颗粒溶素融合蛋白的克隆表达和免疫学鉴定

Cloning expression and identification of granulysin fusion protein

目的采用基因工程方法重组并表达颗粒溶素融合蛋白。方法采用逆转录-聚合酶链反应(RT-PCR)克隆颗粒溶素多肽分子的cDNA,经测序证实后定向插入表达载体pET28a(+),并经双酶切电泳鉴定。进而导入大肠杆菌BL21(DE3)plyS中,经异丙基硫代-β-D半乳糖苷(IPTG)诱导后表达重组融合蛋白。结果经聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western印迹法鉴定,在相对分子质量为9000处有明显的条带。结论经重组表达获得颗粒溶素融合蛋白,有助于进一步探讨颗粒溶素与临床疾病的相关性。

Objective Cloning expression of granulysin recombinant fusion protein in E. coli. Methods The cDNA encoding human granulysin was obtained by RT PCR, confirmed by DNA sequencing, subcloned unidirectionally into the bacterial expression plasmid pET28a（ ＋ ） and then transformed into E. coilBL21 （DE3） plyS to express the recombinant fusion protein induced by IPTG. Results It was shown that there was a marked band at 9 kilodalton by SDS and Western blotting. Conclusion The granulysin recombinant fusion protein so obtained can be used for study of its relevance with clinical diseases.