羊尤氏泰勒虫一个新生多肽复合物α多肽基因的cDNA文库筛选和测序(英文)

MONOCLONAL ANTIBODY SCREENING AND SEQUENCING OF A NASCENT POLYPEPTIDE ASSOCIATED COMPLEX SUBUNIT ALPHA-LIKE SEQUENCE FROM THEILERIA UILENBERGI CDNA LIBRARY

为筛选羊尤氏泰勒虫裂殖子功能基因,用羊尤氏泰勒虫裂殖子提取物免疫BALB/c小鼠,将小鼠脾脏细胞与鼠骨髓瘤SP2/0-Ag14细胞融合,融合细胞培养上清用酶联免疫吸附法和免疫印记法检测。提取羊尤氏泰勒虫裂殖子mRNA后,进行cDNA合成与文库构建。文库在免疫学噬斑筛选后,将阳性克隆进行测序和序列BLAST搜索。筛选结果显示有两个阳性克隆。所得序列BLAST搜索结果显示,它与12类寄生虫新生多态相关复合物α多肽基因的同源性在39%到78%之间。结果提示,所筛选的序列为羊尤氏泰勒虫裂殖子新生多态相关复合物α多肽基因不完全序列。该研究为今后使用单克隆抗体筛选羊泰勒虫功能性基因提供了参考。

Extracts of Theileria uilenbergi merozoites were used to immunize Balb/c mice and the spleen cells were fused with mouse myeloma SP2/0-Ag14.The culture supernatants were tested for antibody production by ELISA and Western blot.The monoclonal antibodies were used to screened cDNA library of T.uilenbergi merozoites.Plaque screening was carried out with the pico BlueTM Immunoscreening Kit.The positive clones were sequenced and a BLAST search was performed using the obtained sequences.Two positive clones were identified.The BLAST search revealed 39% to 78% homology of the cloned sequences with 12 parasite NAC subunit alpha sequences.This suggested that the sequence screened by mAb was the NAC subunit alpha-like partial sequence of T.uilenbergi.This is the first report on the screening of T.uilenbergi functional antigen gene by using monoclonal antibodies.