神经纤毛蛋白-1的原核表达及兔抗小鼠神经纤毛蛋白-1抗体免疫学活性的研究

目的:在原核系统中分段表达神经纤毛蛋白-1(Nrp1);将纯化的蛋白免疫家兔后获得特异的抗体,并将其应用于检测组织和细胞中Nrp1分子的表达。方法:提取BALB/c胎鼠脑组织总RNA,通过RT-PCR分段扩增获得Nrp1基因片段,将PCR产物插入表达载体pET28a( ),获得含5个Nrp1基因片段的重组质粒,在大肠杆菌BL21(DE3)中诱导蛋白表达并纯化;将纯化的重组蛋白免疫新西兰大白兔获得针对目的蛋白的特异性多克隆抗体;利用Nrp1特异性多抗检测胎鼠脑组织和HeLa细胞中Nrp1的表达。结果:在原核系统中分段表达了Nrp1蛋白,通过Ni-NTA纯化了Nrp1蛋白片段;纯化的Nrp1蛋白免疫新西兰大白兔获得了具有免疫活性的多抗;兔抗小鼠Nrp1特异性多抗可用于检测组织、真核细胞中Nrp1的表达。结论:应用原核系统成功地表达了Nrp1蛋白,兔抗小鼠Nrp1特异性多抗可用于免疫学检测Nrp1分子的表达。

Procaryotic Expression of Neuropilin-1 Fragments and Study on the Immunological Characteristics of Rabbit-Anti-Mouse Neuropilin-1 Antibody

Objective:To express neuropilin-1(Nrp1)in prokaryotic system fragmentally and to identify the expression of Nrp1 in tissue and in cells with rabbit-anti-mouse Nrp1 antibodies which were gained from New Zealand rabbits immunized with the purified Nrp1 fragments.Methods:Brain tissue of fetus BALB/c mice were used for RNA extraction,and gene fragments of Nrp1 were obtained successfully by RT-PCR,recombinant plasmids were constructed and then E.coli BL21(DE3)strain were transformed with these Nrp1 gene fragments-contained plasmids and induced for protein expression,all the five fragments were purified by metal chelation affinity chromatography with purity over 80%.Purified recombinant protein fragments were used subsequently as immunogens for immunizing New Zealand rabbits subcutaneously for three times continuously to gain specific antibody;the immunological characteristics of rabbit-anti-mouse Nrp1 antibodies were studied by ELISA and Western blot.Results:It showed that Nrp1 protein fragments were high-level expressed in E.coli and could be purified by Ni-NTA affinity chromatography.The purified proteins were immunogenic and they could induce high-titer antibodies in rabbits.Anti-Nrp1 multi-clone antibodies could be used for the identification of surface molecule Nrp1 by Western-blot assay in brain tissue of fetus BALB/C mice and in HeLa cells.Conclusion:Nrp1 fragments were expressed in procaryotic system and were purified successfully,rabbit anti-mouse Nrp1 multi-clone antibodies were obtained and could be used in immunological assay.