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Neurocan基因蛋白表达及其抗体制备与检测的实验研究

ISSN号：1671-8925
期刊名称：《中华神经医学杂志》
时间：0
分类：Q78[生物学—分子生物学]
作者机构：[1]南方医科大学珠江医院神经外科,广东神经外科研究所,广州510282, [2]北京军区总医院神经外科,北京100700
相关基金：国家基金重点项目（U0632008）;广东省科技计划重点项目[粤财教（2008）258-2008A030201019,粤科基办（2004）08、（2007）05/06-7005206]

作者：杜谋选[1], 姜晓丹[1], 徐如祥[1,2], 陈镇洲[1], 吕俊[1]

关键词： Neurocan基因, 原核表达, 抗血清, Neurocan gene, Prokaryotic expression, Antiserum

中文摘要：

目的制备Neurocan蛋白，用此蛋白免疫动物制备抗血清，并对抗血清进行检测，最终使待参与DNA疫苗构成的Neurocan蛋白所产生的抗体能与脑内创伤区的相应抑制性蛋白抗原结合，从而减轻抑制因子对神经生长的抑制作用，促进神经再生。方法合成Neurocan基因，构建原核表达质粒PET30a-Neurocan，转化大肠杆菌BL21，异丙基-β—D-硫代半乳糖苷（IPTG）诱导表达目的蛋白，并经SDS—PAGE、Western blot鉴定。Neurocan蛋白免疫兔制备免疫血清。ELISA法检测抗血清效价，以兔免疫前血清为阴性对照：Western blot检测抗血清特异性，以免疫血清为-抗，山羊抗兔-AP为二抗，NBT／BCIP显色。结果合成的Areurocart基因经酶切鉴定、PCR鉴定及测序鉴定，显示序列正确。原核表达Neurocan蛋白经过SDS-PAGE鉴定，条带大小约为55000，与计算出来的大小一致；经Westem blot鉴定，目的蛋白能与his抗体特异性结合，说明是Neurocan基因的表达产物。抗血清经ELISA法检测，抗血清效价达到1：100万；经Western blot检测，目的条带明显。结论Neurocan蛋白原核表达成功；通过Neurocan蛋白免疫兔可得到特异性抗体，此抗体能与Neurocan蛋白特异性结合，为DNA疫苗的进一步研制奠定了基础。

英文摘要：

Objective To offered some prophase works by preparing Neurocan protein, antiserum, and assaying their characteristics, in order to construct the isopropy-β-D-thiogalaetoside （IPTG）-participated DNA vaccine, which can neutralize the inhibitors in the injured CNS following the immune administration and then promote the nerve regeneration. Methods Neurocan gene was synthetized with HisTag 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, and induced by IPTG to express positively. The interest protein was identified by SDS-PAGE and Western blot respectively. The preimmune serum was as the negative control during the ELISA assay of antiserum valency. The immune serum was 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. Results The correct sequence of the synthetic Neurocan gene was clearly showed by identification with enzyme-cut, PCR and sequencing. The Neurocan protein expressed by prokaryotic showed its molecular weigh as 55 000 following the SDS-PAGE identification, and it could specifically bind with anti-HisTag, which implied the interesting protein just as the expression product of Neurocan gene. The valency of antiserum was shown by ELISA as 1 ： 1 000 000, the purpose strap of which was confirmed by Western blot. Conclusions Neurocan protein could be successfully expressed in prokaryotic, the antibody of which could be specifically obtained by immune administration to the rabbit. The Neuroncan antibody could bind with the Neurocan protein specifically.

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