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目的克隆与乳腺癌MCF-7细胞相关的旋毛虫TS498基因,并进行原核表达。方法从含TS498基因的噬菌体文库中PCR扩增TS498基因,亚克隆入原核表达载体pET-28a(+)中,构建重组表达质粒pET-28a-TS498,转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE和Western blot分析表达产物。结果扩增的旋毛虫TS498基因与GenBank中登录的基因序列同源性为100%,重组表达质粒pET-28a-TS498经双酶切和测序证明构建正确;表达的重组蛋白相对分子质量约23 000,表达量约占菌体总蛋白的20%,能够被兔抗旋毛虫肌幼虫阳性血清识别。结论成功克隆并在大肠杆菌中表达了与MCF-7细胞相关的旋毛虫TS498基因,为进一步研究其特性和功能奠定了基础。
Objective To clone TS498 gene of Trichinella spiralis associated with breast cancer MCF-7 cells and express in prokaryotic cells. Methods The TS498 gene was amplified by PCR from the phage library containing TS498 gene and subcloned into the prokaryotic expression vector pET-28a (+). The recombinant plasmid pET-28a-TS498 was transformed into E. coli BL21 (DE3) The products were analyzed by SDS-PAGE and Western blot. Results The amplified sequence of Trichinella spiralis TS498 gene was 100% identical to that of GenBank. The recombinant plasmid pET-28a-TS498 was verified by double enzyme digestion and sequencing. The recombinant protein had a relative molecular mass of about 23 000 , The expression of about 20% of the total bacterial protein, can be positive for rabbit anti-Trichinella spiralis larvae sera. Conclusion The TS498 gene of Trichinella spiralis related to MCF-7 cells was successfully cloned and expressed in Escherichia coli, which laid the foundation for further study of its characteristics and functions.