犬干扰素α的原核表达及其抗病毒活性

Prokaryotic Expression and Antiviral Activity of Recombinant Canine Interferon α

目的原核表达犬重组干扰素α(rCaIFNα),并检测其抗病毒活性。方法PCR扩增CaIFNα成熟蛋白基因,并克隆至pGEX-5X-1表达载体中,转化大肠杆菌BL21(DE3),IPTG低温诱导表达。表达的重组蛋白经亲和层析纯化后,进行SDS-PAGE及Westernblot鉴定,并检测其效价及抗犬细小病毒活性。结果PCR、双酶切及序列分析证实CaIFNα成熟序列已插入pGEX-5X-1载体中。表达产物经SDS-PAGE分析,可见相对分子质量约45000的目的蛋白条带,表达量约占菌体总蛋白的(32±2.4)%。纯化的重组蛋白纯度约为85%,且具有良好的反应原性。制备的3批rCaIFNα效价均达到1.1×106IU/ml以上。并可抑制犬细小病毒对MDCK细胞的致病变作用。结论已成功地在大肠杆菌中高效表达了rCaIFNα,表达产物具有良好的抗病毒活性。

Objective To express recombinant canine interferon α （rCaIFNα） in prokaryotic cells and determine its antiviral activity. Methods The gene encoding mature CAIFNα protein was amplified by PCR and cloned into expression vector pGEX-5X-1. The constructed recombinant plasmid was transformed to E. coli BI221 （ DE3 ） for expression at low temperature under induction of IPTG. The expressed recombinant protein was purified by affinity chromatography, identified by SDS-PAGE and Western blot, and determined for potency and activity in inhibiting canine parvovirus. Results PCR, restriction analysis and sequencing proved that the gene encoding mature rCAIFNα protein was inserted into vector pGEX-5X-1. SDS-PAGE showed that the expressed product, with a relative molecular mass of about 45 000, contained （32 ±2. 4 ）% of total somatic protein. The purified recombinant protein reached a purity of about 85% and showed good reactogenicity. All the 3 batches of prepared rCAIFNα reached potencies of more than 1. 1 ×10^6IU/ml and inhibited the CPE of MDCK cells caused by canine parvovirus. Conclusion Recombinant canine interferon α was highly expressed in E. coil, which showed high antiviral activity.