鸡γ干扰素的可溶性表达及纯化产物的抗病毒活性

Soluble expression of chicken interferon-γ and antiviral activity of purified expression product

摘要:

【目的】克隆鸡γ-干扰素（chIFN-γ）基因，大肠杆菌表达及产物纯化与活性检测。【方法】通过RT-PCR方法用ConA刺激的20日龄SPF鸡的脾脏淋巴细胞中扩增出chIFN-γ成熟蛋白基因cDNA，克隆至原核表达载体pET-32a (+)，构建重组表达质粒pET-32a (+)-chIFN-γ，在大肠杆菌BL21（DE3）中经IPTG诱导表达，利用镍柱亲和层析法纯化可溶性蛋白，并进行SDS-PAGE、Western blot鉴定。利用MDCK-VSV系统测定抗病毒活性。【结果】克隆得到456 bp 的chIFN-γ成熟蛋白编码基因，大肠杆菌中成功表达chIFN-γ蛋白，分子量约为31.0 kDa，能与抗His的单克隆抗体和兔源多抗血清发生特异性反应。表达蛋白一部分形成包涵体，另一部分以可溶形式存在，可溶性蛋白经镍柱在天然条件下的纯化得率为3.0 mg/mL。生物学活性试验表明，1:32 稀释纯化的重组chIFN-γ （rchIFN-γ）孵育MDCK细胞后能抵抗100TCID50的VSV攻击。【结论】通过镍柱天然纯化获得的rchIFN-γ具有较好的抗病毒活性，为研制新型抗病毒干扰素制剂奠定基础。

Abstract:

[Objective] We cloned and expressed chicken interferon-gamma gene (chIFN-γ), and detected the bioactivity of chIFN-γ expressed in Escherichia coli (E.coli) . [Methods] The chIFN-γ mature protein gene was amplified by RT-PCR from spleen lymphocytes of chicken which were stimulated with concanavalin A (ConA) and then cloned into the prokaryotic expression vector pET-32a (+). Recombinant expression plasmid of pET-32a (+)-chIFN-γ was constructed and then transformed into the competent E. coli BL21 (DE3) cells for expression with IPTG induction. Purified soluble rchIFN-γ proteins were obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blot assay. The antiviral activity was detected by MDCK-VSV system. [Results] A 456 bp cDNA encoding chIFN-γ mature protein gene was cloned and successfully expressed in E.coli with approximate molecular weight of 31.0 kDa, which could be recognized by anti-His mAb and rabbit polyclonal antibody against chIFN-γ. The recombinant chIFN-γ proteins were expressed to form inclusion bodies with a portion of soluble protein. The soluble rchIFN-γ proteins were purified by Ni-NTA column under a native condition with the yield of 3.0 mg/L. The purified recombinant chIFN-γ (rchIFN-γ) proteins by 1:32 dilution could resist 100 TCID50 VSV virus attack. [Conclusion] The purified rchIFN-γ proteins by Ni-NTA column under a native condition had better antiviral activity, which will establish a basis for further developing new type antiviral interferon praeparatum.