三株交叉反应性的流感病毒HA抗体与H1N1流感病毒的致病机制分析

目的:结合临床甲型流感病例分析流感病毒可能的致病机理。方法:收集87份陕西省2009年甲型H1N1流感重症,危重症及死亡病例的血常规参数,对其淋巴细胞、红细胞和血小板三个指标分析。制备针对甲型H1N1的单克隆抗体,采用抗体亚类鉴定试剂盒分析其抗体轻链和重链的亚型,通过血凝活性实验检测三株抗体的血凝抑制活性,通过ELISA检测三株抗体与人和小鼠的血红蛋白、红细胞、白细胞膜和血小板膜的反应,通过免疫组化分析三株流感病毒抗体与正常小鼠肺组织的结合。结果:流感病毒感染后的死亡病例中淋巴细胞、红细胞和血小板均明显降低。三株抗体与人和小鼠的淋巴细胞、红细胞和血小板均有不同程度的交叉反应;免疫组化结果同时也证实三株HA抗体与小鼠的肺组织有不同的结合力。结论:流感病毒致病的原因可能与流感病毒感染机体后产生的抗体可与血液和组织中的成分结合有关。

Pathogenic Mechanism of Three Cross Reactive Influenza Virus HA Antibodies and H1N1 Influenza Virus

ABSTRACT Objective: To analyze the possible pathogenesis of influenza virus based on clinical influenza A cases and three mAbs against H1N1-HA. Methods: The binding sites of the two antibodies with HA were analyzed by phage display library. Polypeptide synthesis based on the selected antigenic sites and HA sequence, monoclonal antibodies prepared by immunization mice with two polypeptide, light chain and heavy chain variable region of four antibodies were obtained by means of molecular cloning. Moreover, the binding domain of antibodies and antigen was predicted by computer simulation. Blood routine parameters of 87 severe, critical and fatal cases of influenza A (H1N1) in Shaanxi Province in 2009 were collected and their lymphocyte, erythrocyte and platelet indices were analyzed. Three mAbs against H1N1-HA were prepared by our lab, the subtypes of light and heavy chains of antibodies were analyzed by antibody subclass identification kit. The hemagglutination inhibition of the three antibodies was tested by hemagglutination activity test. The reactions of the three antibodies with human and mouse hemoglobin, erythrocyte, leukocyte and platelet membranes were detected by ELISA. The binding of three influenza virus antibodies to normal mice lung tissue was analyzed by immunohistochemistry. Results: The lymphocytes, red blood cells and platelets in the death cases were significantly decreased inafter influenza virus infection. ELISA results showed that the three antibodies had different cross-reactivity with human and mouse lymphocyte, erythrocyte and platelet. The three antibodies had different cross-reactivity with human and mouse lymphocytes, erythrocytes and platelets. The results of immunohistochemistry also confirmed that the three HA antibodies had different binding capacity with mouse lung tissue. Conclusion: The pathogenesis of influenza virus may be related to the combination of antibodies produced by influenza virus infection with blood and tissue components.