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.
李艳芳 李加琪 梅盈洁 陈松玲 谢水华 李仕新 钟万福. 猪TNNC2基因的克隆及其融合蛋白在大肠杆菌中的表达[J]. , 2008, 16(2): 0-.
Yan-Fang LI Jia-Qi LI Ying-Jie MEI Song-Ling CHEN Shui-Hua XIE Shi-Xin LI Wan-Fu ZHONG. Cloning and Expression of Fusion Protein of Porcine TNNC2 Gene in Escherichia coli. , 2008, 16(2): 0-.
