鸡胚成纤维细胞cDNA表达文库的构建

Construction of chicken embryo fibroblasts cDNA expression library

摘要:

鸡胚成纤维细胞(CEF)是研究鸡传染性法氏囊病病毒(IBDV)的主要细胞材料,而构建CEF的cDNA表达文库是筛选IBDV在CEF中的细胞受体,研究细胞嗜性的基础平台。采用Gateway技术构建CEF的表达文库,避免使用限制性内切酶切割cDNA,能够解决常规方法构建cDNA文库的技术缺陷。该技术将CEF的mRNA分离纯化后,以5′端生物素标记的Oligo(dT)primer为引物反转录后连接Adapter,层析柱纯化,通过BP重组反应构建cDNA入门文库,其平均滴度为1.1×10⁶cfu/mL,文库总容量为1.2×10⁷cfu,平均插入片段为2243bp,重组率为100%。通过LR重组反应将入门文库转换为表达文库,经测定平均滴度为5×10⁵cfu/mL,文库总容量为5.5×10⁶cfu,平均插入片段为2411bp,重组率为100%。结果表明,所构建的文库具有较高的重组率和较大的库容量,可作为较高质量的文库来研究IBDV的相关基因,为研究病毒受体和病毒入侵途径,进一步了解IBDV的致病机理奠定了基础。

Abstract:

Chicken embryo fibroblast (CEF) is a primary cellular material to research the infectious bursal disease virus (IBDV). Constructing the cDNA expression library of CEF is the foundation to research cell tropism and find cell receptors of IBDV from CEF. In order to achieve that purpose, a high-quality cDNA expression library of CEF was constructed by Gateway technology, which could avoid using the restriction enzyme for cloning to solve technical limitation of roution method. The mRNA was extracted from chicken embryonic fibroblast. Moreover, single-strand cDNA and double-strand cDNA were synthesized by using biotin-conjugated Oligo (dT) primer in turn. The double-strand cDNA was ligated Adapter and then purified by the cDNA Size Fractionation Columns. After BP recombination reaction, a cDNA entry library was constructed with a titer of 1×10⁶cfu/mL, total clones of 1.2×10⁷cfu and an average insertion size of about 2243bp. After LR recombination reaction, the cDNA entry library was transformed into expression library which took on a titer of 5×10⁵cfu/mL, total clones of 5.5×10⁶cfu and an average insertion size of about 2411bp. The results indicate that the constructed cDNA expression library performs a remarkable high value in both recombination rate and library coverage. As a result, the cDNA expression library, with its good quality, may facilitate to identify the receptors associated with the resistance against IBDV in chicken embryonic fibroblast and to cast new light on the mechanism of cellular tropism. Moreover, it may also provide data of chicken embryonic fibroblast in transcription level and may be helpful to study its biological functions.