关键词: 殊异韦荣菌, S-腺苷同型半胱氨酸水解酶, 基因克隆, 基因表达, 纯化

Abstract:

Abstract:Objective To clone,identify,express and purify the S-adenosyl-L-homocysteine hydrolase (sahH) gene of Veillonella dispar and to provide theoretical basis for  the mechanism of dental caries disease. Methods The DNA  exacted from Veillonella dispar sahH gene were amplified by PCR method,and the recombinant plasmid PET28a-sahH was constructed and transformed into E.coil BL21.The sequencing was performed after PCR identification and restriction enzyme digestion.Then the protein expression was induced by IPTG and the protein was purified.Results The PCR product  was 1 410 bp.The sequencing data was analyzed by BLAST software.The homology of the sequencing result was 99 % compared with the sahH gene of Pseudomonas aeruginosa strain PAO1 which was reported in GenBank.The sequence had been submitted to the NCBI GenBank,and the accession number was KC477409.With SDS-PAGE electrophoresis,the recombinant plasmid PET28a-sahH was expressed and purified in  E.coil JM109,and the molecular weight of 50 000 was noted in sahH protein bands.The highest amount of protein expression was generated 5 h after  induction,The protein concentration was 5.4 g/L after concentrated.The size of protein obtained by Western blotting method was corresponded with target  protein.Conclusion The sahH gene of Veillonella dispar is successfully cloned,and it is proved to have a full reading framework through the analysis on gene sequences and amino acids.And the recombinant vector could  express fusion protein,and the interest protein is obtained by isolation and  purification.

Key words: Veillonella dispar, S-adenosyl-L-homocysteine hydrolase, gene cloning, gene expression, purification
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