Expression, Purification Of Recombinant CT-1/TTC Protein, Target Delivery To CNS And Neurotrophy Biology Ability

Spinal cord injury (SCI) is a catastrophic event that imposes an enormous medical, psychological, social, and economic impact on individuals, families and society. Multiple studies have demonstrated that there is only peripheral nerve regeneration but no regeneration of central nervous system (CNS). Damaged axons in the adult CNS are unable to spontaneously regenerate upon injury, in contrast to neurons in the peripheral or embryonic nervous system. Such a failure in regeneration is believed to arise from both a decline in the intrinsic growth state of mature neurons, as well as the presence of a non-permissive environment in the injured adult CNS preventing axon growth. And the physical barrier formed by the astrocytic scar tissue that develops at the lesion site. Recent researches indicate that CNS regeneration occurs at appropriate condition, which depends on the intrinsic properties of central neurons and the surrounding environment. Although many ways to promote the sprouting of axons, most of the regenerated axons grow surrounding the cell or tissue transplantation are and few nerve fibers will enter the distal end through the injured region. Therefore, it comes to more difficult to establish the connections between functional axons. Drug intervention may be an easy method, if it is useful.Cardiotrophin-1(CT-1) was isolated as a member of the interleukin-6-related cytokine family. It has stronger ability to promote the survival of neurons than ciliary neurotrophic factor (CNTF). CT-1, via coupling through the LIF receptor and gp130, has been shown to activate a number of signaling pathways in cardiac myocytes, including mitogen-activated protein kinases and the Janus kinase(JAK)/signal transducers and activators of translocate to nucleus, and activate expression of target genes. In addition, CT-1 can delay motor defect of mice and exert protective effect against loss of proximal motor axons. Consequently, they share many biological activities on hematopoietic cells, embryonic stem cells, and hepatocytes. Thus, chronic administration of cytokines to animals induces pleiotropic effects including weight loss and synthesis of cute-phase proteins. These results suggested that neuro- protective effects of cytokines might be counter-balanced by their systemic toxicity.Tetanus toxin (TeNT),a potent neurotoxin produced by the anaerobic bacterium Clostridium tetanus, causes spastic paralysis by blocking the neurotransmitter release from spinal cord inter-neurons. TeNT has a well-documented capacity for neurospecific binding and internalization. When administered to animals, the toxin is taken up selectively by nerve endings at the neuromuscular junction and gains access to the CNS via retrograde axonal transport within motoneurons and trans-synaptic migration to interneurons. The 150-kD TeNT protein is composed of a heavy chain which mediates binding and retrograde transport and light chain which is responsible for most of the toxicity. The TTC fragment, corresponding to the carboxy-terminal 451-amino-acid fragment of the heavy chain, retains the neuronal binding and uptake properties of the holotoxin but is nontoxic, Thus these fragments have been shown to be very useful tools to monitor membrane dynamics and retrograde axonal transport in neurons.We therefore hypothesized that fusion of the neurotrophic factor cardiotrophin-1 to TTC fragment might increase its neuronal binding, while leaving intact its genuine function as a survival-promoting protein for neurons. And the fusion protein can be used by local application in CNS, but also can be used by inject to muscle control by idio-conduction fascicular or repetatus subarachnoid space application. It furnishes a unique factor to promote neuron survival and a new method of administration.This research was designed to verify the activities of targeting delivery to CNS and protecting the cornu anterius medullae spinalis motoneurons.Main Methods and Techniques:The experiment was divided to three parts:PART 1 use owned pGEX-CT-1/TTC expression plasmid, expression in Escherichia coli BL21(DE3),and purification;1. Revitalize the DH5αpGEX-CT-1/TTC Escherichia coli stored in -70°refrigerator, Then identified by restriction enzyme digestion assay and sequencing. The results show we have harvested the pGEX-CT1/TTC successfully. And transformed it to Escherichia coli BL21 (DE3), picked monoclonal colony, extracted the plasmid, then assayed the sequencing again. The two cleavage maps show straps match well and it proved the expression plasmid constructed successfully. 2. Optimizing the induct condition by time and temperature. We used transonogram to cleavage the bacteria inducted by IPTG in different conditions, centrifuged. Induction by IPTG, the fusion protein was expressed and then purified by GST affinity agarose. The interest protein was viewed by SDS-PAGE, further characterized by Western Blot. The result shows the CT-1/TTC fusion protein has two existence patterns, including soluble component and inclusion body. We choice 35°C, 4h as induct condition.PART 2 verify the activities of targeting delivery to CNS;Rat sciatic nerve transected model was selected. Using drug by nerve-regeneration-chamber and intramuscular injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained with immunohistochemistry method.PART 3 verify the activities of protecting the cornu anterius medullae spinalis motoneurons.Select the new born SD rat sciatic nerve transected model, using CT-1/TTC fusion protein by muscle injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained by Nissl's staining.Main Results and Conclusion:1. Transformed the pGEX-CT1/TTC plasmid to Escherichia coli BL21 (DE3);2. We found the induct efficacy at 35°,5h is optimal strategy;3. Purified by GST fusion protein column, the interest protein's concentration is 2.7 mg/ml. we harvested 15mg fusion protein totally.4. The CT-1/TTC fusion protein was detected in lumbar intumescentia by immune-histochemistry method.5. after sciatic nerve transected, the numbers of cornu anterius medullae spinalis motoneurons in L4-L6 segments, compared to CT-1/TTC protein grope, have a lower survival rate.