重组灵芝免疫调节蛋白(rLZ-8)单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against recombinant ganoderma lucidum immunoregulatory protein( rLZ-8)

目的:制备重组灵芝免疫调节蛋白(rLZ-8)单克隆抗体,为rLZ-8的药效学及药代动力学研究奠定基础。方法:以纯度99%的rLZ-8免疫BALB/c小鼠;应用常规的细胞融合技术建立一种能够稳定分泌抗rLZ-8单克隆抗体的杂交瘤细胞株;采用小鼠腹腔接种法制备腹水;Protein A Sepharose亲和层析柱在AKTA纯化仪上对抗体进行纯化;间接ELISA方法测定单克隆抗体的效价;Western blot胶片曝光方法对rLZ-8单克隆抗体特异性进行分析。结果:筛选出2株可特异性分泌抗rLZ-8单克隆抗体的杂交瘤细胞株。分别命名为clone13-2-3和clone11-4-4。其抗体亚型分别为IgG1亚型和IgG2a亚型。腹水抗体效价分别为1∶12 000和1∶7 500,细胞培养上清效价分别为1∶3 000和1∶2 800。Western blot检测证明这两株杂交瘤细胞所分泌的单克隆抗体均具有良好的特异性。结论:成功制备出较高效价和较高特异性的鼠抗rLZ-8的单克隆抗体。

Objective：To prepare monoclonal antibody against recombinant ganoderma lucidum immunoregulatory protein （rLZ- 8）, which could pave a way for rLZ-8＇ s further research of pharmacodynamics and pharmacokinetics. Methods： BALB/c mouse were immunized With rLZ-8 of 99% fineness ;routine Cell Fusion Technique was used to devetop a hybridoma line producing antibodies a- gainst rLZ-8 ;antibody was isolated using Protein A Sepharose column on AKTA purifier;the titer of antibody was examined by indirect ELISA, specificity of mAb was analyzed by Western blot. Results：Two hybridomas lines that both secreted antibodies against rLZ-8 were screened out,named clone 13-2-3 and clone 14-4-4 ,their subtype of antibody were IgG1 and IgG2a ,the titer of antibodies purified from ascites were 1：13 000 and 1：7 500, the antibodies from Cell culture were 1：3 000 and 1：2 800 respectively. The results of Western blot indicated that the antibodies from two hybridomas lines both have good performance in examining specificity. Conclusion：The monoclonal antibody against rLZ-8 has been prepared successfully.