摘要
目的构建MAGE-A4的原核表达质粒并表达纯化相应蛋白,为恶性肿瘤的早期诊断和基因治疗疫苗的研制奠定基础。方法根据目的基因序列合成目的基因并与原核表达载体连接,以构建原核表达质粒pET30a(+)/MAGE-A4,将酶切鉴定及测序正确的重组质粒转入E.coli BL21,经IPTG诱导表达并行可溶性分析,Western Blot鉴定重组蛋白特异性后,通过亲和层析法纯化重组蛋白。结果成功构建pET30a(+)/MAGE-A4原核表达质粒,诱导表达得到重组蛋白MAGE-A4,主要以可溶性形式表达,纯化后获得纯度较高的蛋白。结论成功构建pET30a(+)/MAGE-A4原核表达质粒并表达纯化出具有生物学活性的重组蛋白MAGE-A4。
Objective To build the prokaryotic expression plasmid of melanoma antigen-A4 （MAGE- A4）, and to express and purify the corresponding protein, in order to provide the basis for studying early diagnosis and the vaccine treatment against malignant tumors Methods According to the gene sequence of MAGE-A4, the tar- get gene was synthesized and connected with the prokaryotic expression vector to build the plasmid pET30a （＋）/ MAGE-A4, which was transformed into E. coli BL21 and then induced expression by IPTG. The expressed protein was identified by SDS-PAGE and Western-blot , and then purified by Ni-NTA purification system. Results The gene was 954 bp and the sequence was identical to that of the MAGE-A4 gene in GenBank. The SDS-PAGE showed that the relative molecular mass of the protein was consistent with the predicted one and a specific binding band of the protein could be detected at the corresponding site. Conclusion The plasmid pET30a（ ＋）/MAGE-A4 protein was successfully built, and recombinant MAGE-A4 protein with bioactivity was also expressed and purified.
出处
《解放军预防医学杂志》
CAS
2013年第2期109-112,共4页
Journal of Preventive Medicine of Chinese People's Liberation Army
关键词
肿瘤睾丸抗原
MAGE-A4
原核表达
纯化
cancer testis antigen （CTA）
melanoma antigen-A4 （MAGE-A4）
prokaryotic ex- pression
purification
