甘露聚糖结合凝集素相关丝氨酸蛋白酶2 EGF片段多克隆抗体的制备及鉴定  

Preparation and identification of polyclonal antibodies against EGF functional domains from mannan binding lectinassociated serine protease-2

作　　者：张培星 李涓[1] 窦倩倩 贾晓晖[2] ZHANG Pei-xing;Li Juan;DOU Qian-qian;JIA Xiao-hui(Department of Laboratory Diagnostics, Cangzhou Hospital of Traditional Chinese and Western Medicine, Cangzhou 061001, Hebei Province, China)

机构地区：[1]河北省沧州中西医结合医院实验诊断科,河北沧州061001 [2]河北北方学院医学检验学院病原生物与免疫学研究所免疫学教研室,河北张家口075000

出　　处：《微生物学免疫学进展》2018年第2期15-19,共5页Progress In Microbiology and Immunology

摘　　要：目的制备甘露聚糖结合凝集素相关丝氨酸蛋白酶2(MBL-associated serine protease-2,MASP-2)EGF功能区蛋白的多克隆抗体并进行鉴定,为后续研究奠定基础。方法利用带有EGF基因片段的p GEX-6P-2原核载体诱导表达GST-EGF融合蛋白,并采用商品化GST-Beads进行纯化;将纯化的融合蛋白作为抗原,与弗氏完全佐剂充分混合后,免疫5周龄BALB/c雌性健康小鼠,制备多克隆抗体;利用琼脂双扩散法检测多克隆抗体的效价,并进一步应用Western blot鉴定多克隆抗体的特异性及效价。结果成功表达并纯化EGF蛋白,以此成功制备出特异性强的GST-EGF融合蛋白的多克隆抗体,与其他蛋白无交叉反应;琼脂双扩散法检测的EGF抗体效价为1∶8;Western blot检测的EGF抗体效价大于1∶2 000。结论成功制备出具有特异性强且效价高的GST-EGF蛋白的多克隆抗体。Objective To prepare and identify polyclonal antibodies against Epidermal growth factor （EGF） functional domain from mannan-binding lectin associated serine protease-2 （MASP-2） , so as to provide a basis for a further investigation. Methods The pGEX-6P-2 prokaryotic vector carrying the target gene EGF was used to prepare the recombinant protein GST-EGF. The purified fusion protein was thoroughly mixed with Freund complete adjuvant, which was used to immunize the BALB/c mice （female, 5-week-old） and polyelonal antibodies were prepared, and a double diffusion and Western blot were applied to identify specificity and titers of the antibodies. Results The fusion proteins GST-EGF were successful- ly expressed and purified, by which the polyclonal antibodies were obtained from the immunized BALB/c mice.The polyclonal antibodies showed a high specificity no a cross-reaction with other proteins, with a titer of 1 ： 8 by double diffusion and 〉 1 ： 2 000 by Western blotting. Conclusion The polyclonal antibodies against GST-EGF fusion proteins were obtained, with high titers and strong specificity.

关 键 词：甘露聚糖结合凝集素相关丝氨酸蛋白酶2 表皮生长因子功能区 多克隆抗体 琼脂双扩散法 免疫印迹 

分 类 号：R392.11[医药卫生—免疫学]