Cloning,expression And Characterization Of MdMtn1 And MdSERP1 From Musca Domestica

The housefly is an important resource insect, their larvae and adults prefer to feed on mineral-rich mammalian feces and live in the environment with much bacteria, which indicates they have strong adaptive ability to the poor environment. Metallothionein and stress-associated endoplasmic reticulum protein are important stress resistance proteins. Understanding of the function of these proteins from housefly is helpful in realizing the biological resilience mechanism, when they are in stressful environments.In this paper, we use Musca domestica as the research object, the content is divided into five sections:Part 1: With the rapid-amplification of cDNA ends (RACE) technique, the cDNA sequence of metallothionein (MdMtn1) was cloned from the housefly larvae (GenBank accession number GU289398), and its nucleotide and protein sequences were analyzed with bioinformatics methods.Part 2: Withβ-actin gene as a internal control, the expression pattern of MdMtn1 in larvae following stimulation by CdCl₂ was detected using real-time fluorescence quantitative PCR (qRT-PCR).Part 3: The prokaryotic expression vector of MdMtn1 was constructed, and then transformed into Escherichia coli BL21 (DE3). Recombinant protein was induced to expression in E. coli. In addition, the heavy metal binding activity of MdMtn1 was detected in vivo.Part 4: Using bioinformatics technique, we analyzed the housefly MdSERP1(contig 481) gene and amino acid sequence. qRT-PCR was adopted to detect mRNA transcript profile of MdSERP1 in larvae after stimulated by bacteria and cadmium.Part 5: The MdSERP1 prokaryotic expression vector was constructed, and then a fusion protein DsbA-MdSERP1 was expressed and purified, which was used as antigen to make antibody in New Zealand rabbit.The results are as follows:(1) The full-length cDNA of MdMtn1 is 408 bp, containing a 123 bp open reading frame and encoding a protein of 40 amino acid residues. The MdMtn1 peptide sequence included 10 Cysteine residues with a distribution pattern of -C-X-C-. The predicted molecular weight of encoding protein is 3.8 kD with the isoelectric point (pI) of 8.78. The deduced amino acid sequence of MdMtn1 showed 69% homology with MtnA of Drosophila melanogaster and D. simulans.(2) The Cd²⁺ concentration required for 50% larvae viability was 30 mM. The qRT-PCR results showed that when using 30 mmol / L CdCl₂ induced housefly larvae, the expression trend decreased first and then increased. When stimulated for 24 h, the expression level of MdMtn1 was higher than the blank. However, different Cd²⁺ concentrations treatment significantly increased MTs expression in a dose-dependent manner. MdMtn1 mRNA expression increased with dose and reached the highest level at 24 h with exposure to 10 mM Cd.(3) The MdMtn1 prokaryotic expression vector was constructed by prokaryotic vector pET-DsbA. SDS-PAGE protein electrophoresis revealed that the fusion protein of DsbA-MdMtn1 molecular weight is 28.5 kD. In order to detect the MdMtn1 activity in binding heavy metals, the target gene was cloned into a prokaryotic expression vector pET-DsbA and then a fusion protein was expressed in Escherichia coli BL21 (DE3). In the presence of CdCl₂, the expression of MdMtn1 significantly increased the bacteria tolerance to Cd²⁺, suggesting that MdMtn1 may play an active role in housefly adaptation to the environment with heavy metals.(4) MdSERP1 gene contains a 195 bp open reading frame which encodes 64 amino acids. The predicted molecular weight of encoding protein is 7.15 kD with the isoelectric point (pI) of 10.38. There is a hydrophobic region and transmembrane region located on 36-58 amino acid residues, without signal peptide. The result of sequence alignments analysis showed that this protein belongs to RAMP4 superfamily. Homology comparison found that N-terminal sequence of SERP1 is quite different, but the C-terminal containing the transmembrane region is very conservative.(5) To detect the expression profiles of MdSERP1, fly larvae were exposed to bacteria and CdCl₂. The results of qRT-PCR showed that Staphylococcus aureus treatment caused MdSERP1 mRNA rise smoothly, which reached the maximum level at 24 h, and then decreased gradually. After the stimulation of E. coli, the level of MdSERP1 mRNA decreased at first and then gradually restore, it reached the lowest point at 6 h, and more than control groups at 36 h. The MdSERP1 variation tendency of fly larvae stimulated by cadmium is similar to the MdMtn1, but the degree of change is different. (6) The MdSERP1 prokaryotic expression vector was constructed with pET-DsbA, and the recombined plasmid was transfected into DE3. The result of SDS-PAGE showed that the molecular weight of the recombined protein is approximately 31 kD, and the protein is expressed in supernatant. The fusion protein was purified using His affinity chromatography and was used for preparation of SERP1 antibody. Fineness of the antibody was detected via western blot.