Cricket Neuropeptide Gene Grb-ast8 Series Body Construction And Expression

Grb-AST1 is a member of insect Allatostatin neuropeptides family. It was isolated from the brain of Gryllus bimaculatus. In vitro bioassay show Grb-ASTl greatly inhibiting JH (Juvenile Hormone) biosynthesis by insect CA (corpora allata). Even more, comparing with other Grb-ASTs, the halflife of Grb-ASTl is longer. The amino acid sequence of Grb-ASTl is N-AQHQYSFGL, totally nine residues. The cDNA encoding Grb-AST precursor encodes fifteen ASTs. The nucleic acid sequences ofeach AST are termed as Grb-astl, Grb-ast2, ...... Grb-15 by order. NeuropeptideGrb-ASTl is encoded by Grb-ast8. Three methods were tried to construct Grb-ast8 concatemer. The expression analysis of the concatemer was proceeded in the E.coli pET expression system.Base on the encoding sequence of Grb-ASTl and GKR (by which ASTs were concatenated), three pairs of primers were synthesized and used in three constructing methods respectively. The first method was random ligation. Klenow DNA Polymerase and T4 DNA Ligase were used in this method. The second one was gap filling. E.coli DNA Polymerasel and E.coli DNA Ligase were implied. The third one was PCR. In this method, high fidelity DNA Polymerase (Pyrobest?DNA Polymerase) was used to ensure efficient and accurate amplification. After cloning and sequencing of the PCR products, one concatemer contains eight copies of Grb-ast8 was attained.The Grb-ast8 concatemer was cloned into pET22b expression vector. The result of sequencing confirmed the reading frame of the recombinant plasrnid was correct. Then, the recombinant plasmid was transformed into expression host E.coli BL21 strain. Expression of Grb-ast8 concatemer was induced by IPTG. The concentrations of culture (OD600= 0.4, 0.8) and IPTG (0.1mM, 0.5mM, 1.0 mM) were tested to establish an optimum concentration. SDS-PAGE analysis of the expression products indicated that the recombinant E.coli BL21 strain expressed an additional protein( comparing with the control). The molecular mass of the protein equaled to the theoretically deduced value.Bioassay showed the expression products of Grb-ast8 concatemer was toxic to Pieris rapae, especially to the second and third instar .