稳定表达人FcgRII A的293T细胞系的建立及其功能鉴定
首发时间:2016-06-01
摘要:构建稳定表达FcγRIIA的293T细胞系及其相应的对照细胞系(FcγRIIB, FcγRIIA (Y288F/Y304F)),作为进一步研究FcgRIIA介导的信号通路的基础。首先,从人外周血单核细胞cDNA中扩增CD32a和CD32b,并构建pMSCVegfp-CD32a及pMSCVegfp-CD32b逆转录病毒表达载体。另外将构建成功的pMSCVegfp-CD32a 288和304位点的酪氨酸(Y)突变成苯丙氨酸(F),得到pMSCVegfp-CD32a(Y288F/Y304F)表达载体。构建成功的逆转录病毒载体经酶切、测序鉴定后,与pcl-amp辅助载体共同转染293T细胞进行病毒包装。病毒感染293T细胞后通过流式分选得到稳定表达CD32a、CD32b、CD32a(Y288F/Y304F)的293T细胞系,流式检测各细胞株膜表面的CD32分子表达及白色念珠菌考察各细胞株抗体依赖的内吞作用。结果表明:稳定表达CD32a、CD32b、CD32a(Y288F/Y304F)的293T细胞纯度在98%以上,并且膜表面均可检测到CD32的表达,吞噬实验结果表明293T-CD32a细胞呈现对白色念珠菌的吞噬作用,而293T-CD32b、293T-CD32a(Y288F/Y304F)无吞噬作用。
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Establishment and functional identification of 293T cell lines with stable expression of human FcgRII A
Abstract:HEK293T cells stably transfected with CD32a、CD32b or CD32a(Y288F/Y304F) were constructed to detect their phagocytosis,CD32b and CD32a(Y288F/Y304F) as the control of CD32a.The ORF of CD32a and CD32b was amplified from cDNA of human peripheral blood mononuclear cells(PBMC), and retrovirus vectors expressing CD32a and CD32b,we also constructed pMSCVegfp-CD32a(Y288F/Y304F) with mutations in 288/304aa site. After identified by enzyme digestion and sequencing, the plasmids and helper plasmids were co-transfected into 293T cells to produce the virus. To generate stable cell lines of 293T-CD32a,293T-CD32b and 293T-CD32a(Y288F/Y304F),HEK293T cells were infected with these virus and sorted by flow cytometer. The expression of CD32a and the phagocytosis was confirmed by flow cytometer. Our results demonstrated that the CD32 was highly expressed in stable cell lines of 293T-CD32a,293T-CD32b and 293T-CD32a(Y288F/Y304F).The phagocytosis of the cells was then detected and 293T-CD32a, but not 293T-CD32b、293T-CD32a(Y288F/Y304F) exhibited phagocytosis of Monilia albican, suggesting a crucial role of CD32a in phagocytosis.
Keywords: medical immunology phagocytosis vector construction sorting
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