丁型肝炎病毒重组抗原的制备及应用

Establishment and application of recombinant hepatitis delta antigen

目的 :获得能够应用于丁型肝炎病毒 (HDV)酶免试剂的重组丁型肝炎病毒抗原。方法 :采用RT PCR自HDV感染急性期的土拨鼠肝脏扩增编码丁型肝炎病毒抗原的HDVcDNA ,插入质粒pET 3α ,转化大肠杆菌BL2 1,制备丁型肝炎病毒抗原表达菌株 ;对其表达蛋白的特异性、灵敏度、稳定性及活性进行鉴定 ;并将以该重组蛋白为主要原料制备的抗 HD酶免试剂与爱尔兰Noctech抗 HD试剂盒进行比较。结果 :重组质粒PED SHDAg和PED LHDAg经异丙基 硫代 β D 半乳糖苷 (IPTG)诱导表达的 2种蛋白经SDS PAGE电泳后 ,WesternBlot检测显示均与抗 HD发生强烈反应。该表达蛋白对抗 HD阳性血清抑制率为 86 % ,37℃保存 7d稳定性良好 ,比活性为 1:80 0 0 ,检测抗 HD灵敏度与国外同类试剂盒结果一致。结论 :该重组丁型肝炎病毒抗原可用于抗 HD酶免试剂的制备。

Aim: To obtain recombinant hepatitis delta virus antigen and investigate its application to diagnosis to serum antibody to HDV. Methods: The gene encoding HDAg was amplified from woodchuck liver infected HDV by RT-PCR technique. The PCR products were inserted into expression vector pET-3α. The recombinant vectors were cloned into E. coli BL 21 , where HDAg was effectively expressed. The expression products were analyzed by SDS-PAGE method and Western Blot assay. Both specificity and sensitivity were identified. Finally, the EIA diagnostic reagent, of which rHDAg was the major ingredient, was compared with a commercially available EIA kit. Results:Two rHDAg proteins were expressed by the recombinant vector PED-SHDAg and PED-LHDAg induced by IPTG. They particularly reacted with antibody to HDV by SDS-PAGE method and Western Blot. The rHDAg protein kept steady under 37℃ for 7 days and its inhibitory rate was 86% in neutralization inhibitory test. Its activity was 1:8 000 ELISA titre per mg protein and its sensitivity was identical with Notech anti-HD EIA kit. Conclusion: The expression vector containing HDV cDNA has been constructed successfully; the recombinant HDAg could replace antigen by infected liver tissue as a diagnostic reagent used to configure an EIA for detection of anti-HD.