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目的:构建人类促甲状腺激素受体(hTSHR)胞膜外区氨基端的两个片段hTSHRf(aa29~100)、hTSHRe(aa101~278)的真核表达载体pcDNA3.1-hTSHRf和pcDNA3.1-hT-SHRe,并在CHO细胞中进行表达。方法:RT-PCR法从人甲状腺正常组织的cDNA中扩增hTSHRf和hTSHRe,定向插入真核表达载体pcDNA3.1(D)/V5-His-TOPO中,经酶切、PCR和测序鉴定后通过脂质体介导转染至CHO细胞中进行表达,RT-PCR扩增转染细胞的cDNA、Western blot分别鉴定hTSHR在CHO细胞中mRNA和蛋白水平的表达。结果:RT-PCR分别扩增两个片段的阳性克隆株,所得片段大小与预期一致,经Western blot鉴定,相对分子质量(Mr)分别为11900、23600左右,与预期的大小相符。结论:真核表达载体pcDNA3.1-hTSHRf和pcDNA3.1-hTSHRe构建成功,RT-PCR和Western blot检测证实重组质粒能在CHO细胞中高效表达,为进一步研究pcDNA3.1-hTSHRf和pcDNA3.1-hTSHRe在体内的基因表达,建立Graves’病的动物模型奠定了基础。
OBJECTIVE: To construct the eukaryotic expression vectors hTSHRf (aa29 ~ 100) and hTSHRe (aa101 ~ 278) of human TSHR extracellular domain, pcDNA3.1-hTSHRf and pcDNA3.1-hT -SHRe and expressed in CHO cells. Methods: hTSHRf and hTSHRe were amplified by RT-PCR from cDNA of normal human thyroid tissue and inserted into eukaryotic expression vector pcDNA3.1 (D) / V5-His-TOPO. After identification by restriction enzyme digestion, PCR and sequencing The expression of hTSHR mRNA and protein in CHO cells was identified by RT-PCR. The expression of hTSHR in CHO cells was detected by Western blot. Results: The positive clones were amplified by RT-PCR. The size of the fragments was consistent with the expected size. The molecular weight (Mr) was about 11900 and 23600 respectively, which was consistent with the expected size. Conclusion: The eukaryotic expression vectors pcDNA3.1-hTSHRf and pcDNA3.1-hTSHRe were constructed successfully. RT-PCR and Western blot showed that the recombinant plasmids could be efficiently expressed in CHO cells. To further study the effect of pcDNA3.1-hTSHRf and pcDNA3.1 -hTSHRe in vivo gene expression, the establishment of animal models of Graves’ disease laid the foundation.