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

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等人完成了鱼腥藻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。本文通过PCR克隆到鱼腥藻7120中预测的II型FBA基因，插入到质粒pET-32a的相应位点，构建成原核表达载体pET-FBA-II。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40 kDa。酶学活性测定结果表明，其蛋白粗提物的比活力为11.8 U (mg protein)-1，具有标准II型FBA活性。本研究不仅证实了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条件。

Cloning and high expression Anabaena Class-II Fructose-1,6-bisphosphate aldolase gene in Escherichia coli

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. Taken together, 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.