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为建立一种不利用超速离心法纯化浓缩J亚群禽白血病病毒(ALV-J)简便、易行的方法,利用PEG-it~(TM)病毒浓缩试剂与病毒培养细胞上清按比例混合,4℃低速离心,最后用无菌的预冷PBS进行重悬,取100μL病毒液进行TCID50的测定,同时利用电镜负染、SDS-PAGE银染及Western blot的方法对浓缩纯化后的病毒进行鉴定。结果表明:经过PEG-it~(TM)浓缩纯化病毒滴度提高10~100倍,滴度可达到1×10~7TCID50/m L;透射电镜负染观察结果表明,病毒粒子呈卵圆形,直径80~120 nm,并清晰可见囊膜上均匀密布的纤突;Western blot和银染结果显示,经过PEG-it~(TM)浓缩纯化的病毒杂蛋白明显减少,且具有良好的抗原性,能够被鸡多抗血清识别。提示在没有超高速离心机的情况下,可以使用PEG-it~(TM)纯化浓缩ALV-J,该方法使用方便,能满足大多数试验的需求。
In order to establish a simple and easy way to purify concentrated subgroup J subtype avian leukosis virus (ALV-J) without using ultracentrifugation method, the virus was mixed with the virus culture supernatant by PEG-it TM virus concentrated reagent, 4 ℃ low-speed centrifugation, and finally resuspended with sterile pre-cooled PBS, 100μL of the virus solution was TCID50 determination, and electron microscopy negative staining, SDS-PAGE silver staining and Western blot method to identify the concentrated purified virus . The results showed that the titer of recombinant virus was increased by 10 ~ 100 times and the titer reached 1 × 10 ~ 7 TCID50 / m L after PEG-it ™ concentration purification. The negative staining results of TEM showed that the virus particles were oval, The diameter of 80 ~ 120 nm, and clearly visible uniform dense capsule membrane; Western blot and silver staining results show that after PEG-it ~ (TM) concentrated purification of viral protein significantly reduced, and has good antigenicity, Chicken anti-serum can be identified. Tip Without the ultra-high speed centrifuge, PEG-it ™ can be used to purify and concentrate ALV-J. This method is easy to use and meets the needs of most tests.