摘要
试验目的是获得S蛋白受体结合域基因,并获得其高效表达,为SARS病毒受体的进一步研究奠定一定的基础。首先通过PCR方法获得了S蛋白起始密码和SalⅠ限制性内切酶之间包含受体结合区的片段,然后将该基因定向克隆到pET-22b原核表达载体,构建了pET-22b-S1重组质粒,转化大肠杆菌并诱导目的蛋白的表达,经SDS-PAGE没有发现明显的目的蛋白带。利用载体和S1基因上的NcoⅠ酶切位点,将S1基因的信号肽序列和部分疏水序列切掉后,构建pET-22b-SNS重组质粒。pET-22b-SNS重组质粒仍然包含受体结合域序列,并且阅读框没有改变。将pET-22b-SNS转化大肠杆菌,发现明显的目的蛋白带。Western blot结果表明表达蛋白为SARS病毒S1蛋白的一部分。
In order to get an efficient way to express SARS receptor binding domain, the S1 gene fragment was amplified through PCR and ligated to pEr- 22b directly. The recombinant vector was transformed into E. coli BL21 and induced with IPTG, but the target protein was not detected. Taking advantage of the restriction endonudease Nco Ⅰ of S1 gene and pEr- 22b, the signal sequence and some hydrophobic part of S1 gene were cut off to get another recombinant vector pEr- 22b- SNS. The recombinant vector which contained the gene fragment between restriction endonucleaso Nco Ⅰ and Sal Ⅰ of S1 gent was expressed successfully in E. coli BL21 and the expressed protein was mostly soluble. The Western blot experiment showed it reacted well with the polyolone antibody of SARS.
出处
《生物技术》
CAS
CSCD
2005年第6期20-22,共3页
Biotechnology
