中国家蚕抗菌肽基因的PCR扩增、克隆和序列测定

PCR AMPLIFICATION,CLONING AND SEQUENCING OF THE ANTIBACTERIAL PEPTIDE GENE FROM CHINESE SILKWORM,BOMBYS MORI

本实验采用ＰＣＲ技术，扩增中国家蚕抗菌肽ｃｅｃｒｏｐｉｎＢ基因片段，并以ＰＵＣ１９质粒为载体，将该基因片段转化到大肠杆菌中。从筛选得到的阳性克隆中分离出ｃｅｃｒｏｐｉｎＢ基因，测定其序列，并由此推导出氨基酸序列。结果表明：（１）抗菌肽ｃｅｃｒｏｐｉｎＢ基因片段长为８６６ｂｐ，包括７３ｂｐ的ＥｘｏｎＩ，５５６ｂｐ的Ｉｎｔｒｏｎ，８７ｂｐ的ＥｘｏｎⅡ和１５０ｂｐ的３’端非编码区。其中，非内含子部分的碱基序列与已克隆的日本家蚕的两个ｃｅｒｏｐｉｎＢｃＤＮＡ序列比较，同源性分别为８７．５％和９５％；（２）推导出的成熟抗菌肽由３７个氨基酸残基组成，其氨基酸序列与日本家蚕和天蚕的氨基酸序列相比较，同源性为９１．９％和８０．６％。

The genomic DNA fragment of cecropin B,an antibacterial peptide,is amplified from Chinese silkworm by means of PCR,and transformed into E.coli by plasmid vector of PUC19.From the positive clones screened,cecropin B gene is isolated and sequenced.Also,the amino acid sequence is deduced from its nucleotide sequence.The results show that:(1)The cecropin B gene is composed of 866 bp,including 73bp EXon Ⅰ,556bp Intron,87bp EXon Ⅱ and 150bp 3'-end non-coding structure.The coding sequence of the genomic DNA fragment is compared with two known cecropin B cDNA sequences from Japanese silkworm,and show honologue of 87.5% and 95%,respectively;(2)The deduced amino acid sequence is compared with that of cecropins B from Japanese silksworm and Hyalophora cecropia,and show homologue of 91.9% and 80.6% .