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目的利用基因工程手段建立表皮生长因子受体(EGFR)二聚化B细胞表位抗原肽MVF-ER的高效制备方法。方法采用重叠延伸PCR扩增MVF-ER后克隆于表达载体pET32a,于大肠杆菌BL21(DE3)进行表达,产物采用肠激酶酶切和镍离子螯合亲和层析法纯化。结果通过10对引物5步重叠延伸得到273 bp的扩增产物,目的基因与硫氧环蛋白(Trx)融合后可高效表达,经酶切和层析后得到纯度为95%的抗原肽。结论成功建立抗原肽“MVF-ER”的高效制备方法,为进一步研究EGFR过表达恶性肿瘤的治疗性疫苗打下了坚实基础。
OBJECTIVE: To establish an efficient method for the preparation of epidermal growth factor receptor (EGFR) dimerized B cell epitope antigen peptide MVF-ER by genetic engineering. Methods The MVF-ER was amplified by overlap extension PCR and cloned into the expression vector pET32a. The recombinant plasmid was expressed in E. coli BL21 (DE3). The product was purified by enterokinase digestion and nickel chelate affinity chromatography. Results The amplified product of 273 bp was amplified by 10 pairs of primers. The target gene was highly expressed after fusion with thioredoxin (Trx). The purity of the antigen peptide was 95% after digestion and chromatography. Conclusion The successful preparation of the antigen peptide “MVF-ER” efficient preparation method for further study of EGFR over-expression of malignant tumor therapeutic vaccine laid a solid foundation.