Purification Of Adno-associated Virus Vector Of β-galactosidase Gene

Objective: Accompanied by the development of industry, agriculture andtransport, an increasing number of high-energy trauma, peripheral nerve injuryhas become a common disabling injury in the clinical treatment for peripheralnerve injury has been a thorny issue in the clinical, despite themicroscopicsurgical repair technique is superb, but has long been difficult toobtain satisfactory results. With the development of molecular biology andgenetic engineering for gene therapy research today has become a hot topic inthe field of peripheral nerve injury. The study found that in the central andperipheral nervous system to carry the target gene of adenovirus vectors(Adenovirus, AdV) after import target gene expression product in a timelymanner, so as to achieve the purpose of treatment or research.The moment, nerve repair hot area of research is integrated into a variety ofneurotrophic factor gene recombinant adenovirus (Adenovirus, AdV), andthen carry the target gene of adenovirus transferred to the spinal cord orperipheral nerves to the right by neuron loss protection or in the peripheralnervous system induced by peripheral nerve regeneration process. Adenovirusvector has a high infection efficiency, broad host range can be dividing orresting cells infected with a huge amount of genetic load, easy to extract highconcentrations of viral components, the toxicity of low-level expression.Molecular biotechnology cloning lactose operon fragment of LacZ constructadenovirus vector, preparation of viral particles for further study of adenovirusvector mediated LacZ gene in peripheral nerve injury.LacZ gene is widely used in gene expression of a gene in the regulationof research. LacZ gene encoding beta-galactose Cape enzyme (referred to asbeta-gal) by the four-subunit tetramer, Hugh, can catalyze the hydrolysis oflactose. Beta-gal is relatively stable, the substrate X-Gal staining, blue, tofacilitate the detection and observation. Many advantages of the lacZ gene to make it become a commonly used marker gene in the genetic engineeringexperiments, space-time it was used for gene expression, detecting theposition and role of effective segments of known regulatory sequences,looking for unknown sequences.Therefore, the use of adenovirus secure, stable, efficient and LacZ gene-specific expression characteristics of the current use of adenovirus containingthe LacZ gene has become the forefront of gene therapy for peripheral nerverepair. Adenovirus neutralizing antibodies rapidly cleared, this is due to thestrong immunogenicity of the adenoviral vector, adenovirus in the daily worklife would induce the body to produce anti-adenovirus neutralizing antibodyinto the human body. If you accept the adenovirus vector-mediated genetherapy because the neutralizing antibody after elimination of adenovirusvectors, resulting in leaving the adenovirus vector reduced the number of invivo gene therapy fail. In order to achieve long duration, expressing the exacteffect of such effects, this experiment will have been adeno-virus bytransfection of293cells by propagation in293cells, which extract theconcentrated high-purity virus infectivity. Lay the foundation for the next andfuture research will LacZ gene into the AdV expression in the central andperipheral nervous system to promote peripheral nerve regeneration.Methods:1HEK293cell culture frozen cells quickly from-196°Cliquid nitrogen into a37℃water bath to melt into slow shaking vials, so thatthe extracellular ice crystals melt.3000r/min centrifuge3min to melt a goodcell suspension. Upper supernatant to absorb and put into10ml culturemedium to a centrifuge tube, gently wind and percussion into a suspensioninto the cell culture flasks. Containing cells and culture medium flasks in theincubator. Incubator environment close to the human environment is37℃and5%CO2. When cell growth is uniform, you can count the number of cellsin a certain volume of cell suspension. Then converted the amount of cells permilliliter of cell suspension containing the formula. Cell growth to90%of thetotal bottom area2-3times subculture. Cells were observed under a microscope, the selection of the full shape of the growth ability of cellsinoculated with the virus.2Virus transfected293cells into75cm2culture flasks adding10mlDMEM5%for culture. When the cells grew to60%-70%medium tossedaway, carefully add the virus mixture. Cross gently shaking the flasks to coverthe cell layer with the medium. Into the incubator for90minutes adding9mlDMEM5%, and again after72hours incubation, MOI determination todetermine the virus particles.3Adenovirus was amplified cells at-20°C to37°C were placed onhalf an hour, and repeated three times. Using the centrifuge tube aftercentrifugation, supernatants were collected frozen stored in-20°C or-80°C.Take three bottles of293cells develop ideal, drained inside the cell culturemedium,5ml mixture into each culture flask, and Gently mix. Into theincubator and incubated for about90minutes. By adding to the culture flasksDMEM, and cultured for about48-72hours. The same way again after thefirst-transfected cells in the supernatant of transfected cells. Will be entitled tore-amplification of the virus.4Virus purification centrifugal cells until the supernatant after theprecipitation of cell debris.5ml of PEG8000into the10ml supernatant andthe supernatant placed on ice one hour to precipitate. The above mixture,about20min, centrifuged at a speed of12,000rpm. Tossed away after thesupernatant, the sediment in CsCl solution. After5minutes of centrifugation,collect the virus suspension.550%tissue culture infective dose method for the determination of virusTake the293cells in a bottle, and2%FCS/in DMEM cell concentration wasadjusted to7.5×105/ml;100μl cell suspension into two96-well plates,respectively.10holes for a dilution, the two holes as a negative control; wereobserved after10days incubation, count each row of cytopathic phenomenon(CPE) the number of holes. And Karber equation to calculate the titer T=101+d (s-0.5)TCID50was measured AdLacZ virus solution:1010TCID50/ml. Results:1A normal dynamic HEK293cells were polygonal, adherentloose and between cells connected to each other into a mesh, and tend togather into a group.2Adenovirus containing Lacz gene transfected293cells cultured for24hours can be observed under an inverted microscope cell degeneration CPEphenomenon observed by X-gal staining cells appear blue dye, that is detected,the report gene lacZ expression, cells expressing adenovirus particles thatcarry the target gene, the control group did not find blue dye expression.Showed that transfection success.3293cells cultured in96-well plates, and counting CPE hole number forvirus infection of293cells, the virus titer. Finally acquire the virus titer1010TCID50/ml (1×109pfu/ml)Conclusions: Purified AdLacZ adenovirus, and by increasing the degree ofvirus droplets, and to lay the foundation for gene therapy of the mechanism ofperipheral nerve regeneration and nerve damage.50%tissue culture infectivedose method for the determination of virus titer and final titer of the virussolution1010TCID50/ml (1×109pfu/ml), opened the way for furtherexperiments.