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为研究抗甲状腺多肽的特异性与亲和力,本研究建立了人促甲状腺素受体(TSHR)α亚基(hTSHRA)稳定表达株。构建hTSHRA到慢病毒质粒GV218上形成GV218-hTSHRA,挑取GV218-hTSHRA构建物阳性大肠杆菌克隆,提取DNA测序结果与人TSHRA序列比对,100%吻合。结果显示:GV218-hTSHRA转染到HEK 293T后,Western blot显示,HEK 293T细胞提取蛋白出现52kD的hTSHRA目的条带。GV218-hTSHRA、辅助质粒在HEK 293T细胞中包装后,HEK 293T细胞表达绿色荧光蛋白。收取浓缩的包装病毒,qPCR测定滴度达到了2×108 TU/mL。再将包装病毒转染到HEK 293T细胞中,hTSHRA即表达在HEK 293T细胞上,细胞传代后hTSHRA仍然稳定表达。结果表明获得GV218-hTSHRA包装质粒,并建立了hTSHRA-HEK 293T稳定表达株,为研究抗甲状腺多肽与人TSHRα亚基特异性与亲和力提供了必要的实验材料。
In order to study the specificity and affinity of antithyroid peptides, a stable expression of human thyrotropin receptor (TSHR) alpha subunit (hTSHRA) was established in this study. Construction of hTSHRA to lentiviral plasmid GV218 to form GV218-hTSHRA, pick GV218-hTSHRA construct positive E. coli clones, DNA sequencing and human TSHRA sequence alignment, 100% match. The results showed that: After transfection of GV218-hTSHRA into HEK 293T, Western blot showed that the band of 52 kD hTSHRA was extracted from HEK 293T cells. GV218-hTSHRA, helper plasmids After packaging in HEK 293T cells, HEK 293T cells express green fluorescent protein. The concentrated packaging virus was collected and the titer of qPCR reached 2 × 108 TU / mL. The packaged virus was then transfected into HEK 293T cells, and hTSHRA was expressed on HEK 293T cells. After passage, hTSHRA was still stably expressed. The results showed that GV218-hTSHRA packaging plasmid was obtained and the hTSHRA-HEK 293T stable expression strain was established, which provided the necessary experimental material for studying the specificity and affinity of anti-thyroid polypeptide and human TSHRα subunit.