构建胶质原纤维酸性蛋白原核表达载体及蛋白表达鉴定

Protein identification following construct glial fibillary acidic protein prokaryotic expression vector

背景:胶质原纤维酸性蛋白在神经损伤的发生发展过程中具有重要作用。目的:对胶质纤维酸性蛋白进行基因克隆,构建原核表达质粒并加以鉴定。设计、时间及地点:单一样本观察,于2008-09/11在上海市长征医院神经外科实验室完成。材料:中间载体pGEM-TEasy质粒购于Promega公司,原核表达质粒pGEX-4T-2由上海市长征医院神经外科实验室保存。方法:从人脑胶质瘤组织提取mRNA,采用反转录-聚合酶链反应的方法扩增胶质纤维酸性蛋白序列并表达,然后与载体pGEX-4T-2连接,转化宿主菌BL21构建重组质粒,诱导表达后纯化,Western-blot鉴定。主要观察指标:总RNA鉴定结果,聚合酶链反应扩增产物分子质量的鉴定,重组表达载体的酶切鉴定,Western-blot鉴定。结果:抽提肿瘤组织总RNA后,电泳清晰可见rRNA28S、18S、5S3条带。且总RNA的A260nm/A280nm值为1.903。聚合酶链反应结果显示,在约1300bp左右有一条特异性条带,与预期的胶质原纤维酸性蛋白基因大小一致。重组表达载体的酶切鉴定及测序结果与GenBank的胶质原纤维酸性蛋白序列一致,经Western-blot验证为目的蛋白。结论:用双酶切、DNA测序与Western-blot的方法证实原核表达载体构建成功。

BACKGROUND： Glial fibrillary acidic protein （GFAP） plays an important role in the nerve injury. OBJECTIVE： To perform gene clone to the whole gene of GFAP, and to construct and identify the prokaryotic expression plasmid, DESIGN, TIME AND SETTING： The single sample experiment was performed at Laboratory of Neurosurgery Department, Changzheng Hospital from September to November 2008. MATIERIALS： The pGEM-T Easy vector was purchased from the Promega Corporation, and the original expression plasmid pGEX-4T-2 was provided by Laboratory of Neurosurgery Department, Changzheng Hospital. METHODS： The mRNA was isolated from human brain glioma tissue, and subjected to RT-PCR to obtain GFAP fragment, then express, link it with prokaryotic expression plasmid pGEX-4T-2 to express and purify GFAP on the basis of E.Coli BL21. Finally the expected protein was identified by Western-blot. MAIN OUTCOME MEASURES： Identification of total RNA; PCR amplification product; enzyme digestion of recombinant expression vector; and Western-blot results. RESULTS： After extracting total RNA of glioma tissue, 3 strip of rRNA28 S, 18 S, 5 S could be found in electrophoresis. The A260 ore/A280 or, value of total RNA was 1.903. PCR detected a specific band at the 1300 bp, which was identical with the size of the GFAP gene. The enzyme digestion and DNA sequencing showed that the recombinant expression vector was coincided with the GFAP sequence of GeneBank, which was verified as objective protein by Western-blot. CONCLUSION： The constructed GFAP in prokaryotic expression vector was successful identified by double digestion, DNA sequencing and Western-blot.