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
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