【作者】 杜谋选；

【导师】 姜晓丹；

【作者基本信息】 南方医科大学 ， 神经外科学， 2009， 硕士

【摘要】 中枢神经系统（Central Nervous System,CNS）损伤后的修复受多种因素影响,其中抑制因子释放和胶质瘢痕形成为主要原因之一。目前已经分离和鉴定的轴突生长抑制因子有:Nogo-A、髓磷脂相关糖蛋白（myelin-associatedglycoprotein,MAG）、少突胶质细胞髓磷脂糖蛋白（oligodendrocyte-myelinglycoprotein,OMgp）等。研究发现,Nogo-A、MAG、OMgp发生作用时都有LINGO-1蛋白的参与。在胶质瘢痕对轴突再生的影响中,硫酸软骨素蛋白多糖（Chondroitin Sulphate Proteoglycans,GSPGs）是目前已知的一个阻碍轴突再生的重要因子,特异存在于CNS,而Neurocan则为GSPGs的核心蛋白组分之一,并且显示了对视网膜神经节细胞神经突生长的抑制作用。腱糖蛋白-R（tenascin-R,TN-R）是瘢痕组织中所含有的另一种有轴突生长抑制作用的细胞外基质成分。把LINGO-1、Neurocan和TN-R设计成三基因联合DNA疫苗,将会使疫苗在体内发挥作用后、产生抗体并与相关抑制因子结合,以消除抑制作用、促进神经再生。本研究则主要是进行Neurocan基因的蛋白表达及其抗体制备,并且对其进行检测,探讨DNA疫苗制作前期技术方案的可行性,为后续CNS损伤修复实验工作奠定基础。目的制备Neurocan蛋白,用此蛋白免疫动物制备抗血清,并对抗血清的效价和特异性进行检测,最终使待参与DNA疫苗构成的Neurocan蛋白所产生抗体能与脑内创伤区的相应抑制性蛋白抗原结合。方法从基因库调出Neurocan的基因序列,合成Neurocan基因,序列起始端加His标签,两端设计酶切位点。构建原核表达质粒PET30a（+）-Neurocan,对合成的Neurocan基因进行测序鉴定。将质粒转化到大肠杆菌BL21感受态,挑菌培养,使用OMEGA公司质粒提取试剂盒(E.Z.N.A.^TMPlasmid Mini KitⅡ50)提取质粒。对质粒进行酶切鉴定和PCR鉴定,鉴定正确后,对菌株进行异丙基-β-D-硫代半乳糖苷（Isopropylβ-D-1- Thiogalactopyranoside,IPTG）诱导表达,采用不同的诱导时间和IPTG浓度,确定最佳表达条件。对目的蛋白进行纯化,使用PIERCE公司的BCA^TM Protein Assay Kit试剂盒测定蛋白浓度。对目的蛋白进行SDS-PAGE检测,观察目的蛋白的纯度和分子量大小;对目的蛋白进行Western blot鉴定,一抗使用小鼠抗His抗体,二抗使用马抗小鼠—AP抗体,显色底物为NBT/BCIP。Neurocan蛋白免疫兔制备免疫血清,分4次免疫,皮内注射。Elisa法检测抗血清效价,以Neurocan蛋白包被酶标板,兔免疫前血清为阴性对照,二抗为山羊抗兔-HRP,底物显色后酶标仪检测;Western blot检测抗血清特异性,以免疫血清为一抗,山羊抗兔-AP为二抗,NBT/BCIP显色。结果合成的Neurocan基因经测序鉴定,显示序列正确,且开放读码框正确。Neurocan基因经连接、转化、诱导表达后,测得蛋白浓度为0.673ug╱ul。目的蛋白经SDS-PAGE鉴定,目的条带分子量大小约为55kD;经Western blot鉴定,在55kD处有特异性条带,与SDS-PAGE鉴定结果一致,目的蛋白能与His抗体特异性结合。制备的抗血清经Elisa法检测,效价达到1:100万;经Westernblot检测,在55kD处的目的条带明显。讨论本研究中,首先对合成的Neurocan基因进行了测序鉴定,显示其序列正确,且开放读码框和酶切位点与本课题设计的一致。接着将包含Neurocan基因的质粒PET30a（+）-Neurocan转化到大肠杆菌BL21,进行Neurocan蛋白的原核表达,对表达出的蛋白进行SDS-PAGE鉴定,其分子量大小与通过碱基数计算出来的大小一致。进一步对其进行Western blot鉴定,因为本实验在Neurocan基因的起始端设计了His标签,故一抗用His抗体,结果显示目的蛋白能与His抗体特异性结合,说明该目的蛋白是Neurocan基因的表达产物,而Neurocan基因经过测序鉴定正确,提示Neurocan蛋白的原核表达成功。用此蛋白免疫兔制备的抗血清,经Elisa检测,效价达到1:100万;进一步用Western blot检测,都证明特异性良好,所制备的多克隆抗体能与Neurocan蛋白特异性结合。因此提示,Neurocan蛋白原核表达成功;用Neurocan蛋白免疫兔,产生的抗体能与Neurocan蛋白特异性结合,为DNA疫苗的进一步研制及CNS损伤后修复策略提供了有意义的参考依据。更多还原

【Abstract】 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.更多还原