Cricket Neuropeptide Gene Grb-of Ast <sub> 7 </ Sub> Concatemer Expression And Preparation Of Antiserum,

Grb-AST7 is a member of insect Allatostatin neuropeptides family.It was isolated from the brain of Gryllus bimaculatus. In vitro bioassay show Grb~AST7 greatly inhibiting JH (Juvenile Hormone) biosynthesis by insect CA (corpora allata). Even more, comparing with other Grb-ASTs, the halflife of Grb-AST/is longer. The amino acid sequence of Grb~AST7is N-AGMYSFGL, totally eight residues. The cDNA encoding Grb-AST precursor encodes fifteen ASTs. The nucleic acid sequences of each AST are termed as Grb-ASTj, Grb-ASTn, Grb~AST,s by order. Neuropeptide Grb-AST? is encoded by Grb~AST7. The expression analysis of the concatemer was proceeded in the E. coli pET expression system. Synthesized the peptide according to the sequence of AST7. The peptide was linked with carried protein BSA and then was used as fusion antigens for immunization.Base on the encoding sequence of AST7 and GKR (by which ASTs were concatenated), two primers were synthesized and constructed them in a proved PCR method. In this method, high fidelity DNA Polymerase (Pyrobesf* 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~AST7 was attained.The Grb-AST?concatemer was cloned into pET22b 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)plysS strain. Expression of Grb~AST7 concatemer was induced by IPTG. The concentrations of culture (OD600=0. 8) and IPTG (1. OmM) were tested to establish an optimum concentration. SDS-PAGE analysis of the expression products indicated that the recombinant E. coli BL21 (DE3) pLysS strain expressed an additional protein( comparing with the control). The molecular mass of the protein equaled to the theoretically deduced value. The protein Accounting for the total was approximately 11.80%.The peptide was synthesized according to the sequence of Grb-AST7. Since AST7 is a hapten, it was conjugated to the carrier proteins bovine serum albumin (BSA) using l-ethyl-3-(dimethyl-aminopropyl) Carbodiimide (EDC). AST7~BSA conjugation was used to immunize the rabbit. After three times, collected the antiserum from the blood. Enzyme-linked immunosorbent assay (ELISA) proved that the titres of the antiserum was 1/400.