In vitro expression of the recombinant fusion protein of Newcastle disease virus from local Indonesian isolates by using a cell-free protein expression system

Q4 Environmental Science Indonesian Journal of Biotechnology Pub Date : 2020-11-10 DOI:10.22146/IJBIOTECH.54703
A. Haryanto, H. Wihadmadyatami, N. Wijayanti
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引用次数: 1

Abstract

The aim of this work was the in vitro expression of the recombinant fusion (F) protein of Newcastle disease virus (NDV).  The pBT7-N-His-Fusion-NDV expression plasmid which carries the recombinant F protein encoding gene from local Indonesian isolates, was prepared and transformed into E. coli BL21 (DE3). To detect bacterial colonies carrying the recombinant plasmid, a restriction endonuclease analysis was performed using the EcoRI restriction endonuclease. These results showed that the pBT-N-His-Fusion-NDV plasmid was successfully isolated with a size of 4.601 bp, and three recombinant plasmids carrying the gene coding for the recombinant F protein of NDV were obtained. Selected recombinant plasmids were then in vitro by using a cell-free protein expression system followed by visualization of the recombinant F protein on a 12% SDS-PAGE gel both by Coomassie Brilliant Blue staining and Western blotting. Recombinant F protein was successfully in vitro expressed by using a cell-free protein expression system as indicated by a specific single protein band with a molecular mass of 25.6 kDa.
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用无细胞蛋白表达系统体外表达印尼本地分离的新城疫病毒重组融合蛋白
本研究的目的是在体外表达新城疫病毒(NDV)重组融合蛋白(F)。制备了携带印尼当地分离株重组F蛋白编码基因的pBT7-N-His-Fusion-NDV表达质粒,并将其转化为大肠杆菌BL21 (DE3)。为了检测携带重组质粒的细菌菌落,使用EcoRI限制性内切酶进行了限制性内切酶分析。上述结果表明,成功分离到了pBT-N-His-Fusion-NDV质粒,质粒大小为4.601 bp,获得了3个携带NDV重组F蛋白编码基因的重组质粒。选择重组质粒,用无细胞蛋白表达系统进行体外表达,然后用12% SDS-PAGE凝胶对重组F蛋白进行考马斯亮蓝染色和Western blotting。重组F蛋白通过无细胞蛋白表达系统在体外成功表达,其特异的单蛋白带分子量为25.6 kDa。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Indonesian Journal of Biotechnology
Indonesian Journal of Biotechnology Environmental Science-Environmental Science (miscellaneous)
CiteScore
1.00
自引率
0.00%
发文量
20
审稿时长
12 weeks
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