真核表达质粒PcDNA3.1(+)-带有白蛋白信号肽的Vasostatin(120-180aa)的构建及其在体内外细胞中的表达  

Including Albium signal peptide Vasostatin(120-180aa) Gene Clone and Its Expression in eukaryotic Cells

作　　者：刘建巨[1] 关继奎[2] 王琳[1] 赵秀梅[1] 郑建秋[1] 李丹[1] 闫庆慧[1] 

机构地区：[1]哈尔滨医科大学第一临床医学院眼科医院,黑龙江哈尔滨150001 [2]大庆油田总医院

出　　处：《中国实验诊断学》2011年第1期11-14,共4页Chinese Journal of Laboratory Diagnosis

基　　金：黑龙江省卫生厅立项项目(2007-221);哈尔滨医科大学附属第一医院科研基金资助项目(2007051)

摘　　要：目的构建真核表达质粒PcDNA3.1(+)-带有白蛋白信号肽的Vasostatin(120-180aa)并了解其在体内外真核细胞中的表达。方法将带有白蛋白信号肽的Vasostatin(120-180aa)的逆转录聚合酶链式反应(RT-PCR)产物克隆至pcDNA3.1(+)真核表达载体上,经酶切鉴定及测序分析证明构建成功后,以脂质体介导法转染体外培养的真核293T细胞及注射入鼠骨骼肌细胞中,了解其在体内外真核细胞中的表达情况。结果我们获取了包含带有白蛋白信号肽的Vasostatin(120-180aa)成熟肽及HA抗原在内的一段cDNA片段,并成功地与真核表达载体PcDNA3.1(+)连接,经测序表明,其中含有的带有白蛋白信号肽的Vasostatin(120-180aa)成熟肽的序列与Genebank上报道的序列完全一致。即我们成功构建了真核表达质粒PcDNA3.1(+)-带有白蛋白信号肽的Vasostatin(120-180aa)。之后,在转染了该质粒的体外真核293T细胞的上清液中及骨骼肌注射了该质粒的鼠血液中,利用Western-blot法检测到了目的基因的蛋白质。结论带有白蛋白信号肽的真核表达质粒PcDNA3.1(+)-Vasostatin(120-180aa)增强了蛋白分泌表达能力,这为进一步应用该质粒治疗全身新生血管类病奠定了一定基础.Objective To construct the recombined plasmid PcDNA3.1（＋）-Vasostatin（120-180aa） including Albium signal peptide and observe its expression in eukaryotic Cells.Methods Vasostatin（120-180aa） gene was amplified by reverse transcription polymerase chain reaction（RT-PCR） method from human liver tissue,then,was inserted into the eukaryotic expression vector of pcDNA3.1（＋） to construct the recombined plasmids.293T cells were transfected mediated by liposome,muscle cells were injected by recombined plasmid,the expressed protein was detected by Western blotting.Results Exact gene order of Vasostatin（120-180aa） was obtained from the human liver tissue by RT-PCR;PcDNA3.1（＋）-Vasostatin（120-180aa） was constructed,and transient expression was going after transfecting and injecting.Conclusion The ability of secretion expression of combined plasmids we constructed was strengthened after we added Albium signal peptide to Vasostatin（120-180aa） Gene.

关 键 词：Vasostatin(120-180aa) 293T细胞 基因克隆 真核表达 

分 类 号：R346[医药卫生—基础医学]