| 猪TNNC2基因的克隆及其融合蛋白在大肠杆菌中的表达 |
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| 引用本文： | 李艳芳,李加琪,梅盈洁,钟万福,陈松玲,谢水华,李仕新.猪TNNC2基因的克隆及其融合蛋白在大肠杆菌中的表达[J].农业生物技术学报,2008,16(2):214-220. |
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| 作者姓名： | 李艳芳 李加琪 梅盈洁 钟万福 陈松玲 谢水华 李仕新 |
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| 作者单位： | 华南农业大学动物科学学院,广州,510642 |
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| 基金项目： | 国家重点基础研究发展计划(973计划)
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国家高技术研究发展计划(863计划)
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国家自然科学基金
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广东省自然科学基金 |
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| 摘 要： | 根据GenBank上猪TNNC2（Fast skeletal muscle troponin C2）基因序列（GenBank accession No. DQ629177）设计一对引物，采用RT-PCR方法克隆得到617 bp TNNC2 cDNA片段（GenBank accession No. EF673726），包括完整的开放阅读框（ORF），与GenBank上公布的猪TNNC2基因（GenBank accession No. AY575058）的ORF核苷酸序列同源性达99 %，并发现开放阅读框内的5个点突变，319位点T→C，320 位点G→A，321位点C→T，导致氨基酸107位Ala（丙氨酸）→Met（蛋氨酸），322位点A→G为同义突变，433位点A→T，导致氨基酸144位Glu（谷氨酸）→Asp（天冬氨酸）。根据已获得的TNNC2基因开放阅读框序列，重新设计引物扩增得到包含BamH I和EcoR I酶切位点的完整阅读框，将其首先克隆到pMD18-T载体中，经菌液PCR筛选和酶切鉴定后，用BamH I和EcoR I将目的片段切下，再克隆到原核表达载体pRSET A中构建重组表达质粒pRSET A-TNNC2。将重组质粒转化大肠杆菌BL21（DE3），IPTG不同诱导条件诱导表达，经SDS-PAGE电泳和Western blot分析证实重组表达质粒pRSET A-TNNC2表达出24 kD左右的融合蛋白，最佳诱导时间为4 h，最佳的IPTG诱导浓度为0.6 mmol/L，表达产物以可溶性蛋白的形式存在。
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| 关 键 词： | 猪 TNNC2基因 融合蛋白 大肠杆菌 |
| 文章编号： | 1006-1304(2008)02-0214-07 |
| 收稿时间： | 2007-9-12 |
| 修稿时间： | 2007年9月12日 |
Cloning of Porcine TNNC2 Gene and Expression of Its Fusion Protein in Escherichia coli |
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| LI Yan-fang,LI Jia-qi,MEI Ying-jie,ZHONG Wan-fu,CHEN Song-ling,XIE Shui-hua,LI Shi-xin.Cloning of Porcine TNNC2 Gene and Expression of Its Fusion Protein in Escherichia coli[J].Journal of Agricultural Biotechnology,2008,16(2):214-220. |
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| Authors: | LI Yan-fang LI Jia-qi MEI Ying-jie ZHONG Wan-fu CHEN Song-ling XIE Shui-hua LI Shi-xin |
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| Abstract: | In order to amplify the complete ORF of the porcine TNNC2 ( Fast skeletal muscle troponin C2 ) gene, one pair of primers was designed according to porcine TNNC2 mRNA ( GenBank accession No. DQ629177 ). A 617 bp TNNC2 cDNA fragment including the complete open reading frame ( ORF ) was obtained by RT-PCR. The nucleotide sequence identity of TNNC2 cDNA of 617 bp ( GenBank accession No. EF673726 ) with the porcine TNNC2 mRNA ( GenBank accession No. AY575058 ) was 99 %. Five nucleotide mutations were found in TNNC2 ORF. 319 T→C, 320 G→A and 321 C→T, resulted in amino acid substitutions 107 Ala→Met. 322 A→G was a silent mutation. 433 A→T resulted in amino acid substitution 144 Glu→Asp. The pig TNNC2 complete ORF, containing BamH I and EcoR I restriction sites, was amplified with one pair of primers designed according to TNNC2 complete ORF in this study. Then the PCR product was cloned into pMD18-T vector. In order to produce the recombinant expression vector pRSET A-TNNC2, the T-TNNC2 recombinant plasmid was digested with BamH I and EcoR I after colony PCR and restriction analysis, and the fragment was inserted into pRSET A vector digested with the same enzymes. The pRSET A-TNNC2 recombinant plasmid was transformed into E. coli BL21 ( DE3 ) and induced to express fusion protein with IPTG on different induction conditions. SDS-PAGE and Western blot of pRSET A -TNNC2 indicated that about 24 kD fusion protein was obtained. Further, the effects of concentration of IPTG and induction time on the yield of pRSET A -TNNC2 fusion protein were studied, the results of SDS-PAGE indicated that the best expression time of pRSET A -TNNC2 fusion protein was 4 hours and the best concentration of IPTG was 0.6 mmol/L. Analysis of the protein solubility showed that pRSET A -TNNC2 fusion protein was expressed in the form of soluble protein. |
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