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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 purify porcine skin cells and matrix xenograft antigen (α-Gal antigen) by using gene recombinant α-galactosidase from Bacteroides fragilis, and to reduce the immunogenicity of porcine matrix dressing. Methods 2-month-old healthy skin-free pigskin skin slices were cut into different sizes and cut into different sizes and then disinfected by iodine solution and 75% ethanol solution respectively, and then cleaned up with PBS for enzymolysis conditions. The α-Gal antigen of porcine skin was detected by fluorometry, and the residual enzyme was detected by double-antibody sandwich ELISA. The enzyme and α-Gal antigen in the pig’s skin were further optimized to improve the enzymolysis conditions and washing conditions, Good enzymatic hydrolysis process. Results The enzymatic hydrolysis method and washing method were established. After the enzymatic hydrolysis, the clearance rate of α-Gal antigen in pigskin was> 90%, and the residual enzyme could not be detected (the limit of detection by indirect ELISA for a-galactosidase was 1 ng / ml). Conclusion The established enzymatic hydrolysis process and washing process can effectively remove the α-Gal antigen of pigskin cells and matrix, and establish a method for preparing low-immunogenic pigskin dressing.