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目的 :将HER_2 neu配体结合区 (RLD)基因重组 ,并在大肠杆菌中表达 ,对以包涵体形式表达的融合蛋白进行变性和复性的研究。方法 :采用PCR技术将HER_2 neuRLD的两段基因分别扩增 ,并利用基因片段末端的共同酶切位点将两段基因相连接 ,连接产物插入原核表达载体 ,在大肠杆菌中实现融合蛋白的表达。表达产物经SDS_PAGE和Western印迹检测后 ,进行蛋白质的变性、纯化和复性等处理。结果与结论 :在大肠杆菌中实现了融合蛋白的高效表达 ,表达产物可被特异性抗体识别。经SDS_PAGE分析 ,表达产物以包涵体形式存在 ,通过变性、纯化和复性等处理 ,获得了含两个RLD的融合蛋白 ,从而为HER_2 neu肿瘤疫苗的研究打下了基础。
OBJECTIVE: To recombine the gene of HER2 neu ligand binding region (RLD) and express it in Escherichia coli (E.coli), to study the degeneration and renaturation of the fusion protein expressed in inclusion bodies. METHODS: The two genes of HER_2 neuRLD were amplified by PCR. The two segments of genes were ligated by the common restriction enzyme at the end of the gene fragment, and inserted into the prokaryotic expression vector to express the fusion protein in E. coli . The expression product was detected by SDS_PAGE and Western blot, the protein denaturation, purification and refolding and other treatments. RESULTS AND CONCLUSION: High expression of the fusion protein was achieved in E. coli and the expressed product was recognized by specific antibodies. SDS-PAGE analysis showed that the expressed product existed in the form of inclusion bodies. The fusion protein containing two RLDs was obtained through denaturation, purification and renaturation, which laid the foundation for the study of HER2 neu tumor vaccine.