猪IL-2和IL-6的原核表达及多克隆抗体的制备

Prokaryotic Expression and Preparation of Polyclonal Antibody for Porcine Interleukin-2 and Interleukin-6

IL-2和IL-6是机体重要的免疫调节因子,在佐剂的应用方面具有很好的发展前景。论文以pTIL-2和pTIL-6为模板进行PCR扩增得到猪白细胞介素2(pIL-2)和猪白细胞介素6(pIL-6)的全基因序列,通过EcoRⅠ和HindⅢ双酶切及连接反应将目的基因亚克隆到原核表达载体pET30a中构建了融合表达质粒pETIL-2和pETIL-6。将重组质粒分别转化大肠埃希菌BL21(DE3)和Rosetta(DE3),37℃在IPTG诱导下高效表达了pIL-2和pIL-6,分子质量分别约为26ku和30ku。将包涵体纯化并复性后免疫家兔,制备的兔抗pIL-2和pIL-6的多克隆抗体经ELISA检测效价在1∶12800以上。

The interleukin-2（IL-2） and interleukin-6 （IL-6） are the important factors for immunoloregulation,and have great development prospects in the application of adjuvants. The genes of porcine IL-2 and IL-6 were amplified from recombinant plasmid pTIL-2 and pTIL-6. After being digested by restriction endonucleases,the IL-2 and IL-6 genes were subcloned into the prokaryotic expression vector pET30a. After restriction enzyme digestion, PCR identification and sequenc＇e analysis of the recombinant plasmids, the positive recombinant plasmids were selected and designated as pETIL-2 and pETIL-6. The pETIL-2 and pETIL-6 were transformed into E. coli BL21（DE3） and Rosetta（DE3） and then induced with IPTG at 37 ℃. SDS-PAGE and Western-blotting analysis showed that the expressed recombinant IL-2 and IL-6 proteins presented in inclusion bodies and were about 26ku and 30ku in molecular weight. The recombinant IL- 2 and IL-6 protein which were purified and refolded were used to immunize rabbit for specific IL-2/IL-6 antibody generation.