摘要
利用差减抑制杂交 (SSH)技术 ,以旋毛虫 5日龄成虫的cDNA为试验方 (tester) ,以肌幼虫 + 3日龄成虫的cDNA为驱动方 (driver) ,制备 5日龄成虫差减cDNA ,并与 pT Adv载体相连接 ,转入大肠埃希氏菌TOP 10F。以试验方的差减cDNAPCR产物 +未差减cDNA为试验方探针 ,以驱动方的差减cDNAPCR产物 +未差减cDNA为驱动方探针 ,对 5日龄成虫差减cDNA克隆进行初步筛选 ,对筛选到的阳性克隆进一步用southernblot进行鉴定 ,并对其测序 ,进行序列分析。结果表明 ,获得 1个大小为 464bp的旋毛虫 5日龄成虫期特异性cDNA片段 ,经BLAST软件分析表明 ,该序列是 1个未见报道的旋毛虫基因序列片段 ,可能编码 1种表皮胶原蛋白。这为旋毛虫 5日龄成虫期特异性基因全长序列的钓取、分析及鉴定奠定了基础。
By means of a suppression subtraction hy bridization (SSH) method the subtracted cDNA of 5 day-old adult worm of Trichinella spiralis was prepared using cDNA of the 5 day-ol d adult worm as Tester and cDNA of the muscle larvae + 3 day-old adult worm as Driver and ligated to pT-Adv vector and then was transformed into Esche richia coli TOP 10F. The subtracted cDNA clone of 5 day-old adult worm was primarily screened using the Tester subtracted cDN_PCR product + unsubtrac ted cDNA as probe of Tester as well as using the Driver subtracted cDN_PCR pro duct + unsubtracted cDNA as probe of Driver, and then the screened positive clon e was further identified by Southern blot and sequenced following analysis of th e sequence. The test result showed that a 464 bp stage-specific cDNA fragment o f 5 day-old adult worm of Trichinella spiralis was obtained, wh ich was a cDNA fragment that had been not reported before by the BLAST software and probably was responsible for coding a cuticle collagen.
出处
《中国兽医科技》
CSCD
北大核心
2001年第11期9-11,共3页
Chinese Journal of Veterinary Science and Technology
