人淋巴毒素cDNA在大肠杆菌表达的研究

Expression of cDNA for human lymphotoxin in E.coli

目的：在大肠杆菌表达人淋巴毒素ｃＤＮＡ。方法：将本室克隆的人淋巴毒素ｃＤＮＡ亚克隆于原核表达载体ｐＢＶ２２０，温控法诱导重组菌，ＳＤＳＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ检测和鉴定结果，ＭＴＴ比色法测定重组ＬＴ活性。结果：成功地表达了人淋巴毒素重组蛋白；重组蛋白占细菌总蛋白的１２％，并具有细胞毒活性，活性单位相当于２×１０５Ｕ／Ｌ菌液。结论：为重组ＬＴ的大量生产、临床应用，为进一步研究人ＬＴ的生物学功能奠定了基础。

Objective:To express human lymphotoxin (LT or TNF β) cDNA in E.coli.Method: A LT cDNA fragment isolated in this laboratory was subcloned into prokaryotic expression plasmid pBV220 and expressed in E.coli DH5α by temperature shifting from 30℃ to 42℃。The expressed protein(rLT) was confirmed by SDS PAGE and Western blotting analysis,and its cytotoxic activity on L929 cells was evaluated by MTT colorimeric assay.Results: Recombinant LT had been successfully expressed in E.coli.It has a molecular weight 18～19 kD,approximating to its molecular mass calculated from the primary amino acid sequence,and showed positive reaction with anti LT monoclonal antibodies and cytotoxic effect on L929 cells. The expressed protein band constituted 12% of total bacterial protein and the cytotoxic activity of rLT was 2×10 5 U /L of E.coli fermentation broth.Conclusion:These results paved the ways for production,clinical use and researches of LT.