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人淋巴毒素cDNA在大肠杆菌表达的研究 |
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张津利 朱锡华
摘 要 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-blotting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2×105 U/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。
关键词 人淋巴毒素 表达 大肠杆菌
中国图书分类号 Q785
Expression of cDNA for human lymphotoxin in E.coli
ZHANG Jin-Li，ZHU Xi-Hua.
Department of Immunology,the Third Military Medical University,Chongqing 400038
Abstract 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 2×105 U /L of E.coli fermentation broth.Conclusion:These results paved the ways for production,clinical use and researches of LT.
Key words Human lymphotoxin Expression E.coli
淋巴毒素(Lymphotoxin,LT)又称肿瘤坏死因子β(TNF-β)，它可在体内或体外特异地杀伤多种肿瘤细胞，并具有抗病毒及免疫调节活性，因其细胞毒作用与TNF-α相似，而毒副作用大大低于TNF-α而倍受重视，是一种非常有前途的抗肿瘤和抗病毒生物制剂［1］。本实验将本室克隆的人淋巴毒素cDNA［2］构建于原核表达载体，以期在大肠杆菌表达人淋巴毒素(rLT)。
1 材料与方法
1.1 pBV-LT表达载体的构建 原核表达载体pBV220由中国预防医学科学院病毒所提供［3］，经内切酶SmaI切成双平端，同时将克隆于pUC18-LT中的去信号肽LTcDNA［2］经HindⅢ/Nco I双酶切，Klenow酶填平，载体与LTcDNA按常规方法连接并转化大肠杆菌DH5α，地高辛标记探针菌落原位杂交法初步筛选阳性重组子，可疑阳性重组子经酶切进一步鉴定并确定插入方向［4］，获得的正向阳性重组子命名为pBV-L[1] [2] [3] 下一页
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