rTNSALP-FcD10重组表达质粒的构建及在CHO-K1细胞的稳定表达
|
摘要
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.
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.
引文
[1]MILLAN J L,NARISAWA S,LEMIRE I,et al.Enzyme replacement therapy for murine hypophosphatasia[J].J Bone&Mineral Res,2008,23(6):777-787.
[2]SCOTT L J.Asfotase alfa:a review in paediatric-onset hypophosphatasia[J].Drugs,2016,76(2):255-262.
[3]CHEN B,LI L,REN W,et al.A novel missense mutation in the ALPL gene causes dysfunction of the protein[J].Mol Med Rep,2017,16(1):710-718.
[4]CHANG K C,LIN P H,SU Y N,et al.Novel heterozygous tissue-nonspecific alkaline phosphatase(TNAP)gene mutations causing lethal perinatal hypophosphatasia[J].J Bone&Mineral Metab,2012,30(1):109-113.
[5]BELACHEW D,KAZMERSKI T,LIBMAN I,et al.Infantile hypophosphatasia secondary to a novel compound heterozygous mutation presenting with pyridoxine-responsive seizures[J].Jimd Rep,2013:17-24.
[6]DUAN X,LIU R,XU W,et al.Effect of cell culture conditions on antibody heterogeneity[J].Chin J Biotechnol,2013,29(12):1880-1886.
[7]WEI L U,KUN J I.Advances in research on hypophosphatasia[J].Chin J Clinicians,2013,16(8):27-29.(in Chinese)冀堃.ALPL基因突变影响ADRB2基因调控BMMSCs成骨分化的实验研究[D].陕西西安:第四军医大学,2013.
[8]SCOTT,LESHLEY J.Asfotase alfa in perinatal/infantile-onset and juvenile-onset hypophosphatasia:a guide to its use in the USA[J].BioDrugs,2016,30(1):41-48.
[9]REMDE H,COOPER M,QUINKLER M.Successful asfotase alpha treatment in an adult dialysis patient with childhood-onset hypophosphatasia[J].J Endocrine Soci,2017(5):488-491.
[10]YU H W,WEI G,XIN W,et al.Advances in research on Fc fusion protein in pharmaceutical field[J].Prog Pharm Sci,2014(33):358-362.(in Chinese)王宇恒,郭薇,王欣,等.Fc融合蛋白在药学领域的研究进展[J].药学进展,2014(6):419-425.
[11]SCOTT L J.Asfotase alfa:a review in paediatric-onset hypophosphatasia[J].Drugs,2016,76(2):255-262.
[12]WHYTE M P,ROCKMAN-GREENBERG C,OZONO K,et al.Asfotase alfa treatment improves survival for perinatal and infantile hypophosphatasia[J].J Clin Endocrinol Metab,2016,101(1):334-342.
[13]OIKAWA H,TOMATSU S,HAUPT B,et al.Enzyme replacement therapy on hypophosphatasia mouse model[J].J Inherited Metab Dis,2014,37(2):309-317.
[14]ZHONG L P,LV Y N,CUI L.Research and application of bone-targeting compounds aspartic acid oligopeptide[J].Qilu Pharm Affairs,2011,35(2):29-31.(in Chinese)仲利萍,吕应年,崔燎.骨靶向化合物天冬氨酸寡肽的研究及应用[J].药学研究,2011,30(4):228-231.
[15]FLANAGAN M L,ARIAS R S,HU P,et al.Soluble Fc fusion proteins for biomedical research[J].Methods Mol Biol,2007,378:33.
[16]WATIER,HERVE.Variability factors in the clinical response to recombinant antibodies and IgG Fc-containing fusion proteins[J].Expert Opin Biol Ther,2005,5(s1):S29-S36.
[17]WHYTE M P,GREENBERG C R,SALMAN N J,et al.Enzyme-replacement therapy in life-threatening hypophosphatasia[J].New Engl J Med,2012,366(10):904-913.
[18]ZHENG H H,JIANG H.Progress of CHO expression system[J].Curr Biotechnol,2016.(in Chinese)郑惠惠,江洪.CHO细胞表达系统研究进展[J].生物技术进展,2016,6(4):239-243.
[19]MILLAN J L,PLOTKIN H.Hypophosphatasia-pathophysiology and treatment[J].Actualizaciones En Osteologia,2012,8(3):164-182.
[20]SHAPIRO J R,LEWIECKI E M.Hypophosphatasia in adults:clinical assessment and treatment considerations[J].J Bone&Mineral Res,2017,25(3):277-289.
[21]LEVIN D,GOLDING B,STROME S E,et al.Fc fusion as a platform technology:potential for modulating immunogenicity[J].Trends Biotechnol,2014,33(1):27-34.
[22]HUANG C.Receptor-Fc fusion therapeutics,traps,and MIME-TIBODY technology[J].Curr Opin Biotechnol,2009,20(6):692-699.
