骨骼肌肌纤维形成机制的研究进展

目的构建鸡快骨骼肌亚型肌钙蛋白I 基因(fast skeletal muscle troponin I，TNNI2)的真核表达载体，并在永生化鸡前脂肪细胞系(immortalized chicken preadipocyte cell line，ICP1)细胞中转染和鉴定。方法利用RT-PCR方法获得鸡TNNI2基因的全长CDS区序列，采用定向克隆的方法构建该基因的真核表达载体，并对基因序列进行功能生物信息学分析，构建系统发育树。结果TNNI2基因ORF全长552 bp，编码183个氨基酸，蛋白质分子量大小为21.24 ku，理论等电点(pI)为9.19；fsTnI蛋白无信号肽和跨膜区，亚细胞定位于细胞核，属于亲水性不稳定蛋白质；磷酸化预测含有14个磷酸化位点；二级结构主要以α螺旋的形式存在；同源性比对结果表明：不同物种间的TNNI2基因具有较高的同源性。结论本研究成功构建了TNNI2基因的真核表达载体pCMV-HA-TNNI2，并成功转染ICP1细胞系，本结果可为进一步研究鸡TNNI2基因的功能奠定基础。

TNNI1, TNNI2 and TNNI3: evolution,regulation, and protein structure-function relationships

PurposeTo construct the eukaryotic expression vector of fast skeletal muscle troponin I (TNNI2) gene, and to transfect and identify in immortalized chicken preadipocyte cell line (ICP1).MethodsThe RT-PCR was used to clone the whole length of CDS sequence of chicken TNNI2 gene. The eukaryotic expression vector of TNNI2 was constructed by one-step cloning method. The functional bioinformatics analysis was applied to construct the phylogeny tree and study the nucleic acid and the protein of TNNI2.ResultsThe full-length ORF of TNNI2 was 552 bp, and it encodes 183 amino acids, the theoretical size of the protein was 21.24 ku, the theoretical isoelectric point (pI) was 9.19. Furthermore, the protein had no transmembrane region and no signal peptide region, and was mainly located in the nucleus. It belongs to hydrophilic protein. The protein contains 14 phosphorylation sites, secondary structure of this protein is mainly composed of α-helix. In addition, the TNNI2 gene shares highly homology among different species.ConclusionWe constructed the pCMV-HA-TNNI2 eukaryotic recombinant vector and transfected it into ICP1 cells successfully, which lays a foundation for further study of TNNI2 function in chicken.