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.
出处
《中国实验诊断学》
北大核心
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)
