鱼腥藻Ⅱ型果糖-1,6-二磷酸醛缩酶基因的克隆及其在大肠杆菌中的高效表达  

Cloning and High Expression Anabaena Class-II Fructose-1, 6-bisphosphate Aldolase Gene in Escherichia coli

作　　者：魏兰珍[1] 崔轶文[1] 马为民[1] 王全喜[1] 

机构地区：[1]上海师范大学生命与环境科学学院,上海200234

出　　处：《中国生物工程杂志》2006年第5期74-77,共4页China Biotechnology

基　　金：上海高校选拔培养优秀青年教师科研专项基金项目(RE569)

摘　　要：随着更多蓝藻全基因组序列测定完成,蓝藻基因工程研究现已进入后基因组时代。2001年Kaneko等完成了鱼腥藻7120全基因组序列测定,随后人们利用生物信息学的方法对其中一些基因的功能进行了预测,包括II型果糖1,6二磷酸醛缩酶(FBA)基因,但该基因是否编码II型果糖1,6二磷酸醛缩酶及该产物的相关酶学特性至今尚未见报道。通过PCR克隆到鱼腥藻7120中预测的II型FBA基因,插入到质粒pET32a的相应位点,构建成原核表达载体pETFBAII。蛋白电泳结果显示,重组蛋白的表达量占总蛋白含量的23.4%,与蛋白分子标记相比,其分子量约为40kDa。酶学活性测定结果表明,其蛋白粗提物的比活力为11.8U(mgprotein)-1,具有标准II型FBA活性。证实了Cyanobase中关于该基因功能的预测,也为进一步研究该基因表达产物的生理生化特性及功能提供了重要资料。The complete genomes of more cyanobacterial strains have been completed for sequence, and genetic engineering of cyanobacteria has evolved in the post-genome era. Since Kaneko and colleagues had completed the sequence for the complete genome of Anabaena sp. PCC 7120 in 2001, functions of some genes in this genome, including fructose-1,6-bisphosphate aldolase （FBA） gene, have been predicated using the method of bioinformatics. However, little information is available regarding whether this gene can encode FBA and its product characteristics of related enzyme. Here, to explore this information, the predicted Ⅱ-FBA gene-encoding region in Cyanobase database was cloned by PCR method and then ligated into pET-32a to generate the expression vector, pET-FBA-Ⅱ. The results of SDS-PAGE indicated that the expression level of the expected target polypeptide was approximate 23.4 percent as compared to total protein and the molecular weight is about 40 kDa as compared to the protein molecular marker. The results of enzyme activity analysis showed that the activity of Ⅱ- FBA was ～11.8 U per mg protein and owned a standard activity of Ⅱ-FBA. To sum up, the results not only prove the functional prediction of this Ⅱ-FBA gene from the Cyanobase database, but also provide the important conditions for further studying its physiological and biochemical characteristics and functions of the gene expression product.

关 键 词：果糖-1 6-二磷酸醛缩酶 鱼腥藻7120 表达 酶活 

分 类 号：Q786[生物学—分子生物学]