Cloning and Prokaryotic Expression Analysis of HnGUA Gene from Hirudo Nipponia
Objective To clone the HnGUA gene from Hirudo nipponia and conduct bioinformatics analysis,protein prokaryotic expression analysis and gene differential expression analysis.Methods Based on the transcriptome data of H.nipponia in the previous study,the full-length cDNA of HnGUA was cloned by rapid amplification of cDNA ends(RACE),and bioinformatics analysis was performed.The prokaryotic expression vector was constructed,transformed into Escherichia coli BL21(DE3)competent cells and the expression of recombinant protein was induced by IPTG.The qPCR was used to further analyze the tissue-specific expression of HnGUA.Results The size of HnGUA gene was 504 bp,containing an open reading frame(ORF)of 231 bp and encoding 76 amino acids.Its protein molecular weight and isoelectric point are 8.17 kDa and 4.44,respectively.Multiple sequence alignment analysis showed that HnGUA was highly homologous to genes in other leech species that encode inhibitory proteins.The results of the prokaryotic expression analysis showed that the constructed pET32a-HnGUA vector could be successfully expressed in E.coli BL21(DE3),and the SDS-PAGE results showed that the induced recombinantly expressed HnGUA protein was around 6 kDa,which was basically consistent with the predicted protein size.The results of the Real-time PCR revealed spatial and temporal differences in the expression profiles of HnGUA,with high levels of expression detected in the skin and crop tissues.Conclusion This study represents the first successful cloning of the HnGUA gene from H.nipponia and the expression of the corresponding recombinant protein in E.coli.It provides a foundation for future exploration of the biosynthetic pathways and molecular regulatory mechanisms of active small anticoagulant molecules in leeches.
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