Objective To Construction of gene regulation of specific human decorin expressed in A 549 lung cancer cell gene expression vector ,the gene for the further application of targeted therapy for lung cancer experimental basis .Methods Application of PCR from human peripheral blood gene was amplified lung cancer cell-specific expression of human secretory leukocyte protease inhibitor promoter ,the use of recombinant DNA technology the promoter inserted into the eukaryotic expression vector given experimental basis .pcDNA3 .1 (+ ) ,replace the carrier's CMV promoter and enhancer sequences ,which were constructed by the secretory leuko-cyte protease inhibitor gene promoter gene expression starts expression vector .The human decorin gene inserted by genetic recombination technology to the carrier who secretory leukocyte protease inhibitor gene promoter downstream .PCR products were identified by sequencing the nucleotide sequence .The recombinant vector was identified by restriction enzyme digestion .Results PCR amplification of human secretory leukocyte protease inhibitor production degree promoter fragment 1 250 bp ;obtained by sequencing the promot-er and the Genebank nucleotide sequence upstream of the gene on the 5 ' end of the transcriptional regulatory region of the sequence was exactly ;recombinant vector The restriction enzyme digestion showed that (1) the success of the promoter inserted into the pcD-NA3 .1 (+ ) vector,and replace the CMV promoter and enhancer sequences ,(2) Human decorin gene was successfully inserted into the vector .Conclusion Successfully constructed by human secsetory leukocyte protease inhibitor gene promoter human decorin gene expression vector ,the vector to achieve control of decorin gene in human lung cancer cell-specific expression .%目的 构建调控人核心蛋白聚糖基因特异性在肺癌细胞A549内表达的基因表达载体,为进一步应用该基因进行肺癌靶向治疗的研究奠定实验基础.方法 应用PCR技术从人外周血基因组中扩增肺癌细胞特异性表达的人分泌型白细胞蛋白酶抑制剂的启动子,利用基因重组技术将该启动子插入真核细胞表达载体pcDNA3.1(+),替换该载体的CMV启动子与增强子序列,从而构建成由人分泌型白细胞蛋白酶抑制剂基因的启动子启动基因表达的基因表达载体.将人核心蛋白聚糖基因通过基因重组技术插入到上述载体人分泌型白细胞蛋白酶抑制剂基因启动子的下游.PCR产物通过测序鉴定核苷酸序列.重组载体通过限制性酶切进行鉴定.结果 PCR扩增的人分泌型白细胞蛋白酶抑制剂的启动子片段长度为1 250 bp;该启动子经测序获得的核苷酸序列与Genebank 上该基因上游5'末端转录调控区的序列完全一致;重组载体的酶切鉴定结果显示(1)该启动子成功插入到pcDNA3.1(+)载体,并替换CMV启动子与增强子序列,(2)人核心蛋白聚糖基因成功插入该载体.结论 成功构建由人分泌型白细胞蛋白酶抑制剂基因启动子调控人核心蛋白聚糖基因表达载体,该载体实现调控人核心蛋白聚糖基因在肺癌细胞内特异性表达.
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