人热休克蛋白70D的基因克隆、表达及蛋白纯化

Gene cloning, expression, and purification of recombinant human heat shock protein 70D

目的 :研究热休克蛋白 70 D(heat shock protein 70 D,HSP70 D)的基因克隆、重组表达及纯化。 方法 :采用 RT- PCR方法从 He L a细胞中克隆 HSP70 D全长编码区 c DNA序列 ,构建原核表达载体 p ET2 4 a(+) - HSP70 D,经过 DNA序列测定证实其序列正确。完成基因工程人 HSP70 D的高表达工程菌的筛选后 ,对其发酵、蛋白表达条件及重组蛋白的纯化条件进行优化。 结果 :重组表达载体经序列测定及酶切鉴定与理论推测结果相符 ,高表达工程菌经优化表达条件后 rh HSP70 D的表达量可占菌体总蛋白的 2 0 %以上。经过低浓度乙醇沉淀、DEAE阴离子交换、疏水柱层析及 S2 0 0凝胶过滤柱层析后 ,获得了纯度大于 95 %的 rh HSP70 D。 结论 :本研究为进一步开展以 HSP70 D为基础的肿瘤免疫治疗研究提供了实验基础。

Objective: To study the cloning, recombinant expression and purification of human HSP70D. Methods: The full length code region of HSP70D was amplified by RT PCR from HeLa cells. Then the PCR product was inserted into pET 24a(+), by NdeⅠ/XhoⅠ double digestion. The recombinant expression vector pET24a HSP70D was transformed into E. coli strain BL21. Positive clones were determined by NdeⅠ/XhoⅠ digestion and sequence analysis, which was used in the expression and purification of HSP70D protein. The engineered bacteria were collected after fermentation. The recombinant HSP70D was purified by using ethanol precipitation, anion exchanger, hydrophobicity interaction chromatography, and finally high resolution gel filtration chromatography. Results: The expression level of HSP70D was over 20% of total protein in the engineered bacteria. After purification, electrophoretically pure (more than 95%) rhHSP70D was obtained. Conclusion: This study setup a satisfied down stream process which can be used for production of large quantity of recombinant HSP70D.