| BackgroundAdeno-asscociated virus is a novel gene vector which could mediate a long term expression of target gene, and has the advantages of high safety factor, good stability,low pathogenic rate, low immunogenicity as well as a wide host range. One of this virus, adeno-asscociated virus 9 could target the myocardial cells and glial cells, as well as penetrate the blood brain barrier, is regarded as a good prospect in clinical application. Because of the wide use, the purification in large scale has become the urgent problem to be solved. The traditional purification method is time-consuming and laborious as well as security risk. In view of this, it is very important to select a kind of purification method with strong specificity and stable operation, which is suitable for the mass production of AAV9.The affinity chromatography is a separation method of virus particles, specific protein and DNA mixture, mainly relied on the reversible binding between the surface of the virus capsid and the bioactive ligands as well as receptors of chromatographic.With good selectivity, high resolution, good combination between virus particles and matrix and the high recovery rate, the AAV vectors are utilized popularly in many fields. In recent years, affinity chromatography has also been used in the field of AAV purification, such as antibody specific affinity and receptor specific affinity. Recent studies have found that AAV9 can specifically bind to the end of beta-1, 4 galactose receptor, which provides a possibility for AAV9 receptor affinity chromatography purification.ObjectiveThe aim of this paper is to screen the specific binding substance of AAV9, and establish a receptor specific affinity purification method of AAV9 and make a preparation for the establishment of AAV9 affinity purification system.Methods(1) Screen and verify the candidate ligand molecules from the literature data;(2) Use the physical experiments, cell inhibition test and bioinformaticsscreening method to verify the specific combination between the candidate ligands and AAV9;(3) Construct the ligand affinity purification column, and use the pure virus to explore the proper balance buffer solution, elution buffer solution and pH for the optimization of purification column efficiency;(4) Using the silver staining, Western Blot and Real-time PCR to detect the purity, genome specificity and titer of virus in the purification of virus production solution.Results(1) Reference the literature and verify 8 candidate ligands: D-galactosamine,N-acetyl-D-galactosamine, N-acetyl-D-lactose, lactose acid amine, galactinol,D-galactose, D-galacturonic acid, galactooligosaccharides, respectively;(2) Use physical experiments, cell inhibition test and bioinformatics method to screen the binding ligand with the AAV9, D-galactose, lactose, D-galacturonic acid,respectively.(3) Successfully construct the D-galactose, lactose acid and D- galacturonic acid purification column, and get the proper buffer and pH condition---10 mM Tris-HCl+4mM CaCl2, pH=8 as the balace buffer, and use the 20 mM NaCl as the starting elution buffer.(4) Use the D-galactose affinity purification column to purify the virus production liquid, and verify the specificity and purity of virus.ConclusionSuccessfully construct the ligand affinity purification column with D-galactose,D-galacturonic acid, lactobionic acid, which could make an early preparation for the affinity purification system of AAV9; but the purification column and AAV9 affinity purification efficiency is not high, need to be further optimized. |
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