人胎盘来源Ⅳ型胶原蛋白的提取与纯化

Extraction and purification of the type IV collagen derived from human placenta

目的从人胎盘中分离纯化Ⅳ型胶原蛋白,为制备其抗体提供抗原。方法选择人胎盘为提取原料,用胃蛋白酶消化、氯化钠盐析、凝胶化、超速离心、离子交换层析等方法,提取纯化Ⅳ型胶原蛋白。采用SDS-PAGE对提取的Ⅳ型胶原蛋白进行鉴定。结果胃蛋白酶消化法,使获得胶原蛋白的胶原链部分断裂,凝胶化方法可预先分离出组织中的Ⅰ、Ⅱ、Ⅲ型胶原蛋白,保留了Ⅳ、Ⅴ胶原蛋白,DEAE-Sephrose层析图表明,层析柱吸附的蛋白经洗脱得一主峰,经SDS-PAGE证实提取的Ⅳ型胶原蛋白的分子量80Kd和40Kd两条带。结论从人胎盘中提取的Ⅳ型胶原蛋白纯度高,符合Ⅳ型胶原的特征。提取材料广泛、实验条件简便,结果可靠。

Objective To isoIate and purify collagen type IV （Col IV ）from human placenta and provide the antigen for the preparation of the antibody. Methods The human placenta was selected as raw material. The digestion of pepsin, salting of sodium chloride, gelation, uhracentrifugation, ion-exchange chromatography were studied for extracting Col IV. The purity identification was made by SDS -PAGE. Results By digestion of pepsin, part of collagen chain was ruptured; By gelation, collagen type I , II , III in the organization can he isolated and collagen type IV, V can be stored;DEAE - Sepharose chromatography figure showed that Adsorption of proteins by elution chromatography column is the main peak;it was found that the molecular weight of extracted collagen type IV was about 40 and 80KD by SDS-PAGE. Conclusion The results suggest that the collagen type IV isolated from human placenta has high purity and accords with the characteristics of collagen type IV . The improved method we adopted has significant advantages such as simple working process, convenient source of raw material and result reliability.