Preliminary Study On AAV Affinity Purification Method Based On Yeast Surface Display AAVR

Research backgroundAAV vector has the advantages of long expression time in vivo,wide host range,low immunogenicity and high safety.It is one of the most promising gene vectors in gene therapy research.Although the first gene drug Glybera has been approved for sale in the European Union,however,there still exist some problems such as complex purification process,low purification efficiency and high cost of AAV virus vector.Affinity purification is an ideal purification method for AAV virus,which has the advantages of high selectivity,high adsorption rate and high recovery.Affinity purification of AAV is generally based on the specific receptor of AAV.Because the receptor previously found only adsorbs a serotype AAV generally,the purification process is more complicated and cost.A new receptor was discovered recently named adeno-associated virus receptor-AAVR,which is the adeno-associated virus receptor of several serotypes AAV.Making AAVR as the ligand of affinity purification column is expected to solve the problem a the receptor affinity purification column only can purified single serotype AAV.Yeast surface display technology is a technology that can transfer the target protein by yeast cell wall protein expression and transport mechanism,and immobilized the target protein on the outer surface of yeast cells.When AAVR is displayed on the yeast surface,the yeast with AAVR on the yeast surface purified AAVR instead of the matrix with ligand used to purify the virus.It could reduce the contamination caused by chemical licrosslinking the gand and matrix,and avoid the low rate of crosslinking the gand and matrix.ObjectiveThe affinity purification system of AAVR on yeast surface was constructed.It is a preliminary study to solve the problem that the affinity column base on the AAV receptor is too simplistic,to tack the low efficiency of ligand and matrix chemical crosslinking and the hardly pollution and the much cost.Method(1)The gene sequences of the AAVR domain were obtained from the literature,and the structural analysis was carried out.(2)Gene fragment extraction of AAVR by primers.(3)Construction of a surface plasmids containing AAVR gene fragments.(4)The yeast transfer,screening and expression of AAVR were displayed on the surface.(5)Detection of AAVR and the effect of virus adsorption.(6)Purification and elution effect of yeast on the surface of AAVR.(7)Optimal purification effect of salt ion concentration and pH value for regulating eluent.Results(1)By consulting literatures,we find that the effective domain of AAVR is PKD(polycystic kidney disease)1-5,and carry out structural analysis to remove the sequence of transmembrane and peptide regions,and finally select 311-788 amino residues.(2)According to the 311-788 amino residues corresponding to DNA sequence(PKD),primers were designed,and all RNA was extracted from L02 cells(human normal hepatocytes),and the corresponding DNA sequences of 311-788 amino residues were extracted by PCR using designed primers.(3)The PKD gene sequence was inserted into the surface plasmids PYD1,and the pkd-PYD1 fusion surface plasmids were constructed.(4)The PKD-PYD1 fusion surface plasmids were successfully introduced into the surface of yeast EBY100 through electrical transformation.(5)The influence factors and elution conditions of yeast adsorbed AAV on the surface of AAVR were preliminarily explored.Conclusion:The researcher constructed the yeast surface display AAVR successfully,which provides a theoretical base for the exploration with the next step purification process,after preliminary experiments,found on the purification with low efficiency,it needs to be further improved on the process of elution.