摘要
目的构建pEGFP-N3-TFPI-2真核表达载体,为研究TFPI-2基因的功能做准备。方法从胎盘组织中提取总RNA,逆转录合成cDNA,再以PCR法进行扩增。将扩增的TFPI-2基因片段克隆到pEGFP-N3真核表达载体上,经XhoⅠ和KpnⅠ双酶切后,行1%琼脂糖凝胶电泳鉴定,同时测定其DNA序列。将pEGFP-N3-TFPI-2真核表达载体以脂质体LipofectamineTM 2000介导的方法转染Top10感受态细胞(转染组),同时设空白对照组(仅转染pEGFP-N3质粒)和未转染组(不予转染)。采用Western blot法检测3组细胞中TFPI-2蛋白的表达情况。结果总RNA经分光光度法验证,其纯度符合PCR法扩增的要求。构建的pEGFP-N3-TFPI-2真核表达载体经XhoⅠ和KpnⅠ双酶切后,行1%琼脂糖凝胶电泳,结果显示,分别在708 bp处和4 700 bp处有特异扩增条带,大小与理论值一致。DNA序列测定结果证实,pEFFP-N3-TFPI-2真核表达载体的序列完全正确。Western blot法结果显示,TFPI-2蛋白的表达量转染组为0.657 3±0.032 5,空白对照组为0.301 7±0.028 7,未转染组为0.314 3±0.026 6,转染组TFPI-2蛋白的表达量高于空白对照组和未转染组,差异有统计学意义(P<0.01)。结论本实验成功构建了pEGFP-N3-TFPI-2真核表达载体,为TFPI-2基因功能的研究奠定了基础。
Objective To construct eukaryotic expression vector ofpEGFP-N3-TFPI-2, and to provide the base of studying the function of TFPI-2 gene. Methods Extraction of total RNA from placental tissue was extracted at first, and then reverse transcriptase synthesis of eDNA was carried out. The eDNA fragment ofTFPI-2 gene which was obtained by real time PCR （RT-PCID was inserted into eukaryotic expression vector ofpEGFP-N3. After double digestion with Xho I and Kpn I , the recombinant vector of pEGFP-N3-TFPI-2 was identified in 1% agarose gel eleetrophoresis and was tested by the sequence analysis. Then, the recombinant vector of pEGFP-N3-TFPI-2 （transfection group） and vector of pEGFP-N3 （blank control group） were transfected into Topl0 competent cells with LipofectamineTM 2000, but no transfeetion-related treatment was performed in cells of untransfection group. Western blot method was used to test the expression of TFPI-2 protein in cells of 3 groups. Results The purity of total RNA which were analysis by agarose gel electrophoresis and spectrophotometry were fit for PCR. After coding of TFPI-2 gene fragment and eukaryotic expression vector of pEGFP-N3, the recombinant plasmid of pEGFP-N3-TFPI-2 were got double digestion with Xho I and Kpn I , and was identified in 1% agarose gel electrophoresis, of which showing that there were 2 specific amplification of strips at 708 bp and 4 700 bp. Result of sequence analysis confirmed that the size of recombinant vector was consistent with the theoretical value. Results of Western blot showed that the expression of TFPI-2 protein in transfection group （0. 657 3 ± 0. 032 5） was higher than those of blank control group （0. 301 7 ± 0. 028 7） and untransfection group （0. 314 3 ± 0. 026 6）, P〈 0. 01. Conclusions The eukaryotic expression vector of pEGFP-N3-TFPI-2 has been constructed successfully, which laiding the founda-tion for the analysis about function of TFPI-2 gene.
出处
《中国普外基础与临床杂志》
CAS
2014年第3期300-304,共5页
Chinese Journal of Bases and Clinics In General Surgery
