Myostatin （MSTN） is a negative regulator of skeletal muscle growth, in order to study the effect of inhibition MSTN expression on the proliferation of bovine skeletal muscle satellite cells, we constructed co-expres...Myostatin （MSTN） is a negative regulator of skeletal muscle growth, in order to study the effect of inhibition MSTN expression on the proliferation of bovine skeletal muscle satellite cells, we constructed co-expression vector pcDNA3.1-Pro- MSTNshRNA, transfected it into muscle satellite cells by Liposome 2000, and detected cell proliferation changes by CCK-8 method and flow cytometry after 48 h. The expressions of P21 and CDK2 were detected by Western blot and real-time PCR. The results showed that the cell vitality of experimental groups significantly increased than that of the negative control, and cells in S phase also increased significantly （P〈0.05）. After knocked down MSTN gene, P21 expression decreased （P〈0.05）, but CDK2 gene expression increased （P〈0.05）. These results indicated that MSTN gene expression was associated with P21 and CDK2, the proliferation of skeletal muscle satellite cells could be promoted while MSTN was inhibited, which provided a theoretical basis for the study on transgenic cattle.展开更多
The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the go...The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.展开更多
【目的】研究旨在对努比亚山羊大脑富集Ras同源物(Ras homolog enriched in brain,Rheb)基因进行克隆和分析,并构建其真核表达载体,为进一步揭示Rheb对努比亚山羊骨骼肌调控的分子机理奠定基础。【方法】试验采用RT-PCR方法从努比亚山...【目的】研究旨在对努比亚山羊大脑富集Ras同源物(Ras homolog enriched in brain,Rheb)基因进行克隆和分析,并构建其真核表达载体,为进一步揭示Rheb对努比亚山羊骨骼肌调控的分子机理奠定基础。【方法】试验采用RT-PCR方法从努比亚山羊背最长肌组织中扩增Rheb基因,经琼脂糖凝胶电泳及测序验证正确后进行生物信息学分析,同时构建该基因真核表达载体,转染细胞后进行实时荧光定量PCR检测来验证所构建载体的正确性。【结果】努比亚山羊Rheb基因编码区序列长度为555 bp,编码184个氨基酸,Rheb蛋白分子式为C 910 H 1456 N 236 O 279 S 6,分子质量为20359.34 u,原子总数为2887,理论等电点为5.93。Rheb蛋白属于稳定的亲水性蛋白,不包含跨膜结构域。蛋白二级结构预测结果显示,努比亚山羊Rheb蛋白中α-螺旋、β-转角、延伸链和无规则卷曲分别占40.76%、7.07%、22.83%和29.35%。构建的真核表达载体pcDNA3.1-Rheb转染山羊骨骼肌细胞后,与空载体组相比,Rheb基因表达量极显著升高(P<0.01)。【结论】本试验成功克隆努比亚山羊Rheb基因编码区序列,并构建pcDNA3.1-Rheb真核表达载体,这为深入理解Rheb基因在努比亚山羊肌肉中的作用提供了理论支持。展开更多
随着生命科学的迅速发展,细胞工程愈来愈受到人们的重视。以昆虫细胞为对象的细胞培养技术在现代实验生物学上具有重要的价值,已经广泛地应用于医学、农业及生物学的各个领域。本文综述了有关昆虫细胞培养的研究进展,包括昆虫细胞培养...随着生命科学的迅速发展,细胞工程愈来愈受到人们的重视。以昆虫细胞为对象的细胞培养技术在现代实验生物学上具有重要的价值,已经广泛地应用于医学、农业及生物学的各个领域。本文综述了有关昆虫细胞培养的研究进展,包括昆虫细胞培养基研究开发,昆虫细胞系的建立和组织培养,利用生物反应器大规模培养昆虫细胞,昆虫细胞-杆状病毒表达系统(B acu lov irus express ion vector system,BEV S),构建基因工程细胞系及其稳定性表达,以及昆虫细胞培养的应用前景和研究展望。展开更多
基金Supported by the Major Special Projects of New Product Training of Transgenic Organisms(zx080072008-2008)
文摘Myostatin （MSTN） is a negative regulator of skeletal muscle growth, in order to study the effect of inhibition MSTN expression on the proliferation of bovine skeletal muscle satellite cells, we constructed co-expression vector pcDNA3.1-Pro- MSTNshRNA, transfected it into muscle satellite cells by Liposome 2000, and detected cell proliferation changes by CCK-8 method and flow cytometry after 48 h. The expressions of P21 and CDK2 were detected by Western blot and real-time PCR. The results showed that the cell vitality of experimental groups significantly increased than that of the negative control, and cells in S phase also increased significantly （P〈0.05）. After knocked down MSTN gene, P21 expression decreased （P〈0.05）, but CDK2 gene expression increased （P〈0.05）. These results indicated that MSTN gene expression was associated with P21 and CDK2, the proliferation of skeletal muscle satellite cells could be promoted while MSTN was inhibited, which provided a theoretical basis for the study on transgenic cattle.
基金Supported by Key Teachers Foundation of Education Office of Heilongjiang Province, 2005 (1055G005)
文摘The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.
文摘【目的】研究旨在对努比亚山羊大脑富集Ras同源物(Ras homolog enriched in brain,Rheb)基因进行克隆和分析,并构建其真核表达载体,为进一步揭示Rheb对努比亚山羊骨骼肌调控的分子机理奠定基础。【方法】试验采用RT-PCR方法从努比亚山羊背最长肌组织中扩增Rheb基因,经琼脂糖凝胶电泳及测序验证正确后进行生物信息学分析,同时构建该基因真核表达载体,转染细胞后进行实时荧光定量PCR检测来验证所构建载体的正确性。【结果】努比亚山羊Rheb基因编码区序列长度为555 bp,编码184个氨基酸,Rheb蛋白分子式为C 910 H 1456 N 236 O 279 S 6,分子质量为20359.34 u,原子总数为2887,理论等电点为5.93。Rheb蛋白属于稳定的亲水性蛋白,不包含跨膜结构域。蛋白二级结构预测结果显示,努比亚山羊Rheb蛋白中α-螺旋、β-转角、延伸链和无规则卷曲分别占40.76%、7.07%、22.83%和29.35%。构建的真核表达载体pcDNA3.1-Rheb转染山羊骨骼肌细胞后,与空载体组相比,Rheb基因表达量极显著升高(P<0.01)。【结论】本试验成功克隆努比亚山羊Rheb基因编码区序列,并构建pcDNA3.1-Rheb真核表达载体,这为深入理解Rheb基因在努比亚山羊肌肉中的作用提供了理论支持。
文摘随着生命科学的迅速发展,细胞工程愈来愈受到人们的重视。以昆虫细胞为对象的细胞培养技术在现代实验生物学上具有重要的价值,已经广泛地应用于医学、农业及生物学的各个领域。本文综述了有关昆虫细胞培养的研究进展,包括昆虫细胞培养基研究开发,昆虫细胞系的建立和组织培养,利用生物反应器大规模培养昆虫细胞,昆虫细胞-杆状病毒表达系统(B acu lov irus express ion vector system,BEV S),构建基因工程细胞系及其稳定性表达,以及昆虫细胞培养的应用前景和研究展望。
