摘要
通过PCR方法扩增不含信号肽的鸡GM-CSF成熟蛋白基因,克隆至原核表达载体pET-32a(+),构建原核表达质粒p32GM-CSF,通过IPTG诱导重组鸡GM-CSF蛋白表达,经镍离子亲和层析纯化后,用MTT法检测表达重组蛋白的生物学活性,并制备兔抗鸡GM-CSF多克隆抗体。结果表明成功地构建了p32GM-CSF原核表达质粒,SDS-PAGE结果显示表达的重组蛋白约34 ku,主要以包涵体形式表达,纯化后得到高纯度目的蛋白。West-ern Blot结果表明,该重组蛋白能与制备的抗鸡GM-CSF抗体特异性结合。MTT试验证实,该重组蛋白具有明显增强鸡骨髓细胞增殖的生物学活性;这些研究结果表明,表达的重组chGM-CSF蛋白及其多克隆抗体拥有相应的生物学功能,这将为进一步研究鸡GM-CSF蛋白的生物学功能及其应用奠定基础。
The ChGM-CSF gene without signal peptide was amplified by PCR method and ligated into the expression plasmid pET-32a（＋） to construct prokaryotic plasmid p32GM-CSF. ChGM- CSF recombinant protein was expressed by induction with IPTG in E. coli and purified with Ni- NTA column. MTT assay was used to detect bioactivity of rChGM-CSF. Polyclonal antibody was produced by rabbit injected with the purified protein. The result showed that expression plasmid p32GM-CSF was successfully constructed. SDS-PAGE demonstrated that the recombinant protein was expressed in form of inclusion body and was approximately 34 ku. Western Blot analysis showed that it could be specifically recognized by the rabbit sera to chicken GM-CSF. Results of MTT assay confirmed that it can enhance chicken bone marrow cell proliferation obviously. These results demonstrated that the E. coli-derived rChGM-CSF and polyclonal antibody against rChGM-CSF have some bioactivities. Our results would provide foundation for further research of biology characteristics and application of ChGM-CSF.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2009年第5期725-729,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家“十一五”863计划资助(2006AA10A205)
