去端肽Ⅰ型胶原蛋白的免疫原性研究

Study on immunogenicity of type I atelocollagen

背景:Ⅰ型胶原蛋白的免疫原性主要分布在分子链的端肽区域,可在胶原蛋白提取过程中通过水解或是去除而使其失活,制备去端肽Ⅰ型胶原蛋白,降低其免疫原性。目的:评价去端肽Ⅰ型胶原蛋白的免疫原性。方法:采用酶切、盐析、透析工艺制备去端肽Ⅰ型胶原蛋白,通过考马斯亮蓝对牛血清白蛋白染色极限的分析,鉴定去端肽Ⅰ型胶原蛋白的纯度,依据YY/T 0606.25-2014中规定的方法,利用Quant-IT PicoGreendsDNA Reagent and Kits试剂盒测定去端肽Ⅰ型胶原蛋白的DNA残留量,利用ELISA试剂盒分析去端肽Ⅰ型胶原蛋白的端肽去除效果,依据YY/T 1561-2017标准检测去端肽Ⅰ型胶原蛋白的α-Gal抗原含量。结果与结论:去端肽Ⅰ型胶原蛋白的纯度可达99.8%,DNA残留量为4μg/g,C、N末端肽浓度低于试剂盒检测限,α-Gal抗原含量低于检测限。通过酶切、盐析、透析工艺制备的去端肽Ⅰ型胶原蛋白,可显著去除天然胶原蛋白的免疫原性根源,有效控制胶原蛋白临床应用的免疫原性风险。

BACKGROUND:The immunogenicity of type I collagen mainly locates in the telopeptide of molecular chain,which can be inactivated through hydrolysis or removal in the process of collagen extraction.Type I atelocollagen decreases its immunogenicity.OBJECTIVE:To evaluate the immunogenicity of type I atelocollagen.METHODS:Type I atelocollagen was prepared by enzyme digestion,salting-out and dialysis.The purity of type I atelocollagen was identified through analyzing the dyeing limit of comassiebule.The residual DNA was detected by Quant-IT PicoGreen dsDNA Reagent and Kits described in YY/T 0606.25-2014.The effect of telopeptide removing was detected by ELISA.The content ofα-Gal antigen was tested according to the criteria in YY/T 1561-2017.RESULTS AND CONCLUSION:The purity of type I atelocollagen reached 99.8%.The residual DNA was below 4μg/g.The contents of N-terminal telopeptide and C-terminal telopeptide were lower than the examination limit.The content ofα-Gal antigen was lower than the detection limit.The immunogenicity of collagen is remarkably decreased by preparing type I atelocollagen using enzyme digestion,salting-out and dialysis,which can effectively control its risk of immunogenicity in clinical application.