rTNSALP-FcD10重组表达质粒的构建及在CHO-K1细胞的稳定表达

Construction of recombinant plasmid rTNSALP-FcD10 and its stable expression in CHO cells

目的构建重组表达质粒pcDNA3. 1-rTNSALP-FcD10及p EF1-rTNSALP-FcD10,获得能够稳定表达rTNSALP-FcD10蛋白的CHO-K1细胞株。方法去除重组人非组织特异碱性磷酸酶(tissue-nonspecific alkaline phosphatase,TNSALP)多钛链C端氨基酸连接IgG1的Fc片段及10个天冬氨酸,构建目的基因rhALP-FcD10,将基因分别克隆至pcDNA3. 1及p EF1/Myc-His B载体上,均转化E. coli DH5α细胞;将签定正确的两组重组质粒均转染CHO-K1细胞,进行G418压力筛选,建立稳定转染的CHO-K1细胞系,采用RT-qPCR及Western blot方法检测细胞中rTNSALPFcD10 mRNA转录水平及蛋白表达量。结果重组质粒pcDNA3. 1-rTNSALP-FcD10及pEF1-rTNSALP-FcD10经双酶切鉴定证明构建正确;G418最低致死浓度确定为800μg/m L;CHO-K1细胞中rTNSALP-FcD10 m RNA转录及蛋白水平均高于空白对照组,差异均有统计学意义(P <0. 01);转染p EF1-rTNSALP-FcD10组的蛋白表达量较转染pcDNA3. 1-rTNSALP-FcD10组高。结论成功构建了rTNSALP-FcD10的重组表达质粒pcDNA3. 1-rTNSALP-FcD10及pEF1-rTNSALP-FcD10,获得了稳定转染的CHO-K1细胞系,成功表达目的蛋白rTNSALP-FcD10,为蛋白进一步纯化奠定了良好的实验基础。

Objective To construct recombinant plasmids pcDNA3.1-rTNSALP-FcD10 and p EF1-rTNSALP-FcD10 so as to obtain the CHO-K1 cell line for stable expression of recombinant tissue-nonspecific alkaline phosphatase(rTNSALP).Methods After deletion of amino acids at N-terminus,TNSALP molecules were linked to IgG1-Fc and ten aspartic acid residues,and the constructed target gene rh ALP-FcD10 was cloned into expression vectors pcDNA3.1 and pEF1/MycHis B respectively. Both the recombinant plasmids constructed correctly as proved by restriction analysis and sequencing were transfected to CHO-K1 cells for pressure screening of stable CHO-K1 cell lines with G418. The m RNA transcription and protein expression levels of r TNSALP-FcD10 in the cells were determined by RT-qPCR and Western blot respectively.Results Restriction analysis showed that recombinant plasmids pcDNA3. 1-rTNSALP-FcD10 and pEF1-rTNSALP-FcD10 were constructed correctly. The minimum lethal concentration of G418 was 800 μg/m L. Both the mRNA transcription and protein expression levels of rTNSALP-FcD10 in CHO-K1 cells transfected with the two plasmids were significantly higher than those in blank control group(each P < 0. 01). However,the protein expression level in cells transfected with p EF1-rTNSALP-FcD10 was higher than that with pcDNA3.1-rTNSALP-FcD10. Conclusion Recombinant plasmids pcDNA3. 1-rTNSALP-FcD10 and pEF1-rTNSALP-FcD10 were correctly constructed,while the stably transfected CHO-K 1 cell line was established,and the target protein r TNSALP-FcD10 was successfully expressed,which provided an experimental basis for further purification of the protein.