柞蚕抗菌肽A基因的克隆和表达

Cloning of Cecropin A Gene from Chinese Oak Silk Moth, Antheraea pernyi and Its Expression by IPTG Induction

注射大肠杆菌E .coliK12 D31诱导的柞蚕滞育蛹在 2 5℃温育条件下 ,蛹体血淋巴可产生一系列的抗菌物质以抵御外源病菌的入侵 .本研究以脂肪体为材料 ,诱导后提取组织总RNA ,纯化mRNA并用AMV逆转录酶合成cDNA .设计并合成抗菌肽A基因 (cecA)的引物 ,通过PCR法扩增获得柞蚕抗菌肽基因 ,克隆至pBluescriptM13(+)质粒 ,获得相对分子质量 5 .9× 10 4 的融合蛋白表达产物 ,序列分析表明柞蚕抗菌肽A基因及其编码的氨基酸和天蚕具有一定的同源性 .

A series of antibacterial substances were induced in the hemolymph of the diapausing pupae of Chinese Oak Silk moth, Antheraea pernyi , when injected with E.coli and incubated at 25 ℃ for several days. Fat body from immunized pupae was used as a typical material in the research. Total RNA were extracted from the tissue of immunized pupae and mRNA were purified. The first strand cDNA were synthesized by AMV reverse transcriptase. Subsequently, PCR were applied to amplificate cecropin A gene according to designed primer and the first strand of cDNA. The cecropin gene was cloned into Sma I site of plasmid M 13 (+) and expressed by IPTG induction. 5.9×10 4 cecropin A and β galactase fusion protein were obtained on the gel of SDS PAGE. Sequence determination showed that certain homology of cecropin A gene from A.pernyi compared with that from Hyalophora cecropia.