Cricket Neuropeptide Gene Grb-of Ast <sub> 3 </ Sub> Series Splicing Of Expression

Grb-AST3 is a member of insect Allatostatin neuropeptides family. It was isolated from the brain of Gryllus bimaculatus. In vitro bioassay show Grb-AST3 greatly inhibiting JH (Juvenile Hormone) biosynthesis by insect CA (corpora allata). The amino acid sequence of Grb-AST3 is N-AGGRQYGFGL, totally thirteen esidues. The cDNA encoding Grb-AST precursor encodes fifteen ASTs. The nucleic acid sequences of each AST are termed as Grb-AST1, Grb-AST2, Grb-AST15 by order. NeuropeptideAST2 is encoded by Grb-AST3. PCR methods were tried to construct Grb-AST3 concatemer. The expression analysis of the concatemer was proceeded in the E. coli pET expression system.A rare code of Arg was in one of the first pair of primer and all of bases of the second pair of primer were changed to biased codes of E.coli. Concatemer were constructed by polymerization with T4 DNA polymerase and primer-templated PCR with high fidelity DNA polymerase. One concatemer of 360bp, 9 copies of Grb-AST3, were obtained from the first pair of primer and three ones of 515bp, 750bp, 792bp, from the second one. Grb-AST3 concatemers were respectively cloned into expression vector, pET21b,pET22b, pET30a, in certain insertion orientation and correct reading frame must be insured. After the reading frame was confirmed correct by sequencing, the recombinant plasmid was transformed into expression host E.coli BL21(DE3) PlysS. By induction with IPTG, interest protein amonting to 6.8% and 14.7% were detected respectively in the expression products of the two strains with Grb-A3C1 and Grb-A3C3 which were cloned into pET30a, however, only amonting to 0.8% in the expression product of the strain with Grb-A38 which were cloned into pET22b by SDS-PAGE. It was established thatGrb-AST3 concatemer are more easily expressed in the way of fusion expession.