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目的:表达和纯化重组人Ⅲ型胶原蛋白（rhCol），并评价其性能。方法:构建重组基因工程菌pET30a（+）-1880/pACYCDuet-hy726/BL21（DE3），稳定共表达rhCol和脯氨酰羟化酶。通过大肠杆菌高密度发酵与盐析和柱层析蛋白纯化技术制备并纯化rhCol。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定rhCol的纯度，全自动蛋白质多肽测序仪测定rhCol的N端氨基酸序列，紫外分光光度法测定rhCol中的羟脯氨酸含量，噻唑蓝法评价rhCol的细胞相容性。结果:高密度发酵最终菌体湿重约200 g/L，表达量约3 g/L，通过亲和层析获得的rhCol的纯度大于95%。rhCol的羟脯氨酸含量为11.44%，其水溶性及细胞相容性良好。结论:本研究制备的rhCol可作为一种优良的生物材料广泛用于皮肤护理及生物医药等领域。“,”Objective:To express and purify recombinant human collagen type Ⅲ and evaluate its properties.Methods:The recombinant genetic engineering strain pET30a(+)-1880/pACYCDuet-hy726/bL21(DE3) was constructed to stably co-express recombinant human type Ⅲ collagen (rhCol) and prolyl hydroxylase. rhCol was prepared and purified by n E. coli high-density fermentation, salting out and column chromatography protein purification technology. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to determine the purity of rhCol. The N-terminal amino acid sequences of rhCol were determined by automatic protein polypeptide sequencing instrument. The hydroxyproline content of rhCol was determined by ultraviolet spectrophotometry. The cellular compatibility of rhCol was evaluated by MTT assay.n Results:The final wet weight of high-density fermentation was about 200 g/L. The expression level was about 3 g/L. The purity of rhCol by affinity chromatography was over 95%. The results showed that the hydroxyproline content of rhCol was 11.44%, and the rhCol products have good water solubility and cell compatibility.Conclusions:RhCol can be widely applied to the field of skin care and biomedicine as an excellent biological material.