Gene Express And Antibody Preparation Of Neurocan Protein

The regermination of the Central Nervous Systema(CNS) after injured is influenced by many factors,among which,the releasing of inhibitor and gial scar formation are the main reasons.There are some of inhibitors to restrain the axon growth including Nogo-A,myelin-associated glycoprotein(MAG),and oligodendrocyte-myelin glycoprotein(OMgp).By study,it was found that the LINGO-1 protein participate in all of procedures during the action of Nogo-A, MAG,or OMgp.Among the influence of gial scar on the regeneration of axon, Chondroitin Sulphate Proteoglycans(GSPGs) is one of the important factors to obstruct the re-growth of axon,exists in the CNS specificly,in which the Neurocan is one of the core protein components.Moreover,the Neurocan showed the restrain effect on the neurite growth of retinal ganglion cells.Tenascin-R(TN-R) is another one of the inhibitors,and exists in the outside of cells in scar tissue,which can also restrain the growth of axon.The three-gene DNA vaccine with LINGO-1,Neurocan and TN-R,therefore,will play a neutralized role in the body,by the antibodies to combine with the inhibitors,and then eliminate the inhibitory effect,promote nerve regeneration finally.This study was mainly carried to make the Neurocan gene expression,to prepare its antibodies,and then to detect them,for exploring the feasibility of DNA vaccine and making the foundation as the pre-technology for it. ObjectSome of prophase works are offered by preparing Neurocan protein,antiserum, and assaying their characteristics,in order to construct the Neurocan-participated DNA vaccine,which can neutralize the inhibitors in the injured CNS following the immune administration and then promote the nerve regeneration.MethodsTo get the Gene order of Neurocan from the Gene bank,and then to synthetize Neurocan gene with His Tag label in beginning and enzyme-cut sites at amphi- of the sequence.The prokaryotic expression plasmid,PET30a-Neurocan,was constructed as usual,converted into the Escherichia coli BL21 and selected to culture,using the Plasmid Extraction Kit of OMEGA(E.Z.N.A.^TM Plasmid Mini KitⅡ50) to extract of plasmid.Then the plasmid was identified by Enzyme digestion and PCR.Following it,the Neurocan protein expression was induced by isopropy-β-D-thiogalactoside(IPTG), by different of induced time and concentrations to determine the best conditions of Neurocan protein expression.In order to purificate the object protein and to test its concentration,the BCA^TM Protein Assay Kit of PIERCE Company was used.SDS-PAGE was performed to detect the purification and molecular weight of the objective protein.Western blot was done to identified on objective protein,with mouse-anti-His as the first antibody,and Horse-antimouse labeling with alkaline phosphatase(AP) as the second one.Coloration was with NBT/BCIP method.Neurocan protein Immune serum was prepared in the rabbits by sub-4 of intradermal injection.ELISA was performed to assay titer of antiserum,during which the ELISA plates were Neurocan-coated and the rabbit pre-immune serum was as the negative control,Goat-anti-rabbit labeling with HRP as the second antibody,and the microplate reader was detected to identify the substrate.During the Western blot detection of the anti-serum,the immune serum as the first antibody,and the goat-anti-rabbit labeling with alkaline phosphatase(AP) was employed as the second one.Coloration was with NBT/BCIP method.ResultsThe correct sequence of the synthetic Neurocan gene was clearly showed with sequencing.After link,transfer and inducible expression,the protein concentration of the Neurocan gene were tested as 0.673ug/ul.The Neurocan protein expressed by prokaryotic showed its molecular weigh as 55kD following the SDS-PAGE identification,and it could specifically bind with anti-His Tag,which implied the interesting protein just as the expression product of Neurocan gene.The valency of antiserum was shown by ELISA as 1:1000000,the purpose strap of which was clearly confirmed by Western blot.DiscussIn this research,we carried out a sequencing detection on the synthesized Neurocan gene at first.It was showed that its sequence was correct,the open reading frame and restriction sites were just as same as the designed.Then we transformed the plasmid PET30a(+)-Neurocan,which contained the Neurocan gene,into the Escherichia coli BL21 in order to make prokaryotic expression of Neurocan protein. SDS-PAGE identification on the expressed protein was performed,which showed the molecular weight as same as the one that was calculated out by bases number. Further to take Western blot identification whih His antibody as the first,because the His tags was designed in the start of Neurocan gene.The results showed the object protein could combine with His antibody's specificly.It implied that the object protein was just as the expression product of Neurocan gene.The Neurocan gene was showed correct by sequencing,which implied that the Prokaryotic express of Neurocan protein was successful.By this protein to immune rabbit for preparing antiserum,the titer could reach 1:1000000 by ELISA detection.Further detection by Western blot,it was proved as a good specificity,and the prepared polyclonal antibody could well bind with the Neurocan protein specificly.It was prompted, therefore,the Neurocan protein prokaryotic expression was successful.The antibody produced by Neurocan protein-immuning rabbit could bind with Neurocan protein specificly.It provided a meaningful reference frame for the further development of DNA vaccines.