汉坦病毒重组核蛋白抗体用于鼠肺组织病毒抗原的检测

Detection of Hantavirus antigen in murine lungs by antibodies anginst recombinant Hantavirus nucleocapsid protein

目的纯化汉坦病毒(HV)SEO型L99株重组核蛋白(rNP),制备抗体用于鼠肺组织HV抗原的检测。方法重组菌株E.coliBL21(DE3)/pET32a-L99S经IPTG诱导表达rNP,采用Ni亲和层析方法纯化,纯化蛋白免疫日本大耳白兔,获取抗血清用于间接免疫荧光法(IFA)检测鼠肺组织的HV抗原;并与病人混合血清作为一抗的IFA结果进行比较,部分标本进一步用RT-PCR法进行验证。结果rNP表达量占菌体总蛋白的52.2%,纯化蛋白的总回收率为22.9%;纯化蛋白Western blot分析为单带,纯度达95%以上,用其制备多克隆抗体滴度达1∶512000。IFA检测59份鼠肺标本,用兔rNP抗体和病人混合血清作为一抗其阳性率分别为23.7%和16.9%;两者结果不一致的6份鼠肺经RT-PCR检测HV核酸,均与兔rNP抗体的IFA结果相一致。结论采用rNP抗体进行IFA检测,可显著地提高鼠类HV感染监测的准确性,值得推广使用。

To purify recombinant nucleocapsid protein（rNP） of SEO Hantavirus strain L99 and to prepare antibodies for the detection of Hantavirus antigen in murine lungs, recombinant strain E. coli BL21 （DE3）/pET32a-L99S was induced by IPTG,and then the rNP was purified by Ni-affinity chromatography. Polyclonal antisera prepared by immunizing rabbits with rNP were employed to detect Hantavirus antigen in murine lungs through indirect fluorescence assay （IFA）. The results were compared with IFA using mixed patient antisera and confirmed by RT-PCR. It was found that the expression of rNP accounted for 52.2% of the total bacterial protein and the recovery rate was 22.9% after purification. The purity of rNP was up to 95% and Western blot showed a single band. The titer of polyclonal antisera prepared by immunizing rabbits with rNP was 1 ： 512000. 59 murine lung samples were detected for Hantavirus antigen by IFA and the positive detection rate was 23.7% using rabbit antisera against rNP as the first antibody and 16.9 % using mixed patient antisera as the first antibody. RT-PCR showed all of the six inconsistent samples in the two IFAs corresponded to the results of IFA using rabbit antisera against rNP. It is concluded that IFA using anti- rNP antibodies could improve the correction of monitoring Hantavirus infection in mice, and this method is worth of adopting widely.