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目的:建立CA125串联重复序列(CA125R)原核表达系统,表达纯化重组CA125R蛋白并制备其抗血清。方法:全基因合成一段CA125串联重复序列,并将其克隆至pET-32a(+),构建CA125R蛋白原核表达载体pET-CA125R;将构建好的pET-CA125R载体转化至E.coli BL21(DE3),确定最佳可溶性表达条件,通过Ni-NTA亲和层析系统纯化重组CA125R蛋白;纯化重组蛋白经Western blot方法验证后,免疫家兔制备抗血清。结果:成功构建CA125串联重复序列原核表达载体,确定了最佳可溶性诱导表达条件(0.5 mmol/LIPTG,15℃诱导6 h);Western blot结果证实纯化的重组蛋白正确,纯度高;制备的抗血清能特异识别重组CA125R蛋白和天然CA125糖蛋白。结论:成功建立了CA125R高效原核表达系统,并制备了高纯度重组CA125R蛋白及其抗血清。
Objective: To establish prokaryotic expression system of CA125 tandem repeat (CA125R), express and purify recombinant CA125R protein and prepare its antiserum. METHODS: A CA125 tandem repeat was synthesized and cloned into pET-32a (+) to construct CA125R prokaryotic expression vector pET-CA125R. The constructed pET-CA125R vector was transformed into E. coli BL21 (DE3) , To determine the optimal conditions for soluble expression, purification of recombinant CA125R protein by Ni-NTA affinity chromatography system; purified recombinant protein by Western blot method validation, immunized rabbits to prepare antiserum. Results: The prokaryotic expression vector of CA125 tandem repeat was successfully constructed and the optimal conditions of soluble expression were determined (0.5 mmol / L IPTG, induced at 15 ° C for 6 h). Western blot confirmed that the recombinant protein was correct and of high purity. The prepared antiserum Can specifically recognize recombinant CA125R protein and native CA125 glycoprotein. Conclusion: The highly efficient CA125R prokaryotic expression system was successfully established and a high purity recombinant CA125R protein and its antiserum were prepared.