脑心肌炎病毒鉴定方法的初步研究

Preliminary study on the identification methods for encephalomyocarditis virus

目的摸索建立脑心肌炎病毒（EMCV）鉴定方法。方法根据GenBank中EMCVVR．129B序列设计引物，提取病毒RNA，进行RT．PCR，琼脂糖凝胶电泳检测片段大小。将DNA测序结果与GenBank中相同毒株EMCV序列进行比对。以RK细胞培养的全病毒作为免疫原制备抗血清，用于建立间接免疫荧光试验（IIFA）及中和试验方法，并验证方法精密性和特异性。制备抗GST—VPl和抗GST．VP2多克隆抗体，与EMCV浓缩液进行免疫印迹反应。结果获得的DNA片段与GenBank中上传的相同毒株EMCV序列进行比对，相似性为98％-100％。采用20100901批抗血清160倍稀释作为一抗，建立IIFA方法，特异性好。检测20100901批抗血清中和效价1：30211，建立的固定病毒．稀释血清中和试验方法特异性、精密性好。抗GST—VPl和GST—VP2抗体与EMCV浓缩液于相对分子质量为33x103左右分别出现清晰单一条带，与理论值相符。结论初步建立了用于EMCV毒株鉴定的RT-PCR、间接免疫荧光、中和试验及免疫印迹方法。

Objective To explore and develop methods for eneephalomyocarditis virus （EMCV） identification. Methods According to the genetic sequence VR-129B of EMCV recorded in the GenBank, five gene fragments were selected to design primer sequence pairs. RNA was extracted to run RT-PCR, and then the products of amplification were identified by agarose gel electrophoresis. The results of DNA se- quences were compared with the sequences in GenBank of the same EMCV strains. Antiserum was prepared based on the EMCV cultured in RK cells for establishing indirect immunofluorescence assay （IIFA） and neutralization test method, and verification for precision and specificity of the two methods were carried out after it. Antiserum that was prepared with GST-VP1 and GST-VP2 expressed in E. coli was reacted with the purified EMCV in Western blot test. Results By sequencing and comparing, the similarity of DNA frag- ments between the obtained and the GenBank recorded was reached 98% to 100%. The antiserum of No. 20100901 batch that was chosen as the first antibody at a dilution of 1 ： 160 to develop IIFA brought about a better specificity. The neutralization titers of 20100901 batch antiserum was 1 ： 30 211 measured by fixing virus and diluting serum method, which showed good specificity and precision. The results of the Western blot test showed two clear bands above and under 33 103 respectively, which matched the theoretical value. Conclusion The RT-PCR, indirect immunofluorescence, neutralization test and Western blot method for EMCV strains identification were established initially.