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目的研究对前甲状腺激素原基因进行定点突变,并转染体细胞产生成熟甲状旁腺激素。方法采用聚合酶链反应(PCR)定点突变技术,自组织基因组基因中扩增前甲状旁腺激素原的cDNA,并将野生型甲状旁腺激素编码第28位的密码子GTT突变为AGA,使N端2831氨基酸变为ArgLysLysArg,成为furin酶的酶切位点;将获得的突变表达载体pcDNA31/mPTH通过脂质体法转染体外培养的293细胞,用放射免疫法测定表达水平。结果成功地获得突变型甲状旁腺激素原cDNA,293细胞转染突变表达载体后,每日培养液中甲状旁腺激素含量达2834~5264pg/20×106细胞,远高于空载体转染的细胞培养液。结论利用PCR技术可获得突变型甲状旁腺激素原的cDNA,突变表达载体pcDNA31/mPTH能成功转染体细胞并产生甲状旁腺激素。
Objective To study the site-directed mutagenesis of the pre-thyroid hormone gene and the transfection of somatic cells to generate mature parathyroid hormone. Methods The site-directed mutagenesis of polymerase chain reaction (PCR) was used to amplify the cDNA of parathyroid hormone from the genomic DNA of the tissue and to mutate the codon GTT of wild-type parathyroid hormone at codon 28 to AGA The amino acid of N terminal 2831 was changed into ArgLysLysArg, which became the cleavage site of furin enzyme. The obtained mutant pcDNA31 / mPTH was transfected into 293 cells by lipofectamine and the expression level was determined by radioimmunoassay. Results The mutant parathyroid hormone cDNA was obtained successfully. After the 293 cells were transfected with the mutant vector, the daily parathyroid hormone content in the culture medium reached 2834-5264 pg / 20 × 106 cells, much higher than that of the empty vector Cell culture solution. Conclusion The cDNA of mutant parathyroid hormone can be obtained by PCR. The recombinant plasmid pcDNA31 / mPTH can successfully transfect somatic cells and produce parathyroid hormone.