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目的:探讨人细小病毒B19-XA株VP1蛋白在Bac-to-Bac杆状病毒表达系统中的表达及其反应原性分析。方法:以PCR法扩增VP1全长基因,并将其克隆到pFastBac1载体中,通过转化E.coliDH10Bac筛选阳性克隆,抽提重组VP1-Bacmid。以VP1-Bacmid经Cellfectin介导转染昆虫细胞Sf9,获取重组杆状病毒,扩增后感染Sf9细胞表达VP1蛋白,并以SDS-PAGE进行鉴定。以表达的重组VP1蛋白作为抗原,采用间接ELISA法检测B19病毒感染的患儿血清抗体,并与德国ELISA试剂盒检测的结果比较,分析表达产物的反应原性。结果:(1)获得含VP1全长基因的重组杆状病毒,转染Sf9细胞后能表达相对分子质量(Mr)约87000的VP1重组蛋白。(2)以表达的重组VP1蛋白作抗原,与B19病毒感染的患儿血清的反应,平均A值为0.46,P/N值>2.1,与德国ELISA试剂盒检测的结果(平均A值为0.68)相似。结论:人细小病毒B19-XA株VP1蛋白在Bac-to-Bac表达系统中可成功表达,且表达产物能有效地与抗B19病毒的阳性血清反应,具有良好的反应原性。
Objective: To investigate the expression of VP1 protein of human parvovirus B19-XA strain in Bac-to-Bac baculovirus expression system and its analysis of its antigenicity. Methods: The VP1 full-length gene was amplified by PCR and cloned into pFastBac1 vector. Positive clones were screened by transformation with E.coli DH10Bac and the recombinant VP1-Bacmid was extracted. VP1-Bacmid was transfected into insect cell Sf9 by Cellfectin to obtain recombinant baculovirus. The amplified VP1 protein was expressed in Sf9 cells and identified by SDS-PAGE. The expressed recombinant VP1 protein was used as an antigen, and the serum antibody of B19-infected children was detected by indirect ELISA. The results were compared with the results of the German ELISA kit, and the reaction product of the expressed product was analyzed. Results: (1) The recombinant baculovirus containing full-length VP1 gene was obtained and transfected into Sf9 cells to express VP1 recombinant protein with relative molecular mass (Mr) of about 87000. (2) The expressed recombinant VP1 antigen reacted with serum of B19-infected children with average A value of 0.46 and P / N value of> 2.1. The results of the ELISA test with German ELISA kit (mean A value 0.68 )similar. CONCLUSION: VP1 protein of human parvovirus B19-XA strain can be successfully expressed in Bac-to-Bac expression system. The expressed product can effectively react with the positive serum of anti-B19 virus and has good reactivity.