摘要
目的揭示膜结合的TNF α前体(mTNF α)与分泌型TNF α(sTNF α)生物学活性的不同。方法在克隆出mTNF α基因的基础上,我们采用IL 2信号肽编码序列置换m TNF α前导肽编码序列,构建出只产生分泌型TNF α的真核表达质粒pBK sTNFm,并转染至NIH3T3细胞中。结果筛选出的阳性克隆细胞经Westernblot分析及活性测定显示,pBK sTNFm转染细胞裂解液及培养上清 ,均可检出相对分子质量 (Mr)17000的TNF α,且上清具有sTNF α活性,但细胞固定后其活性丧失。结论成功地构建了在真核细胞中只产生sTNF α的表达质粒,并建立相应的表达细胞株。为比较两种类型的TNF α的生物学活性及其作用机制奠定了基础。
Aim To reveal the differences between membrame bound TNF αprecusor(mTNF α) and secretory TNF α(sTNF α)in the bioactivities. Methods A recombinant TNF with only secretory form(pBK sTNFm) was constructed in which the leader sequence of TNF αwas replaced by the DNA sequence coding the human IL 2 signal peptide and then transfected into NIH3T3 cells. Results G418 resistant colonies and their expression products were identified by bioassay and Western blot. TNF with Mr 17 000 could be demonstrated both in cell lysate and culture supernatants of the pBK sTNFm transfected cells and only the supernatant displayed cytotoxicity. Conclusion A cell line only producing sTNF αis successfully established, which also provides the basis for studying the biologic activity and its mechanism.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
2000年第1期6-9,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金!No.39270314
