摘要
利用 Stratagene公司 Hybri ZAP- 2 .1建库试剂盒构建了经有丝分裂原刺激的鲤鱼外周血白细胞 c DNA文库。采取健康鲤鱼外周血 ,分离白细胞 ,经 L PS、PHA和 Con A分组作用不同时间后 ,分别提取总 RNA,将各组样品混合后 ,以 Oligo(d T)纤维素柱分离纯化 m RNA,经逆转录合成 c DNA的第 1链和第 2链 ,与 Eco R 接头连接 ,酶切后 ,用CHROMA SPIN- 4 0 0柱离心层析纯化 ,与 Hybri ZAP- 2 .1载体连接 ,体外包装后转染 E.coli XL I- Blue宿主菌 ,进行滴度测试和文库扩增。结果构建的经有丝分裂原刺激的鲤鱼外周血白细胞 c DNA文库原始库容量为 1.6 7× 10 6 pfu,插入片段长度在 0 .4× 10 3～ 3.0× 10 3bp,重组率为 98.9% ,扩增后的文库滴度为 6 .16× 10 9pfu/m L ,该文库符合 c DNA文库构建的标准 。
In order to clone and study immune related genes in carp (Cyprinus carpio L.) leucocytes, the cDNA library of mitogen stimulated carp leucocytes was constructed. First, the carp leucocytes were isolated from the peripheral blood of normal carp, the leucocytes samples were stimulated by treatment with 50 mg/L LPS, PHA and Con A for various time at 25℃, respectively, total RNA of leucocytes was then extracted, pooled together and mRNA was isolated with a column of oligo (dT) cellulose. Second, single-strand cDNA and double-strand cDNA were synthesized using Stratagene HybriZAP-2.1 XR cDNA synthesis kit, then ligated to EcoRⅠ adapters, size fractionating with CHROMA Spin-400 column. Finally cDNA were ligated into HybriZAP-2.1 vector and packaged in vitro. The obtained carp leucocyte cDNA library contains 1.67×10~6 recombinants, the percentage of vectors with inserts was 98.9% and the average inserts size were between 0.4×10~3-3.0×10~3 bp. After amplification, the titer of amplified library was 6.16×10~9 pfu/mL. This mitogen stimulated carp leucocytes cDNA library gives an ideal base for further study of carp leucocyte immune related cDNAs and genes.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2004年第6期568-570,共3页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目 ( 3 0 2 0 0 2 10 )
