Cloning And Expression Analysis Of Cecropin Gene Family In Culex Quinquefasciatus

Objective: To clone Cecropin gene family of Culex quinquefasciatus, and then to analyze the animo acid sequences, the protein physicochemical properties, the molecular structures, and the evolution relation of 4 Cecropin genes. Expression analysis of Cecropin genes of Culex quinquefasciatus was performed by semi-quantitative PCR. CecB2 was chose to clone into expression vector for prokaryotic expression, and the mature peptide of CecB2 was synthesized for antibacterial tests in vitro. Methods: Screened out 4 Cecropin genes from genome and transcriptome of Culex quinquefasciatus and analyzed the gene sequences by bioinformatics. Designed specific primers of 4 genes to clone them into pMD-18 T vector, then identified by sequenced. Total RNA were extracted at different development stages of Culex quinquefasciatus and the expression analysis of 4 genes was performed by semi-quantitative PCR. The mature peptide of CecB2 was inserted into the multiple cloning sites of the expression vector pET32 a to construct recombinant plasmid which was identified using double enzyme digestion, PCR and sequenced. The recombinant protein of CecB2 was expressed by IPTG induction in E.coli Rosetta after optimizing the concentration and time of IPTG induction,SDS-PAGE and Western blot were used to detect and indentify the recombinant protein respectively.Then the purified protein was dialysed、concentrated and cutted off by enterokinase, Tricine-SDS-PAGE was used to detect the CecB2 protein.The mature peptide of Cec B2 was chemically synthesized to detect antibacterial activity in vitro. Results: 1. The average of the length, theoretical molecular weight and pI of 4Cecropin genes were 60 aa, 6389.0 and 10.69 respectively, all of Cecropin were small molecule, alkalic proteins. 4 Cecropin genes were consisted of two exons and one intron. 4 Cecropin proteins were composed with one signal peptide and one domain which main components was α-helix. Phylogenetic analysis showed that thegenetic relationship between Culex quinquefasciatus and Aedes aegypti, Anopheles gambiae was relatively close. 2. Semi-quantitative PCR study found that CecB2 expressed throughout the development stages, and CecB1, CecA1, CecA2 were expressed in every stage excepted the egg stage. 3. The recombinant plasmid pET32a-Cec B2 was constructed successfully and optimum induction condition of fusion protein were as follows: IPTG concentration was 0.05mmol/L, induction time was 3h,the purified fusion prorein was about 25 kD,but Tricine-SDS-PAGE had not detected CecB2 protein. 4.The synthetic peptide of CecB2 had bacteriostatic activities to Gram-positive bacterial(Staphyloccocus aureus, Staphylococcus albus,Microcococcus lysodeikticus), gram-negative bacterial(Escherichia coli) and fungi(Candida albicans), the results showed that CecB2 produced obvious inhibition zones and a maximum size of the bacteriostatic and fungistatic zone were achieved 17mm(Microcococcus lysodeikticus) and 8mm(Candida albicans) diameter respectively at the tested amount of the protein. Conclusion: 1. CecA1, CecA2, CecB1, CecB2 were a kind of small molecule, alkalic proteins. 2. CecB2 expressed throughout the development stages, and CecB1, CecA1, CecA2 expressed in every stage except the egg stage. 3. The recombinant prorein of CecB2 could be expressed as a soluble fusion protein in E.coli Rosetta, it was about 25 kD. 4.The synthetic peptide of CecB2 with broadspectrum bacteriostatic activities might be as antibiotic candidate molecules.