Cloning, Expression And Bioassay Of Neurotrophic Factors Of The Giant Panda (Ailuropoda Melanoleuca) And The Crested Ibis (Nipponia Nippon)

The giant panda and the crested ibis are flag species of the rare endangered mammals and birds respectively in the world. Both have been on the verge of extinction due to poaching and habitat fragmentation. In order to enlarge the population size and recover the wild populations, ex situ conservation strategy has been advocated by the governing agents in China through natural or artificial breeding in captivity. However, epilepsy has become one of the most important reasons for abnormal death of the giant pandas, with a proportion of 8% death concerning all the sick individuals. In the meantime, congenital amblyopia resulted from optic nerve lesion was observed in the crested ibis, with an incidence rate of 3-5%. Since the epilepsy and congenital amblyopia are both hereditary, it is feasible to perform genetic engineering studies on the correlative genes, which could protect neurons from the damages of excitation toxin and vision sickness, in order to cure the diseases at gene level. The research will predicatively heighten the therapy level for the suffering individuals in captive populations of both the giant panda and crested ibis, and possess great theoretical and practical significances on enhancing theliving status of the endangered animals.Neurotrophic factors (NTFs) are a group of cytokines, which all play important roles in the differentiation, development, growth and regeneration of nerve system. There are 3 families in the NTFs:the neurotrophins (NTs) , the glial cell line-derived neurotrophic factor (GDNF) and the ciliary neurotrophic factor (CNTF). The NTFs are known to be able to protect neurons from the damages of excitation toxin and traumatic brain injury. In addition, some NTFs could induce the development of normal vision pathway, and even help the injured neuron to regenerate synapse.In this study, the genes coding for mature NTs and GDNF were cloned through polymerase chain reaction. After sequencing, alignment showed the NTFs were highly conserved. The coding sequences for the NTFs were then cloned into the expression vector pGEX-4T-3 under the control of a tac promoter, and expressed in Escherichia coli after induction with EPTG. Some recombinant proteins agreeing with those expected were obtained, including the NGF, NT-4 and GDNF of giant panda, BDNF and GDNF of crested ibis. The expressed fusion proteins were verified with SDS-PAGE, GST activity and Western Blotting. After purification using Glutathione Sepharose 4B affinity chromatography and digestion with thrombin, the recombinant NTFs were found to be biologically active in the PC 12 cells neurite outgrowth assay. The assays demonstrated that thepurified NTFs proteins exhibited normal activity, which is the first step in developing a comprehensive gene therapy for nerve diseases of the giant panda and the crested ibis.Although the gene therapy of the human nerve system diseases are still in exploring so far, and similar studies applied to the wild animals have not even been reported, this study will inevitably provide clues for futher studies on the gene therapy in the giant panda and crested ibis.In addition, the NT-3 and NT-4 coding sequences were used to resolve the taxa problems of the giant panda and the red pandaCAiZurus fulgens), combining with mtDNA genetic marker and other traditional taxonomic data. The result suggested that the giant panda should be classified as a separate subfamily within Ursidae, while the red panda should be included in a monotypic family Ailurdae.