[23]QU M H,YU J Y,HU M R,et al.Expression and purification of human CD28-Ig fusion protein in eukaryotic cells[J].J Med Mol Biol,2005,2(5):337-341.
[2]SCOTT L J.Asfotase alfa:a review in paediatric-onset hypophosphatasia[J].Drugs,2016,76(2):255-262.
[3]CHEN B,LI L,REN W,et al.A novel missense mutation in the ALPL gene causes dysfunction of the protein[J].Mol Med Rep,2017,16(1):710-718.
[4]CHANG K C,LIN P H,SU Y N,et al.Novel heterozygous tissue-nonspecific alkaline phosphatase(TNAP)gene mutations causing lethal perinatal hypophosphatasia[J].J Bone&Mineral Metab,2012,30(1):109-113.
[5]BELACHEW D,KAZMERSKI T,LIBMAN I,et al.Infantile hypophosphatasia secondary to a novel compound heterozygous mutation presenting with pyridoxine-responsive seizures[J].Jimd Rep,2013:17-24.
[6]DUAN X,LIU R,XU W,et al.Effect of cell culture conditions on antibody heterogeneity[J].Chin J Biotechnol,2013,29(12):1880-1886.
[7]WEI L U,KUN J I.Advances in research on hypophosphatasia[J].Chin J Clinicians,2013,16(8):27-29.(in Chinese)冀堃.ALPL基因突变影响ADRB2基因调控BMMSCs成骨分化的实验研究[D].陕西西安:第四军医大学,2013.
[8]SCOTT,LESHLEY J.Asfotase alfa in perinatal/infantile-onset and juvenile-onset hypophosphatasia:a guide to its use in the USA[J].BioDrugs,2016,30(1):41-48.
[9]REMDE H,COOPER M,QUINKLER M.Successful asfotase alpha treatment in an adult dialysis patient with childhood-onset hypophosphatasia[J].J Endocrine Soci,2017(5):488-491.
[10]YU H W,WEI G,XIN W,et al.Advances in research on Fc fusion protein in pharmaceutical field[J].Prog Pharm Sci,2014(33):358-362.(in Chinese)王宇恒,郭薇,王欣,等.Fc融合蛋白在药学领域的研究进展[J].药学进展,2014(6):419-425.
[11]SCOTT L J.Asfotase alfa:a review in paediatric-onset hypophosphatasia[J].Drugs,2016,76(2):255-262.
[12]WHYTE M P,ROCKMAN-GREENBERG C,OZONO K,et al.Asfotase alfa treatment improves survival for perinatal and infantile hypophosphatasia[J].J Clin Endocrinol Metab,2016,101(1):334-342.
[13]OIKAWA H,TOMATSU S,HAUPT B,et al.Enzyme replacement therapy on hypophosphatasia mouse model[J].J Inherited Metab Dis,2014,37(2):309-317.
[14]ZHONG L P,LV Y N,CUI L.Research and application of bone-targeting compounds aspartic acid oligopeptide[J].Qilu Pharm Affairs,2011,35(2):29-31.(in Chinese)仲利萍,吕应年,崔燎.骨靶向化合物天冬氨酸寡肽的研究及应用[J].药学研究,2011,30(4):228-231.
[15]FLANAGAN M L,ARIAS R S,HU P,et al.Soluble Fc fusion proteins for biomedical research[J].Methods Mol Biol,2007,378:33.
[16]WATIER,HERVE.Variability factors in the clinical response to recombinant antibodies and IgG Fc-containing fusion proteins[J].Expert Opin Biol Ther,2005,5(s1):S29-S36.
[17]WHYTE M P,GREENBERG C R,SALMAN N J,et al.Enzyme-replacement therapy in life-threatening hypophosphatasia[J].New Engl J Med,2012,366(10):904-913.
[18]ZHENG H H,JIANG H.Progress of CHO expression system[J].Curr Biotechnol,2016.(in Chinese)郑惠惠,江洪.CHO细胞表达系统研究进展[J].生物技术进展,2016,6(4):239-243.
[19]MILLAN J L,PLOTKIN H.Hypophosphatasia-pathophysiology and treatment[J].Actualizaciones En Osteologia,2012,8(3):164-182.
[20]SHAPIRO J R,LEWIECKI E M.Hypophosphatasia in adults:clinical assessment and treatment considerations[J].J Bone&Mineral Res,2017,25(3):277-289.
[21]LEVIN D,GOLDING B,STROME S E,et al.Fc fusion as a platform technology:potential for modulating immunogenicity[J].Trends Biotechnol,2014,33(1):27-34.
[22]HUANG C.Receptor-Fc fusion therapeutics,traps,and MIME-TIBODY technology[J].Curr Opin Biotechnol,2009,20(6):692-699.
[23]QU M H,YU J Y,HU M R,et al.Expression and purification of human CD28-Ig fusion protein in eukaryotic cells[J].J Med Mol Biol,2005,2(5):337-341.