小鼠全长膜型Klotho蛋白cDNA表达体系的构建与应用

Construction of the express system carrying the full length cDNA fragment coding mouse membrane-form klotho protein and its application

目的克隆编码小鼠膜型Klotho(mKL)蛋白的cDNA片段,构建、包装Klotho重组腺相关病毒表达体系,并检测rAAV/mKL载体表达功能。方法选择RT-PCR扩增小鼠全长mKL蛋白的基因片段,将该片段亚克隆至腺相关病毒载体pAAV-IRES-hnGFP,采用酶切及DNA测序鉴定;利用AAV-293细胞包装rAAV/mKL,经转染7901细胞,检测其Klotho表达情况。结果本文成功克隆出序列信息和读码框完全正确的3 064 bp的小鼠Klotho基因片段,并构建pAAV/mKL克隆。在AAV-293细胞中包装出rAAV/mKL,病毒原液转染7901细胞后其Klotho mRNA表达上调,而细胞上清液Klotho蛋白也明显增加(P<0.01)。结论成功构建小鼠Klotho重组腺相关病毒载体(pAAV/mKL),获得了rAAV/mKL,并经转染7901细胞验证其基因表达功能正常,这就为进一步研究Klotho基因治疗衰老相关性疾病提供了技术基础。

Objective To clone the full length of cDNA fragment coding mouse membrane form klotho （mKL） protein and construct the recombinant adeno-associated virus （ rAAV ） vector carrying the open reading frame （ORF） of the membrane form Klotho protein （pAAV/mKL） and package the virus. The expression of Klotho mRNA and Klotho protein in 7901 cell was tested after rAAV/mKL transfec- tion. Methods The full length of cDNA fragment coding mouse membrane form klotho protein was amplified by reverse transcription poly- merase chain reaction （RT-PCR）. After digestion, ligation and transformation, the targeted cDNA fragment was sub-cloned into pAAV- IRES-hnGFP vector in order to construct the recombinant plasmid pAAV/mKL. The pAAV/mKL was verified by restricted digestion and DNA sequencing. Use the AAV-293 cell to package rAAV/mKL and later transfect it into the 7901 cell. Then detect the expression of Klotho by RT-PCR and ELISA. Results The cDNA fragment with 3 064 bp coding membrane form Klotho protein was obtained by PCR amplifica- tion, the sequence of it was identical by GenBank. The rAAV/mKL was packaged in AAV-293 cell successfully. After transfection by rAAV/mKL, the expression of Klotho mRNA in 7901 cell was increased, and Klotho protein in the supernate of cultured cells significantly upregulated （P 〈 0. 01 ）. Conclusions The rAAV/mKL carrying the full length of cDNA fragment which coding mouse membrane form klotho protein is successfully constructed and packaged in AAV-293 cells, and transfecting it into the 7901 cell verifies it＇s function of gene express. All of these will pave a road for the deep research of gene therapy for aging related diseases.