purified protein was obtained. While high yield of NaOH-solubilized GST protein valid be obtained from the column, suggesting that NaOH-solubi...