Cloning and expression of a chitinase gene from Helicoverpa assulta
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Key Words：Helicoverpa assulta, chitinase, gene cloning, prokaryotic expression
Abstract：The cDNA encoding the chitianse (named as Has-Chit) was isolated from adipose tissue and epidermis of Helicoverpa assulta(Guenee) larvae by reverse transcription polymerase chain reaction (RT-PCR). The cDNA fragment was further cloned into pMD18-T vector, and then constructed into pGEX-4T-2 for expression in prokaryotic cells. Sequence analyses showed that the open reading frame of Has-Chit was 1 338 bp in length, encoding 162 amino acid residues; its predicted molecular weight and isoelectric point were 501 kDa and 9.26, respectively. The deduced amino acid sequence showed a high identity with the reported sequences of chitinases from its sibling species H. armigera (99%) and other 6 species of insects (65%～76%), all of which shared the typical structural features of chitinases from other insects. The fragment containing Has-Chit gene was inserted into pGEX-4T-2 for expression in prokaryotic cells (BL21), and induced by IPTG. The results showedthat the specific protein of about 76 kDa was not observed by SDS polyacrylamide gel electrophoresis, which indicated that extrinsic gene, Has-Chit gene, can not expressed in E. coli.