摘要
目的:对殊异韦荣菌(V·dispar)苹果酸脱氢酶(malate dehydrogenase,MDH)基因进行克隆和重组表达,并对其重组蛋白进行纯化和活性测定。方法:提取殊异韦荣菌基因组DNA,PCR扩增MDH同源区序列片段,克隆入pET-28a载体并转化至大肠杆菌DH5α,酶切及PCR鉴定,测序。将重组质粒转入BL21(DE3)中,选择最佳表达条件,提纯及测定活性。结果:PCR扩增产物特异,全长1139bp。测序结果包含MDH基因,并与GenBank中所报道的大肠杆菌MDH基因序列进行对比分析,其同源性为99%。MDH纯化后的蛋白活性值为0.4403U/mL。结论:成功克隆殊异韦荣菌MDH基因,并通过基因序列和氨基酸分析证明其具有完整的阅读框架。还获得了重组表达MDH蛋白的最适表达条件,并测出韦荣菌苹果酸脱氢酶的活性。
Objective： To clone and express the matate dehydrogenase（MDH） gene of Veillonella dispar（V, dispar） ,and to purify and assay activity in Veillonella of the recombinant protein. Methods： To extract genomic DNA of V. dispar,MDH gene was amplified by PCR method and was inserted into pET--28a vector to transform E. coilDH5a. The sequencing was performed after PCR identification and restriction enzyme digestion. This experiment transform the recombinant plasmid pET-28a-MDH in BL21 （DE3）. Induction condition was optimized to obtain high yield of recombinant Protein. Then to determine the purification and activity of recombinant MDH. Results： The PCR product was specific, and the size of the product was 1139 bp. The sequencing data were analyzed by BLAST software and the result indicated that it contained MDH. The homology of the sequencing result was 99 compared with the MDH gene of Escherichia coli which was reported in GenBank. The recombinant protein after purification with MDH reactive kit it＇ s protein active value of 0. 4403U/ml. Conclusion： The MDH gene of Veil- lonella dispar was successfully cloned, and it is proved to have a full reading framework, through the analysis of the gene sequences and amino acids. The optimum condition for induction of MDH protein was selected.
出处
《口腔医学研究》
CAS
CSCD
2013年第11期1008-1011,1015,共5页
Journal of Oral Science Research
基金
吉林省自然科学基金项目(编号:201015204)
关键词
韦荣菌
苹果酸脱氢酶
基因克隆
重组表达
活性测定
Veillonella dispar Malate dehydrogenase Genecloning Recombinant expression Activity assay
