Cloning And Expression Of Recombinant Moricin-2Gene From Antheraea Pernyi

All animals have their own defense function, which can resist the invasion of bacteria, fungi, viruses and cancer cells. The vertebrates can obtain immunity by "immune memory". Invertebrates, lack of the immune system, however, have congenital immunity, and especially the insects have a very strong congenital immunity, which will quickly induce to produce antimicrobial peptides in the fat body and blood cells when bacterial infection or wound infection occurs. The antimicrobial peptide has the characteristics of good thermal stability and broad antibacterial spectrum. Antimicrobial peptides can inhibit bacterial growth, but some antimicrobial peptides have strong killing effect on some fungi, protozoa, viruses and cancer cells, etc., and immunomodulation function to accelerate wound healing without drug resistance.In this thesis, E. coli as inducer was injected Antheraea pernyi pupa for producing antimicrobial peptides. Isolated fat body mRNA, based on the sequence of the right part of the moricin-2gene fragment,5’and3’RACE were used for obtaining gene fragments, then overlapping Antheraea pernyi moricin-2complete gene sequence. Synthesized cDNA through reverse transcription, according to moricin-2CDS sequence, designed a pair of primers, added Bam H I and Hind III restriction enzyme cutting site at5’terminal, the moricin-2gene of Antheraea pernyi was amplified by PCR, and was cloned into p CR2.1for sequencing. The coding gene fragment of moricin-2is207bp, which contains68amino acid residues, and N-terminal23amino acid residue is signal peptide. The similarity of moricin-2, compared with Bombyx mori antibacterial peptides moricin-2sequence, is53%. pET28a (+) expression vector was used for constructing fusion expression plasmid pET28a (+)-moricin-2, and the recombinant protein contains104amino acid residues with a His tag. The recombinant plasmid was transformed into E. Coli BL21(DE3), after induced by IPTG, the target recombinant moricin-2expression was detected by SDS-PAGE, and molecular weight is about10kD, which is same to theoretical size. The expressed target protein was in the form of inclusion body mostly.