丁型肝炎病毒内蒙古株的原核表达及抗原性分析

目的获得表达量高、抗原性强的基因工程重组抗原。检测不同地区的丁肝患者血清,初步验证其抗原性及在检测不同地区丁肝血清方面的差异。方法从内蒙古某丁肝患者血清中提取丁肝病毒,经RT-PCR与PCR,使用PET43a表达质粒及HDVL-Ag5′和3′端引入His标签获得丁型肝炎病毒L-Ag重组表达质粒,转化BL21(Rosetta)宿主菌,IPTG诱导表达。表达产物经饱和硫酸胺沉淀与亲和层析柱纯化后,采用EIA竞争法分析其抗原性。结果经与ABBOTT Murex anti-Delta(total)试剂盒同步检测15份阳性和10份阴性血清,一致率100%。结论EIA检测,证明具有良好的抗原性,基因工程表达的抗原蛋白在检测不同地区丁肝血清方面未见差异,故可用于丁肝的诊断及相关研究。

Expression of hepatitis delta antigen Inner Mongolian strain in prokaryotic cell and analysis of its antigenicity

OBJECTIVE: To obtain high yield and good antigenic activity of HDV L-Ag and to detect different regional patients' sera to test the purified antigen's antigenicity. METHODS: Hepatitis delta virus' sequence was obtained from Inner Mongolian patient by using RT-PCR and PCR methods, PET43a was used and His-tag was added at the HDV L-Ag 5' and 3' to construct the recombinant expression plasmid, transform the plasmid into host bacterium BL21 and induce it with IPTG. The expression supernatant was purified by saturated (NH4)2SO4 and affinity chromatography. The activity and antigenicity of the expressed product were analyzed by using EIA. RESULTS: Comparison of results obtained with detection by using the expressed protein coated plate and ABBOTT Murex anti-Delta (total) of 15 positive and 10 negative sera, the consistence was good (100%). CONCLUSION: EIA proved that the purified antigen had good antigenicity, no serological difference was found in detection between different region's sera, therefore the purified delta antigen may be useful in diagnostic and other research.