酶解法清除猪皮α-Gal抗原的研究

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英文篇名：Enzymatic removal of α-Gal antigen in porcine skin 作者：贠志敏 ; 李素波 ; 张雪 ; 檀英霞 ; 季守平 ; 高红伟 ; 宫锋 英文作者：YUN Zhi-min;LI Su-bo;ZHANG Xue;TAN Ying-xia;JI Shou-ping;GAO Hong-wei;GONG Feng;Institute of Transfusion Medicine,Academy of Military Medical Sciences; 关键词：猪皮 ; 免疫原性 ; α-半乳糖苷酶 ; α-Gal抗原 ; 残留酶 英文关键词：pig skin;;immunogenicity;;α-galactosidase;;α-Gal antigen;;residual enzyme 中文刊名：JSYX 英文刊名：Military Medical Sciences 机构：军事医学科学院野战输血研究所; 出版日期：2015-12-25 出版单位：军事医学 年：2015 期：v.39;No.223 基金：国家科技重大专项资助项目(2013ZX09J13110B) 语种：中文; 页：JSYX201512015 页数：3 CN：12 ISSN：11-5950/R 分类号：52-54

摘要

目的利用脆弱拟杆菌来源的基因重组α-半乳糖苷酶清除猪皮细胞及基质的异种抗原(α-Gal抗原),降低猪皮基质敷料的免疫原性。方法 2月龄健康无皮肤疾患的猪皮皮片,经剖层裁切成不同的规格后,分别经碘酊和75%乙醇溶液浸泡消毒,随后用PBS清洗后进行酶解条件的摸索;采用免疫荧光法检测猪皮的α-Gal抗原,双抗夹心ELISA法检测残留酶,通过对酶解猪皮的残留酶和α-Gal抗原的检测进而优化、改进酶解条件和洗涤条件,以确立最佳酶解工艺。结果建立了有效的酶解方法和洗涤方法,酶解后猪皮的α-Gal抗原清除率>90%,残留酶不能检出(间接ELISA法检测微量α-半乳糖苷酶的下限为1 ng/ml)。结论所建立的酶解工艺和洗涤工艺可有效清除猪皮细胞及基质的α-Gal抗原,建立了制备低免疫原性猪皮敷料的方法。
        Objective To reduce immunogenicity of porcine skin by removing α-Gal epitopes expressed in cell surface and extracellular matrix using recombinant α-galactosidase produced by Bacteroides fragilis. Methods The porcine skin was harvested from healthy 2-month-old pigs without any skin disorders before being sterilized by iodine and 75% alcohol,respectively. Enzymatic removal of α-Gal antigen was followed by washing with PBS. The α-Gal antigen in the prepared porcine skin was measured with immunofluorostaining of cryosections and the residual enzyme was measured with a doubleantibody sandwich ELISA method. Enzymatic removal procedures were optimized by detecting residual enzyme and the efficacy of α-Gal removal under different enzymatic and washing conditions. Results Efficient enzymatic and washing methods were established to remove α-Gal antigen. The α-Gal removal efficacy was above 90% and residual enzyme was undetectable( α prescribed minimum of α-galactosidase detection with indirect ELISA was 1 ng / ml). Conclusion It is feasible to efficiently remove α-Gal antigen under these enzymatic and washing conditions,and a method of producing low-immunogenicity pig skin dressing for burn is established.

引文

[1]Galili U,Shohet SB,Kobrin E,et al.Man,apes,and old World monkeys differ from other mammals in the expression of alphagalactosyl epitopes on nucleated cells[J].J Biol Chem,1988,263(33):17755-17762.
    [2]Eckermann JM,Buhler LH,Zhu A,et al.Initial investigation of the potential of modified porcine erythrocytes for transfusion in primates[J].Xenotransplantation,2004,11(1):18-26.
    [3]高红伟,李素波,鲍国强,等.原核基因工程制备新型重组α-半乳糖苷酶应用于人B→O血型改造的研究[J].中国实验血液学杂志,2011,19(2):503-507.
    [4]高红伟,张雪,李素波,等.新型α-半乳糖苷酶清除动物红细胞表面αGal抗原的研究[J].中国实验血液学杂志,2012,20(5):1231-1234.
    [5]贠志敏,张雪,李素波,等.α-半乳糖苷酶清除猪细胞表面的αGal抗原[J].生物技术通讯,2014,25(6):52-54.
    [6]李素波,高红伟,张雪,等.新型α-半乳糖苷酶多克隆抗体的制备及特异性鉴定[J].生物技术通讯,2013,24(6):853-855.
    [7]李素波,贠志敏,高红伟,等.制备脆弱拟杆菌来源的新型α-半乳糖苷酶单抗用于检测通用型红细胞中的微量残留酶[J].军事医学,2015,39(4):302-305.
    [8]La Vecchio JA,Dunne AD,Edge AS.Enzymatic removal of alphagalactosyl epitopes from poricine endothelial cells diminishes the cytotoxic effect of natural antibodies[J].Transplantation,1995,60(8):841-847.
    [9]常宏宇,檀英霞,宫锋,等.去除αGal抗原的猪红细胞输入猕猴体内的初步研究[J].中国输血杂志,2008,21(2):110-113.