摘要
目的 :构建白纹伊蚊溴氰菊酯抗性株 4龄幼虫cDNA文库。方法 :抽提、纯化白纹伊蚊溴氰菊酯抗性株 4龄幼虫总RNA ,进行反转录合成第 1链cDNA ;用Clontech公司SMARTTM cDNA文库构建试剂盒进行长距离PCR ,合成全长双链cDNA ;PCR产物经蛋白酶K消化、提纯后 ,进行SfiI酶切 ;用ChromaSpin 40 0柱将酶切产物进行分级分离 ,然后经 1 1%琼脂糖凝胶电泳鉴定 ,回收 40 0bp以上的组分 ,并与λTriplEx2载体连接 ;连接产物经体外蛋白包装 ,产生未扩增文库 ;检测未扩增文库滴度和重组效率后 ,进行文库的扩增 ,并测定扩增文库的滴度 ;随机挑取 9个噬菌体 ,用载体克隆位点两端的引物进行PCR扩增 ,以检测所构建的cDNA文库的质量。结果 :经检测 ,未扩增文库滴度达 2 0× 10 7pfu mL ,重组效率在 10 -4 稀释度时每块平板约 2 10～ 2 5 5个噬菌斑中均未发现任何蓝色噬斑 ,扩增文库滴度达 1 75× 10 9pfu mL ;用载体克隆位点两端的引物进行PCR鉴定 ,结果显示 :所选 9个噬菌体中均含有重组的cDNA ,并且均在 5 0 0bp以上 ,其中 1kb以上的有 2个、 70 0bp的有 5个、 5 0 0bp的有 2个。结论 :已成功地获得一高质量的白纹伊蚊溴氰菊酯抗性株 4龄幼虫cDNA文库。
Objective:To construct a cDNA library from the 4 th larvae of deltamethrin resistant strain of Aedes albopictus. Method:Total RNA from the 4 th larvae was extracted and retro transcribed to synthesize the first strand cDNA. LD PCR was carried out to produce the full length ds cDNA by the use of SMART TM cDNA Library Construction Kit from CLONTECH. The PCR product was digested by proteinase K and Sfi I, and fractionated with the CHROMA SPIN 400 Column. The fractions were characterized by 1 1% agarose/EB gel electrophorese. The cDNAs of more than 400bp were collected and ligated to λTriplEx2 Vector. The λ phage packaging reaction for the ligations was performed to produce an unamplified library. After the unamplified library and the percentage of recombinant clones were respectively titered, the library amplification and the titer of the amplified library were executed, successively. Nine phages were randomly selected and amplified using primers from both ends of clone site on the vector by PCR technique to test the quality of th obtained library. Results:The titers of the unamplified library and the amplified library are 2 0×10 7pfu/mL and 1 75×10 9pfu/mL, respectively. The percentage of recombinant clones was close to 100%. The result of PCR indicated that all the selected phages possessed the recombinated cDNA, and that these cDNAs are beyond 500bp. Conclusion:A qualified cDNA library from the 4 th larvae of deltamethrin resistant strain of Ae. albopictus was successfully obtained.
出处
《寄生虫与医学昆虫学报》
CAS
2001年第2期92-98,T002,共8页
Acta Parasitologica et Medica Entomologica Sinica
基金
广东省自然科学基金资助项目!(970 0 92 )
"2 11工程"重点学科建设基金资助项目!(9812 4)
关键词
白纹伊蚊
杀虫剂
抗性
CDNA文库
Aedes albopictus Insecticide resistance cDNA library
