摘要
采用Trizol试剂法提取鸡柔嫩艾美耳球虫石家庄多重耐药虫株的总RNA,利用3种锚定引物和20种随机引物进行RT-PCR,产物经变性聚丙烯酰胺凝胶电泳后银染,共回收10条差异条带,进行2次PCR扩增,产物回收后与pMDTM18-T Vector连接转化,再进行斑点杂交试验、序列分析和同源性比较。结果表明,石家庄多重耐药性虫株的mRNA的S116序列与GenBank和Sanger上已发表的柔嫩艾美耳球虫第1段染色体上882bp长的序列同源性达99%,为未知蛋白。这为克隆全长cDNA和探索球虫耐药性产生的分子机理奠定了基础。
Total RNA of E.tenelladrug-resistant strain from Shijiazhuang was extracted with Trizol.DDRT-PCR was established by using 3 anchored primers and 20 arbitrary primers.The products of PCR were analyzed on the denaturing polyacrylamide gels by silver-straining.Ten differential fragments were excised from the gels and reampilfied with the same sets of primers.Then the PCR products were purified and ligated with pMDTM18-T vector.The Dot-blot hybridization proved that the one clone from Shijiazhuang drug-resistant strain of E.tenellaappeared the strong signals.After sequence analysis compared with homogenize,the similarity was 99% among the sequence S116 from mRNA of Shijiazhuang drug-resistant strain with the sequence 882 bp length in the first chromosome of E.tenellain GenBank and Sanger,and it was an unknown protein.This study paved the way for cloning the full-length cDNAs and finding the molecular mechanism about the drug-resistance of E.tenella.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2013年第9期1373-1377,共5页
Chinese Journal of Veterinary Science
基金
河北省自然科学基金资助项目(C2006000554)
