猪干扰素γ的原核表达及抗血清的制备

Prokaryotic Expression of Porcine Interferon γ and Preparation of Antisera

目的原核表达猪干扰素γ(poIFNγ),并制备其抗血清。方法PCR扩增poIFNγ成熟多肽基因片段,克隆至原核表达载体pET-28a(+)中,构建重组表达质粒pET-28a-poIFNγ,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达的蛋白纯化后免疫小鼠,制备抗血清,ELISA检测抗血清的效价。结果重组原核表达质粒pET-28a-poIFNγ经双酶切和测序证明构建正确;重组蛋白的表达量占菌体总蛋白的40%,主要以包涵体形式表达;纯化的重组蛋白纯度可达90%;制备的抗血清效价可达10-5。结论已成功地在大肠杆菌中表达了poIFNγ,并制备了较高效价的抗血清。

Objective To express porcine interferon γ（poIFNγ）in prokaryotic cells and prepare the antisera. Methods The gene fragment encoding mature polypeptide of poIFNγ was amplified by PCR and cloned into prokaryotic expression vector pET28a（＋）. The constructed recombinant plasmid pET-28a-poIFNγ was transformed to E. coli BL21（DE3）for expression under induction of IPTG. The expressed protein was purified and inoculated to mice, and the antisera were prepared and determined for titer by ELISA. Results Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-poIFNγ was constructed correctly. The expressed product, mainly existing in a form of inclusion body, contained 40% of total somatic protein and reached a purity of 90% after purification. The titer of prepared antisera was 10-5. Conclusion Porcine interferon γ was successfully expressed in E. coli, and high titer antisera was prepared.