Prokaryotic Expression And Functional Preliminary Analysis Of U93 Gene In Musca Domestica

Objective:Construction the prokaryotic expression system of Musca domestica u93 gene and preliminary identification the activity of recombinant expression products.To prepare polyclonal antibody against protein encoded by the u93 gene from Musca domestica,preliminary validation of Musca domestica u93 protein expression of time and space characteristics.Methods: The u93 gene was screened from the Musca domestica transcriptome library and the physicochemical properties of u93 gene coding protein was analyzed by the bioinformatics methods.The combinant vector pET29a-u93 was transformed intoE.coli BL21/DE3 and inducted with IPTG.The production was analyzed with SDS-PAGE electrophoresis.The recombinant protein was purified by use of Ni-NTA.Antimicrobial activity of u93 recombinant protein was determined with microdilution method and MTT.Using protein immunoassay to immunize New Zealand white rabbits with purified recombinant protein as antigen to obtain polyclonal antibodies.The titer of polyclonal antibody of the u93 protein was determined by ELISA.The immunological specificity of the polyclonal antibody was detected by Western blot.Detection of different tissues(salivary gland,hemolymph,fat body,intestines,trachea,martensitic tube and body wall)expression of normal raising three instar larvae of Musca domestica,Western-Blotting was used to detect the localization of the u93 protein in Musca domestica;Three-day instar larvae infected with Candida albicans by injection instrument were used to detect intestinal expression of Musca domestica u93 protein at 3h,6h,9h,12 h,and 24 h by Western-Blotting method.Results: Bioinformatics analysis showed that ORF of the u93 gene was 363 bp that encoded a putative protein with 120 amino acids.The protein with predicted molecular weight 13.35 kDa and pI of 7.52,the signal peptide is located between amino acids 1-20.intracellular secretory proteins,therefore,it is believed that u93 were belong to the SVWC(Single domain von Willebrand factor type C)superfamily.The u93 recombinant protein is mainly expressed in the form of inclusion body in E.coli BL21/DE3,and the relative molecular mass of the recombinant protein is consistent with the theoretical value.The u93 Recombinant protein can inhibit the growth of C.albicans(MIC 21?g/mL),C.parapsilosis(MIC 12.5?g/mL),C.krusei(MIC 12.5?g/mL)and C.tropicalis(MIC 12.5?g/mL),However,it does not inhibit the growth and reproduction of E.coli and Staphylococcus aureus.The titer of anti-u93 protein polyclonal antibody was higher than 1: 4000,Western-Blottingshowed that the antibody specifically recognized the natural u93 protein.Normal feeding third-instar larvas,dissect their different tissues(salivaryglands,haemolymph,fat bodies,intestines,trachea,malpighiantubules,and body walls),Western-Blotting results showed that theu93 protein was localized in the intestine.After infection with Candida albicans,the expression level of the u93 protein was significantly increased at 6h.As time went by,the expression level of the u93 protein was significantly reduced at 12 h.Compared with the control group,the infection group showed a trend of increasing first,then decreasing and then increasing gradually.Conclusion: The Musca domesticau93 gene was successfully cloned and build the PET-29a-u93-BL21(DE3)prokaryotic expression system,acquire purified protein and immune serum.Microfluid dilution method and MTT colorimetric method confirmed the antifungal activity of the Musca domesticau93 recombinant protein.Western-Blotting results showed that the Musca domestica u93 recombinant protein has better purity and specificity,it was mainly localized in the intestine and was tissue-specific.After three-day instar larvae of Musca domestica infected with Candida albicans,theMusca domestica u93 protein was significantly increased after infection for sixhours.Especially after twelvehours of infection,the expression level of the Musca domesticau93 protein was significantly reduced.It is presumed that it participates in the intestinal innate immune process,resists the invasion of pathogenic microorganisms,and the protein may be depleted,thereby reducing the protein expression level;it may also be Candida albicans secreted some proteases and degraded the Musca domestica u93 protein,resulting in decreased protein expression.This study lays a foundation for the subsequent study of the innate immune system of Musca domestica on the recognition mechanism of fungi.