摘要
目的构建粉尘螨cDNA表达文库。方法提取粉尘螨总RNA和mRNA,以NotIdT18作为引物反转录合成第一链,置换法合成第二链。加EcoRI接头,经NotI酶切后,应用定向克隆技术将相应dscDNA片段插入λExCell/NotI/EcoRI/CIP载体的NotI和EcoRI双酶切位点间,经包装、转染宿主菌,构建成cDNA文库。检测文库库容量和重组效率,并采用PCR方法对插入片段大小进行分析。结果已成功构建一个含4.8×105个重组子的cDNA表达文库,重组率为100%,插入片段大小平均约1.2kb。结论所建文库容量及插入片段大小适合对粉尘螨特异性重组变应原的进一步研究。
To construct the cDNA expression library for Dermatophagoides farinae, the synthesis of the first chain of cDNA was catalyzed by Moloney murine leukemia virus (MMLV) reverse transcriptase with NotI dT18 as primer.After addition of EcoRI adaptor phosphorylation and digestion with NotI restriction enzymes,the cDNA with the 5' EdoRI end the 3' NotI end were directionally ligated with the EcoRI arms of the λExCell/NotI/CIP vector, and then followed by packaging with λDNA and transfection into host cell E.coli NM522. The cloning efficiency and the recombination quantity were evaluated,and the length of the cDNA fragment was assayed by PCR. By means of these procedures, the cDNA expression library containing 4.8x10 5 recombinants was successfully constructed. The efficiency of recombination was 100% and the average length of the inserted cDNA fragments was about 1.2 kb. This cDNA library is qualified for the further studies of the specific recombinant allergens in D.farinae.
出处
《中国人兽共患病杂志》
CAS
CSCD
北大核心
2004年第11期923-925,共3页
Chinese Journal of Zoonoses
基金
国家八六三计划(No.2002AA214011)
国家自然科学基金(No.30271226
30260101)
广东省科技计划重点项目(No.2003(104019)
深圳市科技计划项目。
关键词
粉尘螨
CDNA文库
构建
定向克隆技术
Dermatophagoides farinae
cDNA expression library
characterization
recombinant allergen.
