Construction Of Adno-associated Virus Vector Of Ciliary Neurotrophic Factor Gene

Objective: Peripheral nerve injury is a common disease, peripheral nerve injury repair microsurgical techniques are already very skilled, but difficult to obtain a satisfactory results for a long time. As the molecular biology and gene transfer technology's developing, the research of nerve injury's gene therapy is emerging. The research of application of replication defective recombinant adenovirus (Adenovirus, AdV) for carriers to make variety of neurotrophic factor gene switch into the damaged spinal cord to protect neurons and promote regeneration of peripheral nerve, has become a popular issue in the area of the nerve repair. The development of adenovirus research is very quickly and has been successfully applied to a number of gene therapy clinical trials. The positives of adenovirus vector are high efficiency, wide host range, can infect split or resting phase cells, gene loading capacity, easy preparation high titer virus, do not insert the host genome, low genetic toxicity and so on. CNTF is composed of 200 amino acid protein, no homology with NTF, which was named non- target neurotrophic factor. In the process of nerve regeneration, CNTF plays an important role in nutritional support, the functions of CNTF including: to support the feeling, sympathesis and motor neuron's survival and differentiation, prevent motor neuronal apoptosis after axon was cut off, and prevent nerve cells'proceduraldead during the growth period. CNFT is a nerve factor which was found firstly to promote in vitro and in vivo survival and prevent the neural degenerate. CNTF and its receptor exist widely in the nervous system, possesses a wide range of biological activity, can improve a various types of peripheral neurons to survive. In the peripheral nervous system, myelin and ciliary ganglion's Schwann cells (SC), CNTF is showing a high level of expression, are mainly located in cell body and processes, and no positive reaction. In the central nervous system, CNTF mainly exist in atrocities. CNTF model can promote the motor neuron cell's survival through reverse transmission mechanism ciliary neurotrophic factor (CNTF)'s biological functions, has obvious nutritional function for the central nervous system and peripheral nervous system neural cell growth, differentiation.To construct virus particle for deeply research of the application of adenovirus-mediated ciliary neurotrophic factor in peripheral nerve injury, by interrelated virus vector which is established from molecular biology techniques Cloning of rat ciliary neurotrophic factor (CNTF) gene fragment.Methods: 1 Using the competence to create super-competence which is preserve in the laboratory. Imbibe from competence of Escherichia coli and cultivate overnight, then switch into 200mlLB medium, obtain bacteria when the value of OD600 stay around at 0.4. Add 100ul solution precooling 0.1M CaCL2, use the pipette gun mix round the refrigerating centrifugal to average, so that cell could be re-suspension. Cell suspensions can be immediately used for transformation test, preservation at -80℃after add 15% -30% glycerol . (according the specific experimental steps).2 Though containing the CNTF adenovirus vector which was packaged in company, transfer into super-competence that can be amplified to obtain enough plasmid. Add plasmid 5ul in the competence, iced and thaw it , then cooling it by ice after heating, again add culture medium for 1 hour, cultivate 16-24 hours on flat plate,after the monoclonal bacteria grow up, pick bacteria cultivate, Extract plasmid according the plasmid extraction steps after centrifugation.3 After plasmid amplification, using liposome 2000 transfection kit transfect Human embryonic kidney 293 cells, packaging with CNTF gene recombinant virus, through reduplicate freeze-thaw to make the high titer adenovirus at the temperature of -70℃and 37℃.4 After the virus was amplified in 96 plate cultivate embryonic kidney 293 cells, which can be observed that have obvious CPE phenomenon by the microscope, using 50% tissue culture infectious dose determination to determinate the virus titer.Results:1 collocate the plasmid amplification in accordance by the experimental system, the result is as the same value as the one that provided by the company.2 after recombinant plasmid transfection, 293 cells were trained for 24 hours,can be observed that 293 cells has green fluorescent.By the inverted fluorescence microscope.We can detect report gene of CNTF,in control group we can see nothing.transfection process Successful.3cultivate 293 cells on the 96 plank,using 293 cells which obtain virus infected to calculate the number of CPE, determinate virus titer determination,Finally,obtain virus titer of 2.5×107TCID50/ml.Conclusions:1successful makesuper-competence,use for plasmid's transformation and amplification , and successfully amplified the necessary enough measurement plasmid.2 successful packaging of the CNTF and the associated virus,and through iterative freeze-thaw that enhance virus droplets degrees,establish a foundation for deeply research CNFT in peripheral nerve.3HEK-293 cells can be observed that have obvious CPE phenomenon by the microscope as mentioned above, determinate virus titer by 50% tissue culture method, and finally obtain the 2.5×107TCID50/ml virus fluid, will pave the way for the further test.