摘要
目的 获得粉尘螨Derf 2cDNA克隆及亚克隆 ,并进行测序。方法 设计合成引物 ,从粉尘螨螨体中提取RNA ,经RT -PCR获得cDNA ,PCR扩增目的片段经纯化回收后克隆至 pMD - 18T ,转化大肠杆菌E coliJM10 9,经PCR初筛挑选阳性克隆并测序 ,将PCR筛检阳性重组子及pET - 32a(+)表达载体分别用SacⅠ和NotⅠ双酶切 ,连接转化至E coliCompe tentCellJM 10 9中过夜培养 ,挑选菌落进行酶切鉴定。结果 从粉尘螨基因组RNA中扩增出Derf 2cDNA片段 ,成功构建pET - 32a(+) -Derf 2亚克隆 ,酶切产物的大小与预期相符。 结论 成功地对粉尘螨Derf 2cDNA进行体外扩增并构建pET - 32a(+) -Derf 2亚克隆。
To clone,sequence and subclone the cDNA encoding the group Ⅱ allergen of Dermatophagoides farinae (Der f 2),the cDNA of Der f 2 was amplified by RT-PCR,and PCR. and the gene fragments were cloned into a expression vector pMD-18T.The recombinant plasmid pMD-18T-Der-f 2 was then transformed into E.coli JM109.Positive clones were screened,identified by PCR and digested with restriction endonuclease.The sequence of the inserted Der f 2 cDNA was also determined,and then it was subcloned into the expression vector pET32a(+).It was found that the recombinant plasmids pMD-18T-Der f 2 and pET32a(+)-Der f 2 were constructed and digested by Sac Ⅰ and Not Ⅰ.The size of the gene fragment was 455 bp in accordance with the expected value.It concludes that the pET32a(+)-Der f 2 subclonint has been constructed successfully.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2004年第7期630-632,648,共4页
Chinese Journal of Zoonoses
基金
安徽省自然科学基金 (No 0 3 0 43 3 0 3 )资助
关键词
粉尘蝻
抗原
EDNA
序列分析
Dermatophagoides farinae
antigens
cDNA
Sequence analysis
