摘要
用哺乳动物细胞表达克隆系统克隆了鸡ＩＬ－２ｃＤＮＡ。以ＣｏｎＡ（２０μｇ／ｍＬ）诱导ＳＰＦ鸡脾脏淋巴细胞，第２０小时收集细胞，提取ｍＲＮＡ，以ＳｕｐｅｒＳｃｒｉｐｔＰｌａｓｍｉｄＳｙｓｔｅｍｆｏｒｃＤＮＡＳｙｎｔｈｅｓｉｓ试剂盒合成ｃＤＮＡ，定向连结于穿梭质粒ｐＳＶ·ＳｐｏｒｔⅠ，构建ｃＤＮＡ文库。将文库分组提取重组质粒，转染ＣＯＳ－７细胞，表达ｃＤＮＡｓ。以淋巴细胞增殖试验检测表达产物ＩＬ－２活性。经过３轮筛选，获得１４个具有ＩＬ－２活性的克隆。选择其中４个进行酶切分析，结果显示这４个ｃＤＮＡ大小分别为１．５，１．０，０．８和０．８ｋｂ。
Using mammalian cell expression cloning system chicken IL2 cDNA was identified.Splenic lymphocytes derived from 2025 week old SPF Leghorn chicken were stimulated with Con A.After 20 hours IL2 activity in the medium was identified and mRNA was isolated from the activated spleen cells.Complementary DNA(cDNA)was synthesized using Super Scipt Plasmid System for cDNA Synthesis and directionally ligated to shuttle plasmid pSVSport ,and then used to transform E.coli. Library colones were divided into pools.Recombinant plasmids from each pool were transfected into COS7 cells.IL2 activity in COS7 cell medium was assayed by usage of the colorimetric proliferation assay.Through 3 round screening,14 cDNAs which could express IL2 activity in COS7 cells were identified.Four cDNAs being the IL2 positive were 15,10,08 and 08 kb,respectively,in size when analyzed with restriction endonuclease digestion.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
1999年第2期80-84,共5页
Journal of Nanjing Agricultural University
基金
国家博士后基金
