中文摘要：

以酵母穿梭表达质粒pGADT72Ree为载体，采用CLONTECH SMART（switching mechanism at 5’end of RNA transeript）技术，在酵母细胞中构建鸡法氏囊B细胞的酵母双杂交cDNA文库。通过从分离的鸡法氏囊B细胞中提取总RNA，利用经修饰的CDS引物合成cDNA第一链，在链的末端可自动延伸CCC，在反应体系中加入SMART Ⅲ^TM Oligo作为cDNA第二链合成的引物，进行长距离（LD）2PCR合成双链eDNA，后者经CLONTECHCHROMASPIN＋TE24000柱纯化后，与线性质粒pGADT72Ree共转化感受态酵母菌AH109，二者在酵母细胞中利用酵母细胞内具有较高的同源重组酶活性，进行同源重组成环行的有复制活性的文库质粒，在缺亮氨酸（LEU）培养板上筛选出所有克隆，即为鸡法氏囊B细胞的酵母双杂交cDNA文库。结果：共获得2．2×10^6转化子，文库扩增后，插入cDNA长度在0．5—3kb。研究结论表明可在酵母细胞中利用CLONTECH SMART技术成功构建鸡法氏囊B细胞的酵母双杂交eDNA文库。

英文摘要：

To construct yeast two hybrid cDNA library of chicken B cells Bursa of Fabricius. TotalRNA was extract from chicken B cells Bursa of Fabricius. The first chain of cDNA was synthesized by using the modified CDS Ⅲ as the primer and its terminal was automatically extended by CCC. The second chain was produced by adding SMARTTM Oligo and then ds eDNA was synthesized by long distance 2PCR. After purified by using CLONTECHCHCHROMA SPIN ＋ TE24000 column, the PCR products as well as the linearized plasmid pGADT72Rec were eotransformed into the competent yeast AH109. They were reeombinated by yeast homologous recombinase in the yeast cells and become the active cy iclic plasmid. The transformed yeasts grew in the SD/Leu2plates. All the growing clones were harvested and then constituted the cDNA library. The results showed that 2.25 × 10^6 recombinants were obtained from the eDNA library. The amplified PCR fragments were between 0.5 - 3 kb in size. The conclusion showed that the yeast two hybrid cDNA library of chicken B cells Bursa of Fabricius was successfully constructed by CLONTECH SMART method in yeast ceils.