核糖核酸酶barnase基因的克隆策略研究

比较了目前报道的两种克隆barnase基因的方法——barnase基因的单独克隆和barnase抑制剂基因保护下的barnase基因克隆。分别利用4种质粒进行barnase基因的单独克隆时，得到的阳性克隆数量少。对其中15个阳性克隆进行了测序分析，结果有5个克降出现碱基缺失，1个克隆出现碱基增加，其余9个克隆则出现氨基酸突变，均未得到氨基酸序列完正确的克隆。进一步分析表明这些突变的位点大多直接与保持barnase蛋白的活性位点相关。而在克隆barnase基因之前，预先将barnase基因连同其自身启动了加载于待克隆载体上，随后再进行barnase基因克隆时则容易得到与报道的barnase基因序列完全一致的阳性克隆。分析认为由于barnase基因单独存在时有一定数世的蛋白表达，宿主菌大肠杆菌无法忍受而通过某种方式造成其barnase发生相应突变而无法实现barnase基因的单独克隆。因此有必要利用barnase基因的保护来进行barnase基因相应的克降和遗传操作。

Cloning Strategy of Barnase Gene (Extracelluar Ribonuclease Gene)

Two barnase-cloning methods currently reported,independent cloning and barstar gene-protected cloning of barnase gene were comparatively examined.When four plasmids were respectively used to independently clone barnase gen,a small number of positive clones resulted.Fifteen of these positive clones were analytically sequenced and it was accordingly founded that five showed nucleotide deletion,one showed nucleotide increment and the other nine showed amino acid mutations and did not obtained completely cloned amino acid sequences.It was revealed in further analysis that most of these mutation sites directly related to the active sites that maintained barnase activity.Moreover,the positive clones that had the same gene sequences as reported barnase gene sequence could be easily obtained if barstar gene along with its own promoter was loaded on the carrier to be cloned prior to barnase gene cloning.It was indicated through analysis that when barnase gene independently appeared it presented a certain amount of protein expression that the host Escherichia coli could not tolerate so as to bring about the corresponding mutations in some manner,which made the independent cloning of barnase gene come true.