Identification And Characterization Of Three Cystatins From Musca Domestica

Cysteine proteinase inhibitors (cystatin) involve in many vital cellular processes such as signaling pathway, apoptosis, immune response and development. Musca domestica L. (Diptera: Muscidae), commonly called the housefly, is a major domestic, medical and veterinary pest that causes irritation, spoils food and acts as a vector for many pathogenic organisms. As the house flies are ubiquitous, research on various aspects especially the developmental processes of housefly has been conducted world wide. This study with housefly contains 5 parts.Part 1: Three new cysteine protease inhibitors, named Mdcystatin A (MdCYA) ,Mdcystatin B1 (MdCYB1 ) and Mdcystatin B2 (MdCYB2) respectively, were cloned from housefly by RACE (rapid-amplification of cDNA ends) based on dbEST (the expressed sequence tags database) and transcriptome sequencing. The deduced peptides were analyzed.Part 2:β-actin and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were used as the internal control. Relative expressions of three cystatin genes at different conditions were detected respectively by RT-qPCR analyses.Part 3: To understand the inhibitory activity of cystatins to cysteine protease, three prokaryotic expression vectors were constructed and then expressed in Escherichia coli BL21. These activities of recombinant proteins were detected.Part 4: Immunohistochemical localization of MdCYA in the larvae was investigated using polyclonal antibody.Part 5: Knock down of MdCYA and MdCYB1 respectively by RNA-interference approach. The functions of the genes in metamorphosis were analyzed.Results:(1) A full-length 416 bp cDNA gene sequence of the housefly MdCYA was cloned, which containing a 357 bp complete open reading frame (ORF), which MdCYA peptide of 118 amino acids, N terminal signal peptide consists of 17 amino acids, MdCYA mature peptide contains 101 amino acids, theoretical molecular weight is 10.8 kD, theoretical isoelectric point is 5.37. The result of sequence alignments analysis showed that this gene shares 51% identity with sarcocystatin A of Sarcophaga peregrina.MdCYB1 and MdCYB2 were cloned by transcriptome sequencing. A full-length 607 bp cDNA gene sequence of the housefly MdCYB1 was cloned, which containing a 372 bp complete open reading frame (ORF), according to ORF deduced that MdCYB1 peptide has 123 amino acids, N terminal signal peptide consists of 22 amino acids, MdCYB1 mature peptide contains 101 amino acids, theoretical molecular weight is 11.3 kD, theoretical isoelectric point is 9.23. The result of sequence alignments analysis showed that this gene shares 62% identity with sarcocystatin B of S. crassipalpis.A full-length 400 bp cDNA gene sequence of the housefly MdCYB2 was cloned, which containing a 333 bp complete open reading frame (ORF), according to ORF deduced that MdCYB2 peptide has 110 amino acids and does not contain N terminal signal peptide. The theoretical molecular weight and theoretical isoelectric point of MdCYB2 are 12.2 kD and 6.10 respectively. The result of sequence alignments analysis showed that this gene shares 58% identity with sarcocystatin B of Sarcophaga crassipalpis.(2) The expression data indicated that the MdCYA mRNA expression were constitutively expressed in naive larvae and down-regulated after the larvae were challenged with bacteria. But mRNA expression of MdCYB1 and MdCYB2 were up-regulated, reached the maximum in 48 h and 6 h respectively. The result suggested that MdCYB2 may be involved in defense responses to microbial invasion. Musca larvae may early entry into the pupal stage after infection therefore results in MdCYB1 up-regulated. The three genes were expressed highly in hemocyte and the fat body by RT-qPCR. MdCYA is expressed at a higher level during larval and adult stage, decreased expression during larvae feeding and reached a minimum in larvae moulting and the metamorphically committed stage, another two was opposite. These results suggest that all of these genes participate in developmental processes of house?y. The relative expressions of the three genes in larvaes did not changed significantly at different nutrient conditions.(3) Three functional expression systems for recombinant cystatins in E. coli cells were constructed to obtain purified proteins by His affinity chromatography for characterization of the activities of rMdCYA, rMdCYB1 and rMdCYB2. The purified proteins showed single band of about 11.6 kD,12.1 kD and 13 kD in SDS-PAGE. They are consistent with there theoretical molecular weight. rMdCYA and rMdCYB1 could also effectively inhibited the papain of family C1 cysteine protease, but rMdCYB2 could not effectively inhibited the papain.(4) MdCYA polyclonal anti-serum were prepared by immuning New Zealand rabbit with the recombined protein prepared, Western Blot showed that the anti-serum have good specificity. Immunolocalization analysis reveals that MdCYA is specifically expressed in hemocytes and fat bodies.(5) Genomic fragments cloned into L4440 were transformed into HT115 (DE3). Pick bacteria and feed the larvae. The results suggest that knock down of MdCYA do not affect the larval development, but knock down of MdCYB1 results in delayed pupation.