目的构建N-TIMP3重组腺病毒载体,为深入研究组织金属蛋白酶抑制因子-3(tissue inhibitor of metalloproteinases-3,TIMP-3)N端结构域对胰腺癌的作用奠定基础。方法PCR扩增编码人TIMP3信号肽及其N端结构域的DNA序列,定向克隆至空载穿梭质...目的构建N-TIMP3重组腺病毒载体,为深入研究组织金属蛋白酶抑制因子-3(tissue inhibitor of metalloproteinases-3,TIMP-3)N端结构域对胰腺癌的作用奠定基础。方法PCR扩增编码人TIMP3信号肽及其N端结构域的DNA序列,定向克隆至空载穿梭质粒pShuttle-CMV,构建重组穿梭质粒pShuttle-N-TIMP3,并测序鉴定。将重组pShuttle-N-TIMP3转化BJ5183感受态细菌,与其内的腺病毒骨架质粒pAdEasy-1进行同源重组,构建重组腺病毒载体pAd-N-TIMP3,并经酶切与PCR鉴定。将线性化的重组载体转染QBI-293A细胞包装病毒,TCID50法检测病毒滴度,用Western印迹检测目的蛋白的表达。结果重组腺病毒载体pAd-N-TIMP3经鉴定正确,病毒滴度为5×108PFU/ml,并检测到目的蛋白的表达。结论成功构建了重组腺病毒载体pAd-N-TIMP3。展开更多
The N-terminal domain of heat shock protein 90(Hsp90~N) is responsible for the catalytic activity of Hsp90.The reported inhibitors of Hsp90 bind to this domain and would inhibit tumor growth and progression. Here,we s...The N-terminal domain of heat shock protein 90(Hsp90~N) is responsible for the catalytic activity of Hsp90.The reported inhibitors of Hsp90 bind to this domain and would inhibit tumor growth and progression. Here,we synthesized FS23, a small molecule inhibitor of hsp90 and collected X-ray diffraction data of the complex crystal of Hsp90-FS23. High resolution X-ray crystallography shows that FS23 interacted with Hsp90 Nat the nucleotide binding cleft, and this suggests that FS23 may complete with nucleotides to bind to Hsp90~N. The crystal structure and the interaction between Hsp90 Nand FS23 suggest a rational basis for the design of novel antitumor drugs.展开更多
文摘目的构建N-TIMP3重组腺病毒载体,为深入研究组织金属蛋白酶抑制因子-3(tissue inhibitor of metalloproteinases-3,TIMP-3)N端结构域对胰腺癌的作用奠定基础。方法PCR扩增编码人TIMP3信号肽及其N端结构域的DNA序列,定向克隆至空载穿梭质粒pShuttle-CMV,构建重组穿梭质粒pShuttle-N-TIMP3,并测序鉴定。将重组pShuttle-N-TIMP3转化BJ5183感受态细菌,与其内的腺病毒骨架质粒pAdEasy-1进行同源重组,构建重组腺病毒载体pAd-N-TIMP3,并经酶切与PCR鉴定。将线性化的重组载体转染QBI-293A细胞包装病毒,TCID50法检测病毒滴度,用Western印迹检测目的蛋白的表达。结果重组腺病毒载体pAd-N-TIMP3经鉴定正确,病毒滴度为5×108PFU/ml,并检测到目的蛋白的表达。结论成功构建了重组腺病毒载体pAd-N-TIMP3。
基金Supported by National Natural Science Foundation of China(No.81402850)the Natural Science Basic Research Plan in Shaanxi Province of China(No.2015JM3114)+1 种基金the Introduced talents Foundation of Xi’an Medical University(No.2015 RCYJ01)the Doctorate Foundation of Xi’an Medical University(No.2015 DOC23)
文摘The N-terminal domain of heat shock protein 90(Hsp90~N) is responsible for the catalytic activity of Hsp90.The reported inhibitors of Hsp90 bind to this domain and would inhibit tumor growth and progression. Here,we synthesized FS23, a small molecule inhibitor of hsp90 and collected X-ray diffraction data of the complex crystal of Hsp90-FS23. High resolution X-ray crystallography shows that FS23 interacted with Hsp90 Nat the nucleotide binding cleft, and this suggests that FS23 may complete with nucleotides to bind to Hsp90~N. The crystal structure and the interaction between Hsp90 Nand FS23 suggest a rational basis for the design of novel antitumor drugs.
