Objective: To clone the cDNA sequence of Musca domestica antifungal peptide-1 (MAF-1) and analyze the amino acid sequence of MAF-1 by bioinformatics method.
Methods: Based on the primer designed according to the N-terminal amino acid sequence of MAF-1, the cDNA and amino sequence of MAF-1 were obtained by the methods of RACE and NestPCR. The accuracy of the experiment was confirmed by RT-PCR The characteristic of the sequence was analyzed by bioinformatics software.
Results: The length of the cDNA sequence of MAF-1 was 568 bp by 3'RACE, including an open reading frame (ORF) of 441 bp length and 3'UTR of 127 bp. It was a novel sequence with the submission number of HM178948 in GenBank since none homology was found when compared with other sequences by Blast. Added with the 9 amino acids that were not used to design primer, the whole sequence of MAF-1 was 156 amino acids conferred from its cDNA. 139 bp cDNA sequence was obtained by 5'RACE and the result was consistent to 3'RACE. The result of RT-PCR showed the cDNA of MAF-1 mature peptide was accurate. The bioinformatics analysis deduced that the theoretic molecular weight and isoelectric point of the whole protein sequence of MAF-1 gene were similar to those detected. The ExPASy illustrated that the MAF-1 gene had a signal peptide. There were abundant a-helix in it, the domain located between the 128 and 153 amino acid residuals. Subcellular analysis showed MAF-1 was almost in the nucleus. Predict Protein found two protein kinase C phosphorylation sites and one N-myristoylation site, and predicted that it was not a globular protein. In the end, the three dimension image of MAF-1 was set up with 3D-pssm of ExPASy.
Conclusion: The cDNA sequence and the amino acid sequence of MAF-1 have been obtained and analyzed successfully.