[Construction of chicken embryo fibroblasts cDNA expression library].
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.
Liu
Wei
W
Division of Avian Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China. lw-790613@126.com
Gao
Yu-long
YL
Gao
Hong-lei
HL
Wang
Xiao-mei
XM
Xu
Xiu-hong
XH
D000818:Animals; D018175:Birnaviridae Infections / Q000662:veterinary* / Q000821:virology; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism* / Q000821:virology; D015870:Gene Expression; D015723:Gene Library; D007243:Infectious bursal disease virus / Q000235:genetics*; D011201:Poultry Diseases / Q000821:virology*; D014158:Transcription, Genetic
D018076:DNA, Complementary