Roopashree S D, Chai C, Ho B, Ding J L
Department of Zoology, National University of Singapore, Kent Ridge Crescent.
Biochem Mol Biol Int. 1995 Apr;35(4):841-9.
The cDNA encoding Factor C (FC) from the Singaporean horseshoe crab Carcinoscorpius rotundicauda has been studied for in vitro coupled transcription-translation (TnT) under the T7 promoter. Two species of full length cDNA, CrFC26 and CrFC21 which differ in length and nucleotide sequence at their 5' untranslated regions (UTR) were used in this study. Wild type CrFC26 with a long 5' UTR containing multiple "false" ATGs failed to generate a translated product. With a more accessible ATG codon in CrFC21, the recombinant construct gave a high yield of FC when transcribed and translated in vitro. CrFC26 deletion mutants which lack the entire 5' UTR and portions of the putative leader peptide were translatable, albeit at lower efficiency as compared to CrFC21. In vitro and in vivo expression of truncated portions of the CrFC21-T7 gene 10 fusions have been compared. In vitro reactions yielded single gene products from each of the expression constructs whereas E. coli produced three major immunoreactive bands of FC.
对来自新加坡圆尾鲎（Carcinoscorpius rotundicauda）的编码C因子（FC）的cDNA在T7启动子下进行了体外偶联转录-翻译（TnT）研究。本研究使用了两种全长cDNA，即CrFC26和CrFC21，它们在5'非翻译区（UTR）的长度和核苷酸序列上有所不同。具有长5'UTR且包含多个“假”ATG的野生型CrFC26未能产生翻译产物。由于CrFC21中的ATG密码子更容易接近，该重组构建体在体外转录和翻译时产生了高产率的FC。缺少整个5'UTR和部分推定前导肽的CrFC26缺失突变体是可翻译的，尽管与CrFC21相比效率较低。已比较了CrFC21-T7基因10融合体截短部分的体外和体内表达。体外反应从每个表达构建体产生单一基因产物，而大肠杆菌产生了FC的三条主要免疫反应带。
