Molecular Cloning And Expression Of Clip-Msp And Mdctl From Musca Domestica And Their Clip-Msp Temporal-spatial Expression Pattern In Systemic Infections With Candida Albicans

Objective: To clone Clip-serine protease gene(Clip-Msp)and C-type lectin(Mdctl)of Musca domestica(Musca domestica),and to study Clip-Msp temporal-spatial expression pattern after Candida albicans infection.Methods: Clip-Msp(GeneID: LOC101901626)and Mdctl(GeneID: LOC101890789)were screened from the post-infection immune-related gene library constructed by our research group.Primers were designed according to the predicted sequence on NCBI website,and the target fragments were amplified by PCR.Their sequences and coding proteins were analyzed and predicted by bioinformatics software.The recombinant plasmids pET-28a/Clip-Msp and pET-28a/Mdctl were constructed and transformed into E.coli BL21-DE3 to induce expression.The purified proteins were obtained by Ni-IDA Agarose method.The expressed products and the purified proteins were identified by SDS-PAGE and Western-Blot analysis.Using GAPDH as internal reference gene,RT-PCR detected the expression level of Clip-Msp of the third instar larvae in different tissues(epidermis,fat body,hemolymph,salivary gland,intestine,malpighian tube and trachea)and at different time points(3h,12 h,24h and 48h)after systemic infections with Candida albicans.Results: The length of Clip-Msp ORF from Musca domestica is 1524 bp and the coding protein consists of 507 amino acids,theoretical molecular weight 56320.1 Da and isoelectric point 8.52.The protein is unstable in solution and has high hydrophobicity,lacks a signal peptide belonging to a non-classic secretory protein.There is a Clip domain at the N-terminal and a Tryp_SPc domain at the C-terminal of Clip-Msp protein,which belongs to the Clip-serine proteases superfamily.The Mdctl ORF is 333 bp in length and codes 110 amino acids.Its theoretical molecular weight is 13131.42 Da,isoelectric point 4.49.Mdctl is a secretory protein with a signal peptide between 1-22 amino acid residues.There is a Clect structure in the amino acid sequence of Mdctl protein,which belongs to the C-type lectins superfamily.The expression plasmids of pET-28a/Clip-Msp and pET-28a/Mdctl were successfully constructed.After induced by IPTG,the recombinant proteins were mainly expressed in the form of inclusion bodies in E.coli,and their molecular weight is consistent with the theoretical value.The results of RT-PCR showed that the expression level of Clip-Msp were significantly different(P<0.05)in different tissues at different time points after infection.There was a high expression level in hemolymph at 3 hours.The expression of Clip-Msp in fat body,trachea,salivary gland and intestine increased at 12 hours,and the highest expression in fat body;at 24 hours,there was the highest expression in intestine,followed by fat body;at 48 hours,the expression in hemolymph was higher than that in all other tissues.The results showed that Clip-Msp mainly distributes in hemolymph,fat body and intestine.Conclusion: Clip-Msp is a new member of the Clip-SPs superfamily without a signal peptide,encoding a protein of 507 amino acid residues.There is a tissue expression pattern of increasing first and then decreasing after the third instar larvae on systemic infections with Candida albicans.The high expression level of Clip-Msp shows in hemolymph,fat body and intestine.Mdctl of Musca domestica codes a new housefly lectin belonging to C-type lectins superfamily.The prokaryotic expression systems of pET-28a/Clip-Msp and pET-28a/Mdctl were successfully constructed,and the purified proteins were obtained.It is speculated that Clip-Msp and Mdctl may play a role in the immune response against fungal infection.The results should provide a basis for further research on Clip-Msp and Mdctl of Musca domestica.