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通过原核表达获得HLA-G5重链和轻链(β2m),经初步纯化后,利用稀释法与人工合成的九肽(KGPPAALTL)一起进行体外复性折叠,形成HLA-G5-抗原肽复合物,经非变性PAGE/Western blot和夹心ELISA法鉴定,证实复性后成功获得天然构象的HLA-G5。利用PBMC受LPS刺激后产生TNF的特点,观察原核表达、体外折叠的HLA-G5对人PBMC分泌TNF的影响。TNF分泌量采用TNF敏感的L929细胞进行生物法测定,结果显示原核表达、体外折叠的HLA-G5促进人PBMC分泌TNF-α。
After being purified, the HLA-G5 heavy chain and the light chain (β2m) were obtained by prokaryotic expression. After being purified in vitro, the recombinant human non-peptide (KGPPAALTL) was refolded in vitro to form HLA-G5- Identified by non-denaturing PAGE / Western blot and sandwich ELISA, we confirmed that the native conformation of HLA-G5 was successfully obtained after renaturation. Using the characteristics of PBMC stimulated by LPS to produce TNF, the effect of prokaryotic expression and in vitro folded HLA-G5 on the secretion of TNF by human PBMC was observed. The secretion of TNF was determined by the biological method using TNF-sensitive L929 cells. The results showed that prokaryotic expression of HLA-G5 secreted in vitro stimulated the secretion of TNF-α by human PBMCs.