【摘要】 以经产荷斯坦奶牛外周血白细胞为材料,分离Poly(A)+ RNA,反转录合成单链及双链cDNA,经酶切成平均大小为400~600 bp的片段,将患乳房炎奶牛cDNA分成2 组,分别与2 种不同的接头连接,再与健康奶牛cDNA进行2 次消减杂交和2 次抑制性PCR 扩增,将第2 次PCR 产物与pGEM T 载体连接,转化大肠杆菌TOP10感受态细胞进行文库扩增,构建了具有高消减效率的奶牛乳房炎抗性相关cDNA文库。文库扩增后得到610个白色阳性克隆,随机挑选克隆进行PCR鉴定,插入片段主要分布在250~750 bp 之间。文库的成功构建为进一步筛选、克隆与奶牛乳房炎抗性相关的基因奠定了基础,对研究奶牛乳房炎抗性的分子机制以及乳房炎的综合防制具有重要意义。更多还原

【Abstract】 Poly (A) + RNA were purified using Oligotex mRNA Kits (Qiagen) from peripheral blood leukocytes of 20 Holstein cows with mastitis and 20 health Holstein cows in lactation as control; single- and double-stranded cDNA were synthesized from the poly(A) + RNA using PCR-Select TM cDNA Subtraction Kit (Clontech) and further digested using RsaⅠ into cDNA fragments sized from 400 to 600 bp (dscDNA); dscDNAs from the mastitis cows (as tester) were divided into two portions which were ligated separately with a different cDNA adaptor; the ligated cDNA were hybridized twice with the dscDNA of the healthy cows at 68 ℃ for 8 h each; the products after double hybridizations were diluted by 200 times and then used for suppression PCR twice; the secondary PCR products was inserted into PGEM-T vector and transformed into E.coli TOP10 competent cells; 610 positive clones were obtained; identification of the inserted cDNA fragments in subtractive library was done using PCR. The results showed that there were inserted fragments sized by 250~750 bp in the 16 randomly selected positive clones, which would provide useful baseline for screening and cloning specific mastitis resistant genes and understanding the molecular mechanism of mastitis resistance in dairy cow.更多还原