惠阳胡须鸡IFN-α基因的克隆 表达和重组蛋白的抗病毒活性圆二色性分析  

Cloning and expression of IFN-α gene from Huiyang chickens and studies on the antiviral activity, circular dichroic analysis of its recombinant protein

作　　者：韩春来[1] 汪明[1] 张灿[1] 周建民 史喜菊[1] 

机构地区：[1]中国农业大学动物医学院,北京海淀100094 [2]乾元浩生物股份公司,北京海淀100083

出　　处：《中国兽医杂志》2006年第11期3-6,共4页Chinese Journal of Veterinary Medicine

摘　　要：IFN-α是一种具有很强抗病毒活性的细胞因子。从惠阳胡须鸡肝脏克隆了IFN-α基因,为新的亚型。将IFN-α成熟肽与毕赤酵母pPICZαA连接,经酶切鉴定、PCR鉴定和测序后确定目的基因无突变并且插入方向正确,将重组质粒转化到GS115中,阳性转化子经甲醇诱导表达,获得了高效分泌表达,重组IFN-α分子量约为35kD,表达量达到了0.307mg/ml,在CEF/VSV细胞系上的抗病毒活性为3.2×106U/mg。圆二色实验结果表明IFN-α重组蛋白中含有53.2%α螺旋、3.1%β折叠、10.6%转角和33.1%无规谱卷曲,属于全α型蛋白。结果表明,采用毕赤酵母分泌表达了有活性的鸡IFN-α,并首次证实鸡IFN-α属于全α型蛋白。IFN a is a cytokine with high antiviral activity. Huiyang IFN-α was cloned from liver genomic DNA. The homoligies were from 96.9%-97.9% between Huiyang chicken IFN-α and IFN-α s on Genbank, indicating this IFN-α gene was a new subtype. The recombinant plasmid IFN-α/pPICZαA confirmed by enzyme digestion, PCR and sequencing was transformed into Pichia pastoris GS115. After 1% methanol induction, SDS-PAGE analysis of the culture supernatant of recombinant yeast strains indicated that the yield of recombinant IFN-α was 0. 307 mg/ml and the molecular weight was about 35 kD. The recombinant IFN-α expressed by Pichia pastoris showed antiviral activity of 3.2×10^6 U/mg in CEF/VSV, which was higher than that from E. Coll. The secondary structure of recombinant IFN-γ analyzed by Circular Dichroism showed that the contents of this protein were： 53.2%α-helix; 3. 1% β sheet, 10.6% turn; 33. 1% random, which showed it was a tipical helical protein. This paper reported that recombinant IFN-α with high antiviral activity was highly produced in Pichia pastoris and first confirmed that chicken IFN-α was a helical protein which was similar to IFN-α of mammals.

关 键 词：惠阳胡须鸡 IFN-Α 克隆 表达 抗病毒活性 圆二色 

分 类 号：Q784[生物学—分子生物学]