Abstract：In the present study, the total RNA was extracted from three instar larvae of Musca domestica, the cDNA sequence encoding the ORF of cecropins was amplified by RT-PCR, and the target fragment was further sequenced after being cloned into T vector pUCm-T. Then, the cDNA sequence of the mature cecropins was amplified by PCR with recombinant plasmid pUCm-T/cecropin as template, the N-terminal rare codon GGA of E.coli was changed to the favorable codon GGC, and a Asn codon AAC was added in front of the stop coden TAA in the C-terminus. This mutant gene designated as mCecropin was then ligated with the fusion expression vector pGEX-4T-1. After restriction analysis and DNA sequencing, the positive recombinant plasmid pGEX-4T-1/mCecropin was transformed to different strains of E.coli cells and the fusion protein was expressed after IPTG induction. The fusion protein was assayed by SDS-PAGE and the E.coli BL21(DE3) cell was chosen as the host cell for the expression of the fusion protein. The expressed fusion protein GST-mCecropin was purified by GSTrap affinity coloum and the GST marker was then cleaved by thrombin. In this way, the fusion protein mCecropin with antibacterial activity was obtained after purification with HiTrap benzamidine column.
[1]Kenjiro U, Ayaka S, Morikazu I ,et al . Purification and cDNAcloning of Luxuriosin,a novel antibacterial peptide with Kunitz domainfromthe Longicorn Beetle[J].Acal- oleptaluxuriosaBiochi m Biophys Acta ,2005 ,1722(1) : 36-42 .
[2]Hoff mann J A,Reichhart J M, Hetru C.Innatei mmuni- ty in higher insects[J].Curr OpinI mmunol ,1996 ,8(1) : 8-13 .
[3]Xu J H,Zhu J Y.Status and prospect of the research for insect antibacterial peptides[J]. Bulletin of biology , 2004 ,39(10) :5-7 .
[4]Boman H G, Nilsson I , Rasmuson B,et al.Inducible antibacterial defense system in Drosphila[J]. Nature , 1972 ,237 :232-235 .
[5]Boman H G . Peptide antibiotics and their role in innate i mmunity[J]. Annu Rev I mmunol ,1995 ,13 :61-92 .
[6]Vizioli J , Salzet M. Anti microbial peptides versus para- sitic infections ?[J]. Trends Parasitol ,2002 ,18(11) :475- 476 .
[7]Andra J , Berninghausen O, Leippe M. Cecropins ,anti- bacterial peptides frominsects and mammals ,are potent- ly fungicidal againstCandida albicans[J]. Med-Microbi- ol-I mmunol-(Berl) ,2001 ,189(3) :169-173 .
[8]An RJ , Xing C L, Wang Z Y,et al . Feeding technique ofMusca domesticalinnaous[J].Journal of Zheli mu Ani- mal Husbandry College ,1999 ,9(2) :49-52 .
[9]Xu J H,Zhu J Y,Jin X B,et al .Molecular Cloning and sequence analysis of Cecropin-His _ 6 fusion gene from Musca domesticaand construction of its expression vector [J].Biotechnology ,2005 ,15(3) :3-7 .
[10]Sambrook J , Fritsch EF, Maniatis T. Molecular clo- ning :a laboratory manual[M].2nd ed .Beijing :Science Press ,1992 .852-897 .
[11]Schagger H,Jagow G. Tricine-sodium dodecyl sulfate- polyacrylamide gel electrophoresis for the separation of proteins in the range from1 to 100 kDa[J]. Anal Bio- chem,1987 ,166 :368-379 .
[12]Hult mark D, Steriner HR, Rasmuson T,et al .Insect i mmunity : purification and properties of the inducible bacterial proteins from hemolymph of i mmunized pupae of Hyalophora cecropin[J]. Eur J Biochem,1980 ,106 : 7-16 .
[13]Jin X B,Zhu J Y.The applications of insect antibaeteri- al peptides[J].Chin J Parasit Dis Con,2004 , 17(2) : 125-127 .
[14]Lu J ,Wang J H, Zhong Y,et al .Purfication and char- acterization of weak-acid antibacterial peptide MD7095fromMusca domesticalarvae[J]. Acta Microbiologica Sinica ,2006 ,46(3) :406-411 .
[15]Otvos L Jr . Antibacterial peptides isolated frominsects [J].J-PEPT-SCI ,2000 ,6(10) :497-511 .
[16]Andra J ,Berninghausen O,Leippe M.Cecropins ,anti- bacterial peptides from insects and mammals , are po- tently fungicidal againstCandida albicans[J]. Med Mi- crobiol I mmunol (Berl) ,2001 ,189(3) :169-173 .
[17]Hancock RE, Lehrer R. Cationic peptides : a new source of antibiotics[J].Trends Biotechnol ,1998 ,16 (2) :82-88 .
[18]De Lucca A J , Walsh T J . Antifungal peptides : novel therapeutic compounds against emerging pathogens[J]. Anti microb Agents Chemother ,1999 ,43(1) :1-11 .
[19]Hancock R E. Peptide antibiotics[J]. Lancet , 1997 , 349(9049) :418-422 .
[20]Andersons D,Engstrom A,Josephson S,et al .Biolog- ically active and amidated cecropin producedin a baculo- virus expression systemfroma fusion construct contai- ning the antibody-binding part of protein A[J]. Bio- chemJ ,1991 ,280(Pt 1) :219-224 .
[21]Dou F,Xie W,Deng X Y,et al .The terminal structure plays an i mportant role in the biological activity of CMIV[J].Science in China(Series C) , 1999 ,42(5) : 494-500 .
[22]Peng Z Y, Yu X B, Wu Z D,et al .Cloning and expres- sion inEscherichia coliof a new gene ofSchistosoma japonicumencoding casein kinase II beta subunit[J]. Chin Med J (Engl) ,2004 ,117(9) :1321-1325 .
[23]Ahmad S L, Ali M M, Mustafa A S. Construction of a modified vector for efficient purification of recombinant Mycobacteriumtuberculosisproteins expressed inEsch- erichiacoli[J]. Protein Expr Purif ,2003 ,29(2) :167- 175 .
[24]Smith DB,Corcoran L M.Current protocols in molecu- lar biology[M]. New York: Academic Press ,1990 .23- 30 .
[25]Shen Y, Qiu Y H, Lao X G,et al .Cloning of cecropin X gene and its expression inE.coli[J]. LETTERS IN BIOTECHNOLOGY,2003 ,14(4) :278-281 .
