中文摘要：

目的为研究黄粉虫体内抗菌肽表达调控的分子机制,克隆黄粉虫抗菌肽Tenecin基因,并研究其原核表达产物的抑菌活性。方法用大肠杆菌DH-5α菌液（1×108/ml）腹部注射黄粉虫5龄幼虫,72 h后提取其总RNA,RT-PCR克隆Tenecin基因,测序并进行生物信息学分析;构建原核表达重组载体pET-28a（＋）-Tenecin并转入大肠杆菌BL21菌株,用IPTG诱导观察其表达情况;将4种不同浓度的原核表达产物Tenecin蛋白作用于大肠杆菌DH-5α,测量抑菌圈直径大小,观察其抑菌作用。结果电泳及测序结果显示：Tenecin基因的大小为255 bp左右;用1 mmol/L IPTG诱导后,经SDS-PAGE电泳检测在9 000附近有目的条带,与理论值大小相符;体外抑菌试验表明：试验设3个重复,浓度为80、60、40、20μg/ml的Tenecin蛋白与大肠杆菌DH-5α共培养18 h后,抑菌圈直径（mm）分别为25.1±0.03、20.7±0.06、17.2±0.11、9.3±0.04。结论成功克隆黄粉虫抗菌肽Tenecin基因,并成功表达Tenecin蛋白,该蛋白对大肠杆菌DH-5α有明显抑制作用,为后期临床应用奠定基础。

英文摘要：

Objective To clone tenecin gene,an antibacterial peptide gene,from Tenebrio molitor for its prokaryotic expression and explore the molecular mechanism for regulating the expression of antibacterial peptide in Tenebrio molitor larvae.Methods The antibacterial peptide was induced from the larvae of Tenebrio molitor by intraperitoneal injection of Escherichia coli DH-5α（1×108/ml）.RT-PCR was performed 72 h after the injection to clone Tenecin gene followed by sequencing and bioinformatic analysis.The recombinant expression vector pET-28a（＋）-Tenecin was constructed and transformed into E.coli BL21（DE3） cells and the expression of tenecin protein was observed after IPTG induction.Results Tenecin expression was detected in transformed E.coli using SDS-PAGE after 1 mmol/L IPTG induction.Tenecin gene,which was about 255 bp in length,encoded Tenecin protein with a relative molecular mass of 9 kD.Incubation of E.coli with 80,60,40,and 20 μg/ml tenecin for 18 h resulted in a diameter of the inhibition zone of 25.1±0.03,20.7±0.06,17.2±0.11 and 9.3±0.04 mm,respectively.Conclusion Tenecin protein possesses strong antibacterial activity against E.coli DH-5α,which warrants further study of this protein for its potential as an antibacterial agent in clinical application.