The Preliminary Study About Function Of Epstein-Barr Virus-Induced Gene 3

Objective The preliminary study about function of Epstein-Barr virus-induced gene 3 (EBI3) was done from the following both sides. (1) To identify binding sites of EBB for Sedlin. (2) To learn the location and character of Epstein-Barr virus-induced gene 3 in the mouse placenta and umbilical vessel.Methods (1) In our previous study we identified several proteins interacted with Sedlin by yeast-two-hybrid system screening of a human placenta cDNA library. We found that pACT2-EBI3 is the one of positive clone which codes amino acid regions 54-229 of EBI3 and there are Ncol and BamHI restriction sites in this part of EBI3. pACT2-EBI3 was transformed into the E.coli TG1 and amplified for being prepared and then digested respectively by NcoI or BamHI. We could get two long segments and two short segments and then respectively purified every part. The long segments were respectively ligated by themselves and became the mutants pEBI3m1 and pEBI3m2. The short segments were respectively inserted into pACT2 to construct the mutants pEBI3m3 and pEBI3m4. Then four mutants were amplified and identified by restriction endonucleases mapping analysis. After every mutant was cotransformed with pAS-Sedlin into Y190 yeast competent cells, assay of β-galactosidase activity was performed. The blue clones were positive, which shows protein-protein interactation. (2) For 12.5-18.5d umbilical cord and placenta production, Kunming mice were mated and the day on which a vaginal plug was found was designated 0.5d. (3) EBI3 was detected using EBB polyclone antibody by immunohistochemical SABC. Hematoxylin and eosin (HE) staining as well as victoria blue-ponceau red staining was used to observe the structure of umbilical vessel, which can contribute to preliminary identification of...