Liu Wei, Gao Yu-long, Gao Hong-lei, Wang Xiao-mei, Xu Xiu-hong
Division of Avian Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China.
Wei Sheng Wu Xue Bao. 2007 Jun;47(3):413-7.
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 x 10(6) cfu/mL, total clones of 1.2 x 10(7) cfu and an average insertion size of about 2243 bp. After LR recombination reaction, the cDNA entry library was transformed into expression library which took on a titer of 5 x 10(5) cfu/mL, total clones of 5.5 x 10(6) cfu 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.
鸡胚成纤维细胞（CEF）是研究传染性法氏囊病病毒（IBDV）的一种原代细胞材料。构建CEF的cDNA表达文库是研究IBDV细胞嗜性及从CEF中寻找细胞受体的基础。为实现该目的，利用Gateway技术构建了高质量的CEF cDNA表达文库，该技术可避免使用限制性内切酶进行克隆，解决了常规方法的技术限制。从鸡胚成纤维细胞中提取mRNA。此外，依次使用生物素偶联的寡聚（dT）引物合成单链cDNA和双链cDNA。双链cDNA连接接头后通过cDNA大小分级分离柱进行纯化。经过BP重组反应，构建了一个滴度为1×10⁶ cfu/mL、总克隆数为1.2×10⁷ cfu且平均插入片段大小约为2243 bp的cDNA入门文库。经过LR重组反应，将cDNA入门文库转化为表达文库，其滴度为5×10⁵ cfu/mL、总克隆数为5.5×10⁶ cfu且平均插入片段大小约为2411 bp。结果表明，构建的cDNA表达文库在重组率和文库覆盖率方面均具有显著的高价值。因此，该高质量的cDNA表达文库可能有助于鉴定鸡胚成纤维细胞中与抗IBDV相关的受体，并为细胞嗜性机制提供新的线索。此外，它还可能提供鸡胚成纤维细胞转录水平的数据，并有助于研究其生物学功能。
