胃泌素释放肽前体融合蛋白的制备及其在肿瘤研究中的应用

目的克隆胃泌素释放肽前体(pro-GRP)基因、构建重组质粒pGEM-T-pro-GRP和表达载体PMS-31b-pro-GRP、在大肠杆菌中热诱导表达并制备融合蛋白。方法从BGC823胃癌细胞中提取总RNA,利用pro-GRP特异引物,扩增人pro-GRP分子的cDNA全长。将pro-GRP基因定向克隆于pGEM-T载体转化JM109,DNA测序鉴定基因序列。将pro-GRP基因定向克隆于原核高效表达载体PMS-31b,转化大肠杆菌POP2136经42℃热诱导表达MS2-pro-GRP融合蛋白。将融合蛋白分离纯化后,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)凝胶鉴定。结果经聚合酶链反应(PCR)扩增成功获得210bp的pro-GRP基因,目的基因序列正确。SDS-PAGE显示热诱导后表达的融合蛋白分子量约为18000。结论成功克隆pro-GRP基因,并表达和纯化了PMS-31b-pro-GRP融合蛋白,为建立pro-GRP检测方法奠定了基础。

Prepare and application of recombinant human pro-gastrin releasing peptide

Objective To clone the human pro-gastrin releasing peptide (pro-GRP) gene, construct the pGEM-T-pro-GRP recombinant plasmid and PMS-31b-pro-GRP vector, and to produce the fusion protein by heat-induced expression in Escherichia coli. Methods The total RNA was extracted from BGC823 cells. Using pro-GRP specific primers, wes amplified the full-length cDNA of human pro-GRP. The RT-PCR products were ligated to pGEM vector to transfect JM109 for DNA sequencing. After this, the recombinant pro-GRP was ligated to the expression vector pMS-31b and transfected into E coli (POP2136), where MS2-pro-GRP fusion protein was expressed by heat-induction at 42 degree Celsius. Results The acquired gene was 210 bp and its sequence was correct. The gene product, characterized by SDS-PAGE, appeared to be a fusion protein with molecular weight of 18 000. Conclusion The clone, expression and purification of human pro-gastrin releasing peptide lay a foundation for detection of pro-GRP.