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| Cloning and Expression of Fusion Protein of Porcine TNNC2 Gene in Escherichia coli |
| Yan-Fang LI Jia-Qi LI Ying-Jie MEI Song-Ling CHEN Shui-Hua XIE Shi-Xin LI Wan-Fu ZHONG |
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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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Received: 12 September 2007
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Corresponding Authors:
Jia-Qi LI
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ZHU Xiao-Feng, XU Hou-Qiang, CHEN Wei, CHEN Ying-Lian, NI Kai, WU Xiao-Min, NI Meng-Meng, LU Xian-Jun. Cloning, Expression and Bioinformatics Analysis of IGF-1 and IGF-2 Genes in Congjiangxiang Pigs (Sus scrofa)[J]. 农业生物技术学报, 2019, 27(8): 1382-1391. |
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