钙网蛋白与卵清白蛋白融合蛋白的纯化及其生物学功能

摘要

To express and purify the fusion protein of calreticulin (CRT ) with a fragment of chicken ovalbumin (OVA ) and to make a preliminary study on its biological function .Now the vector containing OVA161‐276‐CRT39‐272 was constructed by overlap PCR .The expression of OVA161‐276‐CRT39‐272 fusion protein was induced in genetic engineering strain .The protein was purified from soluble fraction of bacteria by His‐Trap metal chelation chromatography .The effects of the fusion protein on the function of dendritic cells were investigated .The bio‐activity of this fusion protein was also investigated .The results show that the vector is successfully constructed and OVA161‐276‐CRT39‐272 has a purity over 95% .The fusion protein can promote the maturation of den‐dritic cells and stimulate it produce more cytokines than the control protein .OVA161‐276‐CRT39‐272 has some biological function and the result lay down a certain foundation for future research .%为原核表达纯化钙网蛋白（calreticulin ，CRT）分子与模型抗原鸡卵清白蛋白（ovalbumin ，OVA）片段的融合蛋白，并初步探究其生物学活性。现通过重叠PCR方法人工构建CRT‐peptide复合物OVA161‐276‐CRT39‐272载体，在基因工程菌中诱导表达后，通过N i2＋亲和层析的方法纯化出目的蛋白，并考察融合蛋白对树突状细胞功能的影响，初步探究其生物功能。结果显示，正确构建好载体且所纯化融合蛋白纯度超过95％，融合蛋白相对于作为对照的O V A片段能促进树突状细胞的成熟和细胞因子的释放。OVA161‐276‐CRT39‐272融合蛋白具有一定的生物学活性，为下一步探究奠定基础。