旋毛虫5日龄成虫期特异性基因片段的克隆及鉴定

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cDNA PCR产物+未差减cDNA为试验方探针,以驱动方的差减cDNA PCR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southern blot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464 bp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.

Cloning and identification of the stage-specific gene fragment of 5 day-old adult worm of Trichinella spiralis

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.