昆虫抗真菌肽Thanatin-CAD双价基因的合成、克隆及其表达

为促进昆虫抗菌肽基因在转基因抗病育种和生物工程制药的应用,通过设计嵌合引物结合分段PCR的方法将抗真菌肽Thanatin基因与Cecropin AD基因(CAD基因)融合拼接为Thanatin-CAD双价基因,采用T-A克隆法将其克隆至pGEM-T Easy载体,进行测序,结果证实与预期设计的完全一致;然后再将其亚克隆至表达载体pET-21d中,用IPTG诱导含重组表达质粒的菌株,对表达产物进行生物活性测定,初步结果显示Thanatin-CAD双价基因的融合表达产物对受试细菌无抑菌活性,但对部分病原真菌有较强的抑菌作用。

Synthesis,Cloning and Expression of the Gene of Antifungal Peptide Thanatin-CAD

To prompt utilization of insect antifungal peptides gene in the transgenic disease-resistance breeding and bioengineering pharmacy, in the study, thanatin gene and cecropin AD gene ( CAD gene) were linked through fused primers and the method of step-by-step PCR to form a new gene,named thanatin-CAD gene.The thanatin-CAD gene was cloned into pGEM-T Easy Vector by means of T-A pairing direct molecular cloning method and analyzed by sequencing, and the result proved that thanatin gene and cecropin AD gene were fused as expected. The thanatin-CAD gene was subcloned into expression vector pET-21d and was expressed in fusion form after the host bacterium was induced with IPTG. Assaying the expression product,the prelimilary result indicated that the fused protein product has no antibacterial activity to most of the tested pathogenic bacteria,but has some activity against some kinds of pathogenic fungi.