Abstract：Objective To construct glycosylation site mutant plasmids of human leukocyte-C (HLA-C) gene were, and to preliminarily investigate the effects of glycosylation modification of HLA-C molecule on the function of HLA-C.Methods The HLA-C gene and HLA-C glycosylation site mutation gene were amplified by using the method of polymerase chain reaction (PCR), recovered, and connected to the PCMV-Flag eukaryotic expression vector after double enzyme digestion. The constructed plasmids were transfected into human embryonic kidney cells (293T) to express HLA-C protein and glycosylated mutant HLA-C protein, and were identified by the protein western blot method. The identified correct HLA-C gene and HLA-C glycosylation site mutation gene were connected to the carrier with red fluorescence,and transfected into 293T cells for immunofluorescence experiment to observe the expression of the protein in the cells.Results Agarose gel electrophoresis showed that HLA-C gene and HLA-C mutation gene were successfully amplified.Western Blot results showed successful expression of HLA-C protein and HLA-C glycosylated mutant proteins. Laser confocal results showed that normal HLA-C proteins were mainly expressed in the cytoplasm and peri-membrane,while most HLA-C proteins were expressed in the cytoplasm after glycosylation mutation. Conclusion The HLA-C plasmid and glycosylation site mutant plasmid vectors have been successfully constructed. The results of this experiment provide the basis and ideas for further research on glycosylation modification of HLA-C molecules.
周晓曼. 白细胞抗原糖基化位点突变质粒载体构建及表达[J]. 中国当代医药, 2021, 28(12): 30-033.
ZHOU Xiao-man. Construction and expression of human leukocyte antigen glycosylation site mutant plasmid vector. 中国当代医药, 2021, 28(12): 30-033.
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