摘要
利用自动发酵罐高效分泌表达rPoIFN-α,经硫酸铵沉淀、Q Sepharose FF阴离子交换层析和Superdex200分子筛层析纯化重组猪α干扰素,用纯化的rPoIFN-α进行PRV体外抑制试验.结果表明:随着表达时间的延长,发酵表达液中rPoIFN-α含量增高,其中72h表达液中rPoIFN-α的含量可达0.52μg/μL,约占总蛋白量的57.50%,活性为1.00×106 U/μL.rPoIFN-α提纯液的浓度为7.02μg/μL,纯度为98.1%,活性高达1.00×109U/μL.纯化rPoIFN-α的PRV抑制作用和感染阻断作用的比活均为1.42×105 U/μg,对PRV增殖抑制作用的比活为1.42×103 U/μg.表明发酵表达的rPoIFN-α对伪狂犬病毒病具有良好的抑制活性,为进一步开展重组猪α干扰素临床试验提供依据.
Abstract.The recombinant porcine interferon-α （rPolFN-α） was efficiently expressed by Pichia pasto- ris in a fermentation reactor and the inhibition of rPoIFN-α on Pseudorabies virus （PRV） replication in vitro was tested after the purification by ammonium sulfate precipitation,Q Sepharose FF chromatography and gel filtration in turn. The results showed that the concentration of secreted rPoIFN-α in supernatant reached 0.52 μg/μL after 72 hours induction, which accounted for about 57.50% of the total amount of protein and its activity was 1. 00 × 10^5 U/αL. The purified rPoIFN-α solution contained 7. 02 〉g/μ rPolFN-α with the purity of 98. 1% and the activity of 1.00 × 109 U/μL. The inhibition of the rPolFN-α on PRV was approximately 1.42 × 105 U/μg. The blocking effect of the rPoIFN-α on PRV infection was approximately 1.42 × 105 U/μg. The replication inhibition effects of the rPolFN-α on PRV was approximately 1.42 × 103 U/μg. The conclusion is that the rPoIFN-αexpressed by fermentation has a high inhibition effects on PRV infection in vitro,indicating that the rPolFN-α can be used for clinical practice test.
出处
《甘肃农业大学学报》
CAS
CSCD
北大核心
2012年第4期1-6,13,共7页
Journal of Gansu Agricultural University
基金
浙江省科技计划项目(2010C32087)
关键词
猪Α干扰素
巴斯德毕赤酵母
酵母表达
伪狂犬病毒
抑制作用
porcine interferon-α
Pith ia pastoris
yeast expression
Pseudorabies virus
inhibition effect
