摘要
目的将前期在大肠埃希杆菌中获得表达的A型人呼吸道合胞病毒兰州分离株截短的F1重组蛋白进行纯化和复性,为后期动物免疫制备抗原。方法 37℃诱导重组菌体p ET-42b-F1J/Rossata,诱导完毕后离心收集菌体,高压破碎菌体并收集包涵体后用不同浓度的Triton X-100(细胞裂解液)洗涤包涵体3次。洗涤的包涵体用8 mol/L尿素进行溶解并用镍离子亲和层析方法进行初步纯化,用阳离子交换层析方法对初步纯化蛋白进行最终的纯化。亲和层析纯化蛋白用3种不同的复性液进行了稀释复性。结果 37℃诱导5 000 m L重组菌p ET-42b-F1J/Rossata共收获37 g湿菌体,经过不同浓度Triton X-100洗涤包涵体后纯度可达75%。包涵体用8 mol/L尿素溶解后经镍离子亲和层析纯化纯度约为40%,再用阳离子交换层析介质SP HP进一步纯化样品后纯度可达90%。纯化蛋白以3种不同的复性液都能得到复性,其中复性液3的复性效果相对较好。结论实验中探索了人呼吸道合胞病毒截短F1重组蛋白包涵体的纯化方法及步骤,为后期的蛋白制备及动物免疫奠定了基础。
Objective The truncated F1 recombinant protein from subtype Lanzhou isolate of human respiratory syncytial virus, which was previously expressed in Escherichia coli , was purified and completed of renaturation in preparation antigen for immunization of animal. Methods Recombinant bacterial pET-42b-F1J/Rossata was induced at 37 ℃ and then centri- fuged and collected. Inclusion body was obtained by pressure crush and centrifugation, and washed with different concen- tration of TritonX - 100 for three times. The washed inclusion body was lysised with 8M urea solution and purified with a neckel ion affinity chromatography, then the target protein was further purified with a cation exchange chromatography. The affinity purified recombinant protein was refolded with three different renaturation liquid by dilution. Results 37 g wet bacteria was gained from 5 000 mL recombinant bacteria pET-42b-F1J/Rossata at 37 ℃. The purity of inclusion body was up to 75% after being washed with different concentration Triton X-100. The purity of recombinant protein was about 40% by the neckel ion affinity chromatography, while the purity could reach 90% with the cation exchange chromatography SP HP. Purified protein could be refolded by using three different renaturation liquid. The renaturation liquid 3 is relatively better among them. Conclusion The purification method and procedures for truncated F1 recombinant protein of human respiratory syncytial virus contained in inclusion body were performed in this stuidy, on which a foundation was set up in preparation of protein and immunization of animal in later stage.
出处
《微生物学免疫学进展》
2015年第3期21-25,共5页
Progress In Microbiology and Immunology
关键词
呼吸道合胞病毒截断短F1蛋白
包涵体
纯化
复性
F1 truncated protein of human respiratory syncytial virus
Inclusion body
Purification
Renaturation
