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目的:分别构建人源性抗前列腺特异性膜抗原(PSMA)单链抗体(sc Fv)/鱼精蛋白截短体(tp)、sc Fv/弗林蛋白酶识别位点(Fdt)/流感病毒融合肽结构域(HA2)/tp融合蛋白基因。利用原核表达体系表达、纯化并检测sc Fv、sc Fv-tp、sc Fv-Fdt-HA2-tp融合蛋白的活性。方法:采用PCR的方法,扩增融合基因sc Fv、sc Fv-tp、sc Fv-Fdt-HA2-tp,将获得的基因克隆入原核表达载体p ET28,在大肠杆菌BL21中表达,表达产物经SDS-PAGE和Western印迹鉴定,通过Ni2+-NTA螯合层析纯化。ELISA分析融合蛋白抗原亲和活性。结果:成功构建了人源性抗PSMA融合基因,经IPTG诱导后在M15中以包涵体形式表达。表达的目的蛋白均能与PSMA抗原结合。结论:融合蛋白具有结合抗原的活性,为靶向递送siRNA的研究奠定了基础。
OBJECTIVE: To construct human scFv / protamine truncation (tp), sc Fv / furin recognition site (Fdt) / influenza virus fusion Peptide domain (HA2) / tp fusion protein gene. The prokaryotic expression system was used to express, purify and test the activity of scFv, sc Fv-tp, sc Fv-Fdt-HA2-tp fusion protein. Methods: The fusion genes sc Fv, sc Fv-tp and sc Fv-Fdt-HA2-tp were amplified by PCR. The obtained gene was cloned into the prokaryotic expression vector p ET28 and expressed in E. coli BL21. The expression product SDS-PAGE and Western blot, purified by Ni2 + -NTA chelation chromatography. ELISA analysis of fusion protein antigen affinity activity. Results: The human anti-PSMA fusion gene was successfully constructed and expressed in the form of inclusion bodies in M15 after induced by IPTG. The expressed protein can bind to PSMA antigen. Conclusion: The fusion protein has antigen-binding activity, which lays the foundation for the research of targeted siRNA delivery.