猪传染性胃肠炎病毒纯化方法的选择与优化

Optimization of Porcine Transmissible Gastroenteritis Virus Purification

利用超速离心、饱和硫酸铵、聚乙二醇-6000(PEG-6000)对猪传染性胃肠炎细胞毒进行纯化,收集纯化病毒测定蛋白含量,分别运用PCR方法和作为ELISA包被抗原方法对纯化的效果进行检测,并对最佳纯化方法进行优化。3种纯化方法获得的蛋白浓度分别为1.875、6.435、0.763 mg/mL。PCR检测结果显示PEG-6000纯化后在上清液中未检出病毒,其余2种上清中均检出病毒。3种纯化方法制备的包被抗原ELISA试验阴阳性比值(P/N)分别为2.21、1.27、2.25,最终选择PEG-6000作为纯化病毒的方法,其最佳优化参数为PEG-6000浓度8%、浓缩时间2 h、NaCl浓度1%。研究表明利用PEG-6000纯化TGEV是一种经济实用、操作简单、并有较高纯化效率的方法,纯化后的TGEV为后续建立特异、敏感、快速的ELISA检测方法奠定了良好基础。

Ultra-speed centrifugation,saturated ammonium sulphate and PEG-6000 were respectively used to purify and recover porcine transmissible gastroenteritis virus(TGEV) for quantification.The purification efficiency was tested by PCR and coated antigen for ELISA evaluation.The final concentration of protein in TGEV purified by above 3 ways was 1.875,6.435 and 0.763 mg/mL separately.There was no virus RNA by PCR detection in the supernatant after PEG-6000 purification,while there were detectable virus RNAs in the supernatant after other two ways of purification.The coated antigens made by 3 kinds of purification ways were used for ELISA test,the results showed that P/N was 2.21,1.27 and 2.25 respectively.Finally PEG-6000 was selected to purify TGEV under the condition of 8% PEG-6000 concentration,incubation for 2 h and 1% NaCl.It was demonstrated that PEG-6000 was economic,convenient,efficient way to purify TGEV.This study could provide the purified TGEV for the further research on the specificity and sensitivity of ELISA.