摘要
目的获得大量具有生物活性的人成熟β细胞素(BTC)蛋白。方法以人胰岛细胞瘤cDNA为模板,PCR扩增BTC基因成熟蛋白编码区的全部序列,并按读框克隆入原核细胞表达载体pET32a(+)中,构建重组质粒pET32a(+)-hBTC。重组质粒转化大肠杆菌BL21,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导及亲和层析法纯化获得融合蛋白,运用SDS-PAGE和Western blot进行鉴定。以不同浓度BTC连续5 d作用于NIH3T3细胞,MTT比色法检测细胞增殖能力。结果融合蛋白以水溶性形式分泌于大肠杆菌BL-21胞浆中,其作用于NIH3T3细胞可明显促进细胞增殖。结论人成熟BTC蛋白在pET32a(+)表达系统中得到高效表达,所纯化蛋白具有促进NIH3T3细胞体外增殖的作用。
Objective To obtain abundant human betacellulin （BTC） with biological activity. Methods The whole mature protein coding sequence of BTC gene was amplified by polymerase chain reaction （PCR） method applied to human pancreatic β-cell tumors cDNA. The fragment was cloned into prokaryotic expression vector pET32a（ ＋ ） plasmid. The recombinant plasmid was transformed into E. coli BL21 and the fusion protein was expressed under isopropyl-beta-D-thiogalactopyranoside （ IPTG）. The fusion protein was purified by Ni^2 ＋ affinity chromatography. SDS-PAGE and Western blot were employed to determine the expression and purification of the expected protein. BTC was added to culture NIH3T3 cells for 5 days, and cell proliferation was detected by MTT. Results Lots of fusion protein were produced, and the purified protein can stimulate the proliferation of NIH3T3 cells. Conclusion The human BTC can be successfully obtained from the pET32a（ ＋ ） system with the biological activity of stimulating the proliferation of NIH3T3 cells.
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2007年第2期185-188,共4页
Journal of Shanghai Jiao tong University:Medical Science
关键词
Β细胞素
克隆
表达载体
纯化蛋白质
betacellulin
clone
expression vector
purified protein
