Neurocan基因蛋白表达及其抗体制备与检测的实验研究

发布时间：2018-06-14 20:55

本文选题：Neurocan基因 + 原核表达　；参考：《南方医科大学》2009年硕士论文

【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.~(TM)Plasmid 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%qul。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。
[Abstract]:The repair of Central Nervous System (CNS) after injury is affected by a variety of factors, in which inhibition factor release and glial scar form become one of the main reasons. There are currently isolated and identified axon growth inhibitors: Nogo-A, myelin related glycoprotein (myelin-associatedglycoprotein, MAG), oligodendrocyte pulp Oligodendrocyte-myelinglycoprotein (OMgp) and so on. Studies have found that Nogo-A, MAG, and OMgp are involved in the participation of LINGO-1 protein. In the effect of glial scar on axon regeneration, the chondroitin sulfate proteoglycan (Chondroitin Sulphate Proteoglycans, GSPGs) is an important factor that is known to impede axonal regeneration. It is specific to CNS, and Neurocan is one of the core protein components of GSPGs and shows the inhibitory effect on the growth of the retinal ganglion cells. -R (tenascin-R, TN-R) is another extracellular matrix component of the inhibition of axon growth in scar tissue. LINGO-1, Neurocan and TN-R are set up. The combination of the three gene DNA vaccine will enable the vaccine to play a role in the body, produce antibodies and combine with related inhibitory factors to eliminate inhibition and promote nerve regeneration. This study is mainly about the protein expression and antibody preparation of the Neurocan gene, and the detection of it and the preliminary technical scheme for the preparation of the DNA vaccine. The feasibility lays the foundation for subsequent CNS damage repair experiments.
objective
The Neurocan protein was prepared, the antiserum was prepared by this protein, and the titer and specificity of the serum were detected. Finally, the antibody produced by the Neurocan protein, which is made up of the DNA vaccine, could be combined with the corresponding inhibitory protein antigen in the brain wound area.
Method
The gene sequence of Neurocan was transferred from the gene pool, the Neurocan gene was synthesized, the initial end of the sequence was added with the His label, the enzyme cutting site was designed at both ends. The prokaryotic expression plasmid PET30a (+) -Neurocan was constructed, and the synthesized Neurocan gene was sequenced and identified. The plasmid was transformed into the BL21 sense of Escherichia coli, picked up the bacteria and used the OMEGA company plasmid to extract the kit. The plasmid was extracted (E.Z.N.A.~ (TM) Plasmid Mini Kit II 50). The plasmid was identified by enzyme digestion and PCR identification. After the identification was correct, the strain was induced by isopropyl - beta -D- Thioglucoside (Isopropyl beta -D-1- Thiogalactopyranoside, IPTG). The optimal expression conditions were determined by different inducible time and IPTG concentration. Purified, the protein concentration was measured by PIERCE's BCA~ (TM) Protein Assay Kit kit. The pureness and molecular weight of the target protein were detected by SDS-PAGE and the purity and molecular weight of the target protein were observed. The target protein was identified with Western blot, the first anti His antibody was used, the two anti horse anti mouse mouse AP antibody was used, and the chromogenic substrate was NBT/BCIP.Neur. Ocan protein immunized rabbits were immunized to prepare immunized serum, 4 times of immunization, intradermal injection of.Elisa to detect antiserum titer, Neurocan protein was coated with enzyme labeled plate, rabbit pre immunized serum was negative control, two anti Rabbit Rabbit anti rabbit -HRP, the substrate chromogenic enzyme labeling instrument was detected, Western blot was used to detect anti serum specificity, immune serum was one anti, Goat anti rabbit -AP was Two anti, NBT/BCIP color.
Result
The synthesized Neurocan gene was sequenced and identified, and the sequence was correct, and the correct.Neurocan gene of the open reading frame was connected, transformed and induced, and the protein concentration was identified as 0.673ug%qul. by SDS-PAGE. The molecular weight of the target band was about 55kD; the specific bands at 55kD were identified by Western blot and identified with SDS-PAGE. The result is consistent. The target protein can be specifically combined with His antibody. The antiserum prepared by Elisa is detected by Elisa method, the titer reaches 1:100 million, and the target strip in 55kD is obvious by Westernblot detection.
discuss
In this study, we first sequenced the synthesized Neurocan gene, showing that the sequence was correct, and the open reading frame and the enzyme cutting site were consistent with the design. Then, the plasmid PET30a (+) -Neurocan containing the Neurocan gene was transformed into the Escherichia coli BL21, the expression of the Neurocan protein was expressed, and the expressed protein was SDS- PAGE identification, the size of its molecular weight is in accordance with the size calculated by the base number of the base. Further Western blot identification is carried out, because the experiment has designed the His tag at the beginning of the Neurocan gene, so the anti His antibody is used. The result shows that the target protein can specifically bind to the His antibody, indicating that the target protein is the table of the Neurocan gene. The Neurocan gene was correctly identified by sequencing, suggesting that the prokaryotic expression of Neurocan protein was successful. The antiserum prepared by this protein was detected by Elisa, and the potency reached 1:100 million. Further detection by Western blot proved that the specificity was good and the prepared polyclonal antibody could be specifically combined with the Neurocan protein. Therefore, the antibody could be specifically combined with the Neurocan protein. The prokaryotic expression of Neurocan protein is successful, and the antibody produced by Neurocan protein can specifically bind to the Neurocan protein. It provides a useful reference for the further development of the DNA vaccine and the strategy for the repair of CNS after the injury.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R651.3;R392

【参考文献】