CAJ | 학술논문

目的：构建重组质粒 pET32a - hIL -7，诱导表达，纯化并鉴定目的蛋白。方法：将人白介素-7基因克隆到原核表达载体 pET32a（＋）上，得到重组质粒 pET32a - hIL -7，转化大肠杆菌 BL21（DE3），经 IPTG 诱导得到融合蛋白 Trx - hIL -7。融合蛋白主要以包涵体形式存在。包涵体通过复性、酶切、纯化等步骤处理。利用 Western blot 鉴定重组蛋白，对其生物学活性用其依赖性细胞株2E8增殖实验测定。结果：成功构建重组质粒 pET32a - hIL -7。包涵体通过透析复性，肠激酶切，镍柱纯化等步骤得到重组蛋白人 IL -7。纯化的重组蛋白经 Western blot 鉴定能与抗人的 IL -7抗体特异性结合。经体外细胞实验检测证明，纯化的重组蛋白具有生物学活性。结论：得到了有活性的重组蛋白 IL -7，为研究其功能奠定了基础。
목적：구건중조질립 pET32a - hIL -7，유도표체，순화병감정목적단백。방법：장인백개소-7기인극륭도원핵표체재체 pET32a（＋）상，득도중조질립 pET32a - hIL -7，전화대장간균 BL21（DE3），경 IPTG 유도득도융합단백 Trx - hIL -7。융합단백주요이포함체형식존재。포함체통과복성、매절、순화등보취처리。이용 Western blot 감정중조단백，대기생물학활성용기의뢰성세포주2E8증식실험측정。결과：성공구건중조질립 pET32a - hIL -7。포함체통과투석복성，장격매절，얼주순화등보취득도중조단백인 IL -7。순화적중조단백경 Western blot 감정능여항인적 IL -7항체특이성결합。경체외세포실험검측증명，순화적중조단백구유생물학활성。결론：득도료유활성적중조단백 IL -7，위연구기공능전정료기출。
Objective:To acquire recombinant human interleukin 7 prokaryotic expressing plasmid,fusion protein is expressed,purified and identified. Methods:The hIL - 7 gene was inserted into the prokaryotic expressing vector pET - 32a( + )to acquire pET32 - hIL - 7 and transformed into BL21(DE3)cells. IPTG induced the expression of fusion protein Trx - IL - 7. The induced product was washed,dissolved and purified by affinity chromatogarphy under renaturing condition. IL - 7 was identified by Western blot,and the biologic activity was detected by proliferation of IL - 7 dependence cell 2E8. Results:The recombinant plasmid pET32 / rhlL - 7 has been constructed correctly. The recombinant hlL - 7 was gained after gradient dialysis,enzyme digestion and purified,which has the right immunology specificity and biologic activity. Conclusion:The recombinant human interleukin - 7 with biologic activity has been acquired,which lay the foundation for the further study of function of IL - 7.