| Metabolism is the foundation of life activity,includes material metabolism and energy metabolism.Microsporida are intracellular,eukaryotes parasites and have a widely host range.Different parasitic environment makes microsporidia different metabolic status.Genome analysis of Microsporidia found that its genome encoding protein sequence,on the length and quantity,has greatly reduced compression,especially the genes associated in metabolism path,so the metabolic research about Microsporidia has always been the hot topic of scholars.Genome analysis of Nosema bombycis found it has a complete glycolytic pathway,and can get a part of the energy required to life activities through this way.Enolase(ENO)is the rate-limiting enzyme in glycolytic pathway,the study of enolase enzyme can help us understand the energy metabolism of microsporidia better.While new features of have been discovered about enolase in recent years.So,whether the enolase in the life activities of Nosema bombycis has other functions or not also is what we are interested in.In this study,the Nb ENO gene was cloned with primers designed based on genomic data got from Microsporidia DB.The ORF of gene Nb ENO has 1239 bp and encoding 413 amino acids.The molecular weight and isoelectric point of Nb ENO were predicted as 46.323 k D and 6.13.The protein contained 3 kinds of secondary structures,alpha helix(28.33%),extended strand(22.52%)and random coil(49.15%).The protein contained 25 phosphorylation sites and a glycosylation site,without signal peptide and transmembrane domain.Sequence comparison and phylogenetic analysis with other 12 microsporidia from Microsporidia DB showed that there had a close relationship between Nb ENO and Nc ENO with 62.10% identity.Nb ENO was inserted into the prokaryotic expression vector of p ET-28 to construct prokaryotic expression vector p ET-28a-Nb ENO,recombine Nb ENO with 6×His tag in the N-terminal;Transformed Rosatta(DE3)cells with recombinant plasmid,Induced with IPTG.The results of SDS-PAGE and Western Blot test show that the recombinant bacteria could express a target protein Nb ENO with predicted molecular weight 50 k D,proved that recombinant protein Nb ENO was expressed successfully.Purify the recombinant Protein Nb ENO with Nickel ion affinity colμmn,immune the New Zealand white rabbit by Nb ENO and prepare antibody by Protein A antibody affinity.The valence of antibody anti-Nb ENO-Ab is 1:512,000,tested by ELISA.And the result of Western Blot shows that anti-Nb ENO – Ab can combined with Nb ENO specifically.So the antibody can satisfy the requirement of the experiment later.From the result of semi-quantitative analysis of the expression of Nb ENO,after silkworm was infected by Nosema bombycis,we know that Nb ENO was expressed all the time in the whole progress of Nosema bombycis infected silkworm.And the relatively quantitative analysis results of the expression of Nb ENO show that Nb ENO was expressed significantly higher at the early times after infect silkworm than other times,and expression level tended to be stable after the third day.The results of sub-cellular localization of Nb ENO showed that: Nb ENO was distributed in the surface of the spores,and in the spore coat,but there was not an obvious fluorescence signal in the sporoplasm.In this study we analysis gene Nb ENO and recombinant protein Nb ENO by bioinformatics methods,laid the foundation for further study of the functions and subcellular location of Nb ENO.Combined with the results of the relatively quantitative analysis of expression and sub-cellular localization of Nb ENO,we presμmed that the glycolytic pathway is the main source of energy at the early time after Nosema bombycis infected silkworm,and there may be another functions of Nb ENO in the life history of Nosema bombycis. |
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