fgl2凝血酶原酶多肽抗体的制备和应用

Preparation and application of fgl2 prothrombinase peptide antibody

目的 利用人工合成多肽制备针对人源、鼠源fgl2凝血酶原酶 (hfgl2、mfgl2 )的特异性多克隆抗体 ,以期用于与fgl2表达异常密切相关的疾病的研究和诊治。方法 根据mfgl2、hfgl2基因编码的氨基酸序列合成多肽 3(Pep3)和多肽 4 (Pep4 ) ,并用化学方法与匙孔血蓝蛋白 (KLH)连接 ,纯品Pep3 KLH和Pep4 KLH免疫动物 ,所制备的抗血清用ELISA、Westernblot和前凝血质活性 (PCA)实验鉴定 ,并采用免疫组化法应用于乙型肝炎患者肝组织中hfgl2的检测。结果 ELISA检测表明 ,所制备的抗血清可分别与Pep3和Pep4发生特异性免疫反应 ;Westernblot结果显示 ,抗血清可识别MHV 3感染BALB cJ小鼠腹腔巨噬细胞特异性条带 ,相对分子质量 (Mr)为 6 5× 10 3;PCA实验提示 ,抗血清具有中和mfgl2分子酶活性的作用。对病毒性乙型肝炎患者肝组织免疫组织化学染色发现hfgl2仅在重型乙型肝炎患者高表达 ,而在慢性乙型肝炎和肝硬化患者的肝组织中无表达。结论 所制备的fgl2的多肽抗体 (多克隆抗体 )可识别mfgl2和hfgl2分子 ,具有与抗原特异性结合、中和其酶活性的特征 ,为深入研究这一新发现的分子提供了有力的科学手段。

ObjectiveTo prepare a peptide polyclonal antibody against both murine and human fgl2 prothrombinase/fibroleukin based on the chemically synthesized peptide 3 (Pep3) and peptide 4 (Pep4) from exon 1 and 2 of the predicted amino acid sequence of mfgl2 protein separately. MethodsThe Pep3 and Pep4 were coupled to keyhole limpet hemocyanin (KLH) to form a complex of Pep3-KLH and Pep4-KLH. The anti sera were developed by immunizing rabbit with Pep3-KLH or Pep4-KLH after emulsified in Freund′s complete adjuvant (FCA). The purified anti sera were also utilized to detect the hfgl2 expression in the liver tissue of hepatitis B patients. ResultsThe anti sera react with Pep3 and Pep4,and the titer around 1∶320 000 (Pep3) and 1∶160 000 (Pep4) respectively. Western blot displayed a specifically M_r (molecular mass relatively) 65 000 reactive band in murine hepatitis virus strain 3 infected macrophages from BALB/cJ mice when incubated with anti sera. PCA assay showed that the anti sera inhibited functional activity of mfgl2 prothrombinase expressed on the MHV-3 infected BALB/cJ macrophages in a dose-dependent fashion. The purified anti sera also recognized hfgl2 molecule highly expressed in liver tissue from patients with active acute on chronic hepatitis B. ConclusionPeptide polyclonal antibody against fgl2 prothrombinase was successfully raised with expected specificity,titers and neutralization activity. This study provided an important tool to explore the roles of this newly discovered molecule from human disease and animal model.