Cricket Neuropeptide Gene Grb-ast <sub> The 2 </ Sub> Concatemer Expression Insects Efficiency Study

Grb-AST is a member of insect Allatostatin neuropeptides family. It was isolated from the brain of Gryllus bimaculatus. In vitro bioassay showed Grb-AST couldgreatly inhibit JH (Juvenile Hormone) biosynthesis by insect CA (corpora allata). The cDNA encoding Grb-ast precursor encodes fifteen ASTs. Grb-ast2 encoded the second polypeptide. The amino acid sequence of Grb-AST2 is N-LPVYNFGL, totally eight residues. So it was named as Grb-AST2.Base on the encoding sequence of Grb-AST2 and GKR (by which ASTs were concatenated), two primers were synthesized and constructed in a proved PCR method. In this method, high fidelity DNA Polymerase (PyrobestTM DNA Polymerase) was used to ensure efficient and accurate amplification. After cloning and sequencing of the PCR products, one concatemer contains seventeen copies of Grb-asti was attained.The Grb-ast2 concatemer was cloned into pET-44a expression vector. The result of sequencing confirmed the reading frame of the recombinant plasmid was correct. Then, the recombinant plasmid was transformed into expression host E.coli BL21(DE3) strain. Expression of Grb-asti concatemer was induced by IPTG. The concentrations of culture (OD600= 0. 8) inducer IPTG (1.0mM) and 37 induce for five hours. The expression quantity were most highest est tested to establish an optimum concentration. Using Uvisoft the amount of the expression yield of Grb-asti accounting for the total protein of the cells was 37.2%.Using Ni-NTA Agarose kit of QIAGEN company, the expression products was purified. Then it was used to insect-resistant experiment. Primarily, results showed that purified protein has obvious effect on inhibiting beet armyworm to grow and kill them to some extent.