仙台病毒抗原的制备及初步应用

目的纯化和制备仙台病毒(Sendai Virus,SeV)抗原,初步应用于间接酶联免疫吸附试验(ELISA)及免疫印迹(Western-blot)试验对SeV抗体进行检测。方法通过鸡胚尿囊腔及BHK-21细胞增殖法培养增殖SeV,低温冻融去除细胞碎片后,用差速离心及超声破碎法纯化并制备SeV抗原,以SDS-PAGE电泳进行纯度鉴定;优化确定间接SeV-ELISA抗原的最佳包被浓度,对比检测间接免疫荧光试验(IFA)初筛小鼠SeV阳性血清,同时用Western-blot检测法进行特异性验证。结果用BHK-21细胞培养增殖的SeV,病毒滴度HA为1∶128,SeV抗原的电泳纯度达80%;确定SeV-ELISA抗原最佳包被浓度为2.5μg/mL时,测得小鼠SeV抗体ELISA与IFA的阳性符合率为100%,并由Western-blot检测法得到了证实。结论通过BHK-21细胞增殖和差速离心及超声破碎法制备的SeV抗原,可用于ELISA及Western-blot法对小鼠SeV抗体的检测,进而验证了SeV具有较强的抗原特异性及蛋白活性。

Preparation and Preliminary Application of Sendai Virus Antigen

Objective To prepare and purify the murine Sendai virus （SeV） antigen so as to establish the indirect ELISA and Western blot test for detecting the antibody against SeV. Methods SeV was inoculate into the allantoic cavity of SPFchick embryo, and then the allantoic fluid was added to the BHK-21 cell culture to proliferate SeV. Sendai virus antigen was prepared and purified by the differential centrifugation and uhrasonication, and its purity was identified by SDS-PAGE. The best coating antigen concentration for the indirect SeV-ELISA was selected and confirmed. SeV antibody in murine serum was screened by the indirect immunofluorescence assay （IFA） , and the SeV-ELISA specificity was verified by Western-blot test. Results The HA titer of Sendai Virus from BHK-21 cell culture was 1： 128, and SeV antigen electrophoretic purity reached to 80 percent. While the best coating antigen concentration for the indirect SeV-ELISA was assigned to 2.5 p~g/mL, the detection rate of positive Sev antibody in murine serum by the SeV-ELISA was absolutely compatible with that by IFA. Conclusions The SeV antigen prepared by differential centrifugation and sonication broken method can be used for detection of Sendai Virus antibody in mice, and the antigen-specific and activity of Sendai Virus have been verified by the Western-blot test.