脊髓灰质炎病毒衣壳多肽VP1在大肠杆菌中的表达。

Expression of poliovirus capsid polypeptide VP1 in Escherichia coli.

作者信息

van der Werf S, Dréano M, Bruneau P, Kopecka H, Girard M

出版信息

Gene. 1983 Jul;23(1):85-93. doi: 10.1016/0378-1119(83)90219-6.

Abstract

A recombinant plasmid (pPV1-958) carrying a poliovirus cDNA insert corresponding to nucleotides 1-5750 of the poliovirus RNA genome was constructed through in vitro recombination of two plasmids carrying overlapping poliovirus cDNA inserts (Van der Werf et al., Proc. Natl. Acad. Sci. USA 78 (1981) 5983-5987). pPV1-958 was then cleaved with PstI and the fragment carrying the sequence encoding capsid polypeptide VP1 was isolated and subcloned at the PstI site of pBR322. The VP1 sequence was successfully fused with the 5' sequence of the beta-lactamase gene by deleting 300-350 bp from each side of the PvuI site of the plasmid with nuclease BAL31. One of the resulting plasmids, pSW119, expressed an Mr 49 000 VP1-beta-lactamase-fusion protein, which was specifically immunoprecipitated with anti-VP1 immune serum but not by hyperimmune sera raised against native or heat-inactivated virus.

摘要

通过对两个携带重叠脊髓灰质炎病毒cDNA插入片段的质粒进行体外重组，构建了一个重组质粒（pPV1 - 958），该质粒携带与脊髓灰质炎病毒RNA基因组第1至5750个核苷酸相对应的脊髓灰质炎病毒cDNA插入片段（范德韦夫等人，《美国国家科学院院刊》78 (1981) 5983 - 5987）。然后用PstI切割pPV1 - 958，并分离出携带编码衣壳多肽VP1序列的片段，将其亚克隆到pBR322的PstI位点。通过用核酸酶BAL31从质粒的PvuI位点两侧各删除300 - 350 bp，VP1序列成功地与β-内酰胺酶基因的5'序列融合。所得到的质粒之一pSW119表达了一种分子量为49000的VP1 - β-内酰胺酶融合蛋白，该蛋白能被抗VP1免疫血清特异性免疫沉淀，但不能被针对天然或热灭活病毒产生的超免疫血清免疫沉淀。