摘要
运用反转录聚合酶链式反应 (RT PCR)技术从家蝇体内扩增出抗菌肽天蚕素 (cecropin)基因的开放阅读框(ORF) ,与pMD 18T载体重组 ,经限制性酶切片段分析和核苷酸序列分析 ,与GenBank中报道的序列一致。根据此ORF ,重新合成 1对引物 ,并在碳末端进行定点突变 ,加上Asn编码 ,使其末端酰氨化 ,再利用半嵌套式PCR扩增出家蝇cecropin基因的成熟肽 ,与双酶切的酵母表达载体pPICZαA连接 ,经PCR和双酶切鉴定 ,成功构建了分泌型表达质粒。
The total RNAs were isolated from the larvage of houseflies (Musca domestica). The open reading frame (ORF) of Cecropin gene were cloned by using reverse transcriptase polymerse chain reaction (RT-PCR) from larvae of houseflies (Musca domestica). The PCR products were ligated into pMD-18T vector. According to biological softwares and the ORF of cecropin, the mature peptide were cloned by using RT-PCR, Nested PCR methods. The nucleotide sequence of the fragment, mature cecropin, which consists of 111 bp , encods 37 amino acid residues. The PCR products were ligated into pMD-18T vector too. The positive recombinant were digested by XhoⅠ/NotⅠ, the digested PCR products can be ligated into pPICZαA vector which were also digested by XhoⅠ/NotⅠ, then were transformed into DH5 α. It is indicated that we have succeeded constructing a productive expression vector, pPICZαA-cecropin.
出处
《蚕业科学》
CAS
CSCD
2004年第1期90-94,共5页
ACTA SERICOLOGICA SINICA
基金
珠海市科技计划项目 (编号PC2 0 0 3 10 0 85 )
