摘要
采用液氮冷冻研磨法破碎吉陶单极虫（Ｔｈｅｌｏｈａｎｅｌｕｓｋｉｔａｕｅｉ）孢子，按常规法提取其基因组ＤＮＡ。取ＤＮＡＥｃｏＲＩ消化产物，采用低熔点琼脂糖回收法制备大（３．０～１５．０ｋｂ）、小（０．５～３．０ｋｂ）片段，将其克隆于ｐＢｌｕｅｓｃｒｉｐｔｋｓＥｃｏＲＩ位点，经α－互补现象、酶切鉴定和虫体ＤＮＡ生物素标记探针筛选，构建了含２３个克隆的基因文库，克隆片段介于０．９～６．８ｋｂ之间；经阳性克隆标记筛选及特性分析，制备了长度为０．９ｋｂ（１９号质粒克隆片段）的探针，该探针具有较强的敏感性和特异性。对该探针两端ＤＮＡ序列进行分析，设计出１对引物。上游引物序列为：５′ＴＴＴＣＧＡＡＣＣＣＧＣＡＣＡＡＣＡＡＧ３′，下游引物序列为：５′ＴＧＡＧＴＧＧＡＴＡＧ－ＴＡＴＣＧＣＴＧＣ３′。
T.kitauei spores were ruptured ultrasonically and its DNA isolated, then the fragment of DNA digested by Eco RI were cloned into pBluscript ks Eco RI site. Through α complementation, Eco RI digesting identification and selection of T.kitauei DNA probe which was labelled with biotin, the gene library of T.kitauei containing 23 clones were established with the length of cloning fragments to be from 0.9 kb to 6.8 kb. Through the selection of T.kitauei DNA probe and the analyses of sensitivity and specifility of positive clones, the fragment that was about 0.9 kb of No.19 clone was labelled to prepare gene probe which could detect 5 mg/L of parasite DNA. Both ends of this gene probe were sequenced. Through sequence analysis by computer, upstream primer (P 1) and downstream one (P 2) were designed. P 1 sequence was 5′TTTCGAACCCGCACAACAAG3′, P 2 sequence was 5′TGAGTGGATAGTATCGCTGC3′. P 1 and P 2 primers were specific to myxosporidia. The detective methods for T.kitauei with gene probe and PCR were tested and discussed.
出处
《中国兽医学报》
CAS
CSCD
1996年第6期580-584,共5页
Chinese Journal of Veterinary Science
