黑腹果蝇抗真菌肽基因Drs和Drs-lC的原核可溶性表达及抗真菌活性测定

Soluble expression of the antifungal peptide genes,Drs and Drs-lC,of Drosophila melanogaster in the prokaryote and the detection of their antifungal activity

Drosomycin (Drs)是第1个从黑腹果蝇Drosophila melanogaster体内鉴定发现的昆虫抗真菌肽因子。它对细菌无明显的抗性,但对丝状真菌具有高效广谱的抑杀作用。此外,在黑腹果蝇基因组还存在着Drs的另外6个同系物的基因序列,其中同系物Drosomycin-lC(Drs-lC)的抗真菌谱仅次于Drs。将Drs抗真菌肽基因(Drs)和同系物Drs-lC基因(Drs-lC)进行可溶性表达,对果蔬等农产品防腐保鲜的研究有应用前景。本实验将Drs和Drs-lC分别克隆到硫氧还蛋白(Trx)融合表达载体pThiohis A中,转化宿主菌TOP10 ,进行可溶性表达,并从诱导表达的菌液起始浓度、IPTG的诱导浓度及诱导时间等方面进行了表达条件的优化。结果表明2种融合蛋白Trx-Drs和Trx-Drs-lC大部分以可溶形式表达,可溶性表达的Trx-Drs在上清液中约占菌体总蛋白的22 %。2种融合蛋白的表达产物经Ni-NTA亲和层析得到纯化。生测结果表明,2种融合蛋白分别对8种供试真菌中的5种真菌显示明显的抗性。

Drosomycin （Drs） is the first insect antifungal peptide from Drosophila melanogaster which showed a broad activity against filamentous fungi but no activity to bacteria. Besides the Drs, there are six gene sequences of Drs isoforms in the genome of D. melanogaster. Among them, only the activity of Drosomycinlike C （Drs-lC） was close to Drs. The soluble expression of the antifungal peptide genes Drs and Drs-lC may be applied in the preservation of agricultural products such as fruits and vegetables. In this paper, the genes of Drs and Drs-lC were respectively inserted into the fusion vector pThiohis A containing thioredoxin （Trx） and solubly expressed after the recombinant vectors were transformed into TOP10. The conditions of the expression including the starting concentration of the bacteria induced, the inductive concentration of IPTG and the incubation time after induction were optimized. The results showed that the fusion proteins, Trx-Drs and Trx- Drs-lC, were expressed as soluble proteins. The soluble expression level of Trx-Drs in the supernate amounted to 22 % of the total proteins of bacteria. The expressed products of fusion proteins were purified by Ni-NTA. The results of antifungal activity detection indicated that the fusion proteins, Trx-Drs and Trx-Drs-lC, showed obvious activity against five of the eight tested fungi.