| Human ?-Galactosidase A(?-Gal A)is a lysosomal glycoprotein hydrolase encoded by the GALA gene on human X chromosomes,which is responsible for hydrolyzing the oligosaccharides,glycolipids,and glycopeptide ends ?-galactose residues in the lysosome.?-Gal A deficiency leads to the accumulation of ceramide trihexoside(GL3)in various tissues of the human body and induces Fabry disease.As an X chromosome-linked hereditary lysosomal storage disease,the clinical symptoms of Fabry disease are stroke in youth,left ventricular hypertrophy,etc.,which seriously threaten patients' lives.Enzyme replacement therapy(ERT)is currently the most effective method for treating Fabry disease.The human ?-Galactosidase currently approved for the clinics is expressed in Chinese hamster ovary cells or human fibroblasts.However,the low expression level and the high cost of the ?-Gal A expression system lead to the expensive treatment of patients with Fabry disease.It is therefore a long time goal to express high levels of human ?-Galactosidase in a system economically suitable for the treatment of Fabry disease.Trichoderma reesei(RUT-C30)is an asexual Hypocrea jecorina,a mesophilic filamentous fungus,has a robust protein synthesis and secretion system and mechanism that is eukaryotic in nature.Its N-Glycation and high mannose contents are similar to mammalian expression systems.T.reesei has also obtained "Generally recognized as safe(GRAS)" certification from the US Food and Drug Administration(FDA),which is non-pathogenic,free of mycotoxins and antibiotics during production,which provides a practical system for the efficient expression and production of human ?-Galactosidase in T.reesei.Although T.reesei has a good potential for industrialized protein producti on,the research in this field is still hampered by the few commercial expression vectors available and the expression levels of exogenous proteins are still low.Thus,the application of T.reesei expression system is very limited.Therefore,this study in tends to construct a universal and efficient expression vector suitable for T.reesei,and to apply this vector for the expression of human ?-Galactosidase.Objective: To construct a universal expression vector for T.reesei to provide a new option for the treatment of Fabry disease by achieving high expression of ?-Gal A.Methods:(1)Construction of a T.reesei expression vector:(1)After designing specific primers and extracting T.reesei genomic DNA,we performed PCR amplification and purification of the required vector elements.The purified vector DNA fragments were ligated with recombinase,sequenced,and run on gel electrophoresis to determine whether the T.reesei expression vector pTRUC was successfully constructed.(2)Using m Cherry red fluorescent protein as the reporter gene,we constructed pTRUC-m Cherry plasmid,and the function of which was analyze and identified using fluorescence microscope,PCR and Western blotting analysis.(2)Study on the expression of human ?-Galactosidase fusion protein in T.reesei:(1)The codon-optimized GALA cDNA was fused to the CBH I gene in the pTRUC expression vector to construct a pTRUC-CBH I-HGal plasmid and transformed into T.reesei and the corresponding protein expression was detected by Western blotting.(2)Detection of transcript levels of the genes related to unfolded protein response(UPR)& ER-associated degradation(ERAD)pathway in ?-Gal A recombinant bacteria by q PCR.Results:(1)Construction of the T.reesei expression vector:(1)Sequencing and gel electrophoresis showed that the T.reesei expression vector pTRUC was successfully constructed;(2)A strong red fluorescent signal was observed under a fluorescence microscope for m Cherry recombinant bacteria;m Cherry was detected in the host genome through PCR assays;Western blotting assay further showed that the red fluorescent protein m Cherry was correctly expressed in T.reesei.The above results indicate that the T.reesei expression vector pTRUC was successful constructed,and the foreign gene was correctly expressed in T.reesei.(2)Study on the expression of ?-Gal A fusion protein in T.reesei:(1)Western blotting results showed that CBH I and ?-Gal A fusion protein(CBH I-HGal)were successfully expressed in T.reesei,and accumulated the most in the fermentation supernatant 2 days during fermentation.The optimal ammonium sulfate concentration for precipitation was 70%.GALA gene could be stably passaged in the T.reesei genome and human ?-Galactosidase fusion protein was successfully expressed during the passage.(2)The q PCR results showed that the transcription of immunoglobulin binding protein Bip gene in the UPR was not significantly different from that of the parent strain;the transcription levels of disulfide isomerase gene,Pdi,and oxidoreductase gene,Ero,were significantly higher than those of the parent strain.The expression of ?-Gal A by ?-Gal A recombinant bacteria was still low,indicating that the recombinant bacteria express the CBH I-HGal fusion protein did not stimulate UPR.However,the transcription level of ubiquitin ligase Hrd1,Der1,Hrd3 genes in the ERAD pathway are not significantly different from the parental strain;the transcription of glucosyltransferase Gpt,glucosyl hydrolase Gls2,?-mannosidase hydrolase Mns1 gene of ?-Gal A recombinant bacteria was 4-6 times higher than those of the parent strain,indicating that the expression of CBH I-HGal fusion protein by ?-Gal A recombinant bacteria stimulated the ERAD.The transcriptional level of genes related to UPR response and ERAD provide a theoretical basis for the subsequent study of ?-Gal A expression and regulation mechanisms in T.reesei.Conclusion: The successful construction of the universal expression vector achieves the expression of ?-Gal A in T.reesei,which provides a possible scientific basis for the treatment of Fabry disease. |
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