Objective To detect the reliability of α-1，3-galactosyl（α-Gal）antigen knockout and the expression of human protein CD46（hCD46）on the surface of genetically modified pigs at the protein level, to provide reliable donors for xenotransplantation. Methods A total of 500 μl blood from wild type（WT），α-1，3-galactosyl transferase knockout（GTKO），hCD46 and GTKO/hCD46 Bama miniature swine were collected for peripheral blood mononuclear cell（PBMC）isolation. Expression of α-Gal antigen was detected by flow cytometry after coincubation with fluorescein isothiocyanate-Griffonia simplicifolia isolectin B4（FITC-GSIB4）and PBMC. Expression of hCD46 protein was detected by flow cytometry after co-incubation of PBMC with fluorescein isothiocyanate-CD46 membrane protein antibody（anti-CD46-FITC）. Results The α-Gal expression on the surface of PBMC were negative in GTKO and GTKO/hCD46 Bama pigs and positive in WT and hCD46 Bama pigs，consistent with the sequencing results. The hCD46 protein was detected positively on the surface of PBMC of hCD46 and GTKO/hCD46 Bama pigs and was negative in WT and GTKO Bama pigs，which was consistent with the results of genotype identification after birth. Conclusion A method for visually detecting the expression of α-Gal antigen and hCD46 protein in donor porcine using microscale blood was established.

目的 检测基因修饰猪细胞表面 α-1，3- 半乳糖（α-Gal）抗原敲除情况和人源蛋白 CD46 （hCD46）的表达情况，为异种器官移植提供可靠的供体。方法 采集 500 μl 野生型（wild type，WT）、 α-1，3- 半乳糖基转移酶基因敲除（GTKO）、hCD46 和 GTKO/hCD46 巴马小型猪血液，分离外周血单核细 胞（peripheral blood mononuclear cell，PBMC）。异硫氰酸荧光素标记的植物凝集素（FITC-GSIB4）与 PBMC 共孵育后采用流式细胞术检测 α-Gal 抗原敲除情况，异硫氰酸荧光素标记的 hCD46 膜辅蛋白抗体（antiCD46-FITC）与 PBMC 共孵育后采用流式细胞术检测 hCD46 蛋白表达情况。结果 GTKO 和 GTKO/hCD46 巴 马猪 PBMC 表面 α-Gal 检测呈阴性，WT 和 hCD46 巴马猪检测呈阳性，与测序结果一致 ；hCD46 和 GTKO/ hCD46 巴马猪 PBMC 表面 hCD46 蛋白检测呈阳性，WT 和 GTKO 巴马猪检测呈阴性，与 PCR 鉴定结果一致。 结论 建立了微量血液流式细胞术直观检测供体猪 α-Gal 抗原和人源 CD46 蛋白表达的方法。