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当前位置: 首页 > 文章 > 杂交猪白介素IL-18全基因克隆及原核表达 中国动物检疫 2009,26 (9) 28-30
Position: Home > Articles > Cloning and Prokaryotic Expression of Inbred Pig Interleukin-18 Full Gene China Animal Health Inspection 2009,26 (9) 28-30

杂交猪白介素IL-18全基因克隆及原核表达

作  者：
王金良;沈志强;董林;祖立闯
单  位：
山东省滨州畜牧兽医研究院;山东绿都生物科技有限公司
关键词：
基因克隆;原核表达;IL-18基因;杂交猪
摘  要：
为研究杂交猪白细胞介素-18(IL-18)蛋白的结构与功能,将杂交猪外周血淋巴细胞体外刺激培养后,提取淋巴细胞总RNA,通过RT-PCR扩增的方法,获得IL-18全基因,克隆到pMD18-T载体中进行测序,测得核苷酸序列与GenBank登录的参考序列的同源性在99.0%～99.8%之间,推到氨基酸同源性在99.7%～100%之间;然后亚克隆到pET30a(+)表达载体中,构建并筛选出阳性重组子,标记为pET30a/IL-18。将阳性重组质粒转化进RossettaTM宿主菌中,通过改变IPTG浓度、诱导温度和诱导时间,使重组蛋白获得表达,并确定最佳诱导条件为:IPTG终浓度1.0mM、诱导温度37℃、诱导时间为3-4h。经SDS-PAGE和Western-blotting分析表明该重组蛋白相对分子量约为35Ku,表达量约为38%,主要存于包涵体中,具有良好的免疫学活性。
译  名：
Cloning and Prokaryotic Expression of Inbred Pig Interleukin-18 Full Gene
作  者：
Wang Jin-liang1,Shen Zhi-qiang1,2,Dong Lin1,Zu Li-chuang2 (1. Shandong Binzhou Animal Science&Veterinary Medicine Academy,Binzhou Shan dong 256600; 2.Shandong Lvdu Bio-Industry Co.,Ltd.,Binzhou Shan dong 256600)
关键词：
Gene cloning; Prokaryotic Expression; Interleukin-18; Inbred pig
摘  要：
In order to study the inbred pig interleukin-18(IL-18)protein structure and function,RT-PCR amplification of IL-18complete gene was performed with primers designed from total RNA extracts of periphe ral-blood lymphocyte from an inbred pig,then was cloned into pMD18-T and sequenced. The sequence analysis showed that the nucleotide sequences of the IL-18 in the study shared 99.0%～99.8% homology with that of pig published in GenBank,and the identity of amino acid was 99.7% ～100%. The positive recombinant,termed pET30a/IL-18,was constructed and screened by subcloning into expression vector pET30a (+). It was transfered into RossettaTM,resulting in the recombinant protein and the optimal inducing conditions were detemined:37℃with 1.0mM IPTG for 3-4h. This recombinant protein with molecular weight of about 35ku and mainly presenting in inclusion bodies,was approved to have immunological activity,by SDS-PAGE and Western-blotting analysis.

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