昆虫抗真菌肽Thanatin基因的合成及原核表达

Synthesis and Prokaryotic Expression of Insect Antifungal Peptide Thanatin Gene

斑腹刺益蝽 (Podisusmaculiventris)抗真菌肽Thanatin是目前发现的 6种昆虫抗真菌肽之一 ,对真菌具有广谱的免疫诱导杀菌活性 ,由 2 1个氨基酸残基组成 ,对革兰氏阳性和阴性细菌也有一定的杀菌作用。根据已报道的抗真菌肽Thanatin基因的氨基酸序列 ,推导出cDNA序列 ,采用基因片段合成结合PCR扩增的策略 ,获得大量完整的Thanatin基因片段 ,通过T A克隆法克隆至pGEM TEasy载体 ,进行测序 ,结果证实合成的Thanatin基因与预期设计的完全一致。然后将其亚克隆至表达载体pET 2 1d中。经IPTG诱导表达后 ,采用RNA DNA点杂交和RT PCR检测具Thanatin基因插段的RNA转录表达活性 ,初步结果显示该融合表达产物对烟曲霉菌 (Aspergillusfumigatus)和绿色木霉菌 (Trichodermariricle)

Thanatin of Podisus maculiventr is one of the six insect antifugal peptides that have been found in the recent years.It is an induced peptide composed of 21 amino acids not only exhibits a large antifungal spectrum,but shows antimicrobial activity against Gram positive bacteria and Gram negative bacteria as well.The cDNA sequence was designed based on the amino acid sequence of Thanatin.The Thanatin gene was obtained through oligodeoxynucletides synthesis and PCR amplifying.The PCR product was cloned into the pGEM T Easy vector by means of T A pairing direct molecular cloning method.The synthesized thanatin gene was proved correct by DNA sequence analysis .The thanatin gene of 87 bp was subcloned into the pET 21d vector through the linkage of the cohesive ends.The recombinant expression vector pET 21d th was constructed.The recombinant expression plasimid pET 21d th was transformed into E.coli BL21(DE3) and the thanatin gene was expressed in fusion form when induced by IPTG.The transcript activity of the thanatin gene in induced cells was verified by two method of RT PCR and Dot blotting.We determined bio activity of its expression product by agar plate assay.The results showed that the expression products of thanatin gene exhibit antifungal activity against the two pathogenic fungi: Aspergillus fumigatus and Tricholderma riricle.