人胰腺癌抑制消减cDNA文库的构建

目的应用抑制消减杂交技术(SSH)构建胰腺癌和正常胰腺组织间差异表达的抑制消减cDNA文库。方法分别提取胰腺癌(tester)和癌旁正常胰腺组织(driver)中的总RNA和mRNA．合成双链cDNA，经RsaI酶切后，将胰腺癌双链cDNA分为两组，分别加上不同的接头，再与正常胰腺组织cDNA进行两次消减杂交及两次抑制性PCR，分离出胰腺癌差异表达基因的cDNA片段。将该差异表达片段克隆至T／A载体，并转化大肠杆菌TOP10F’，经蓝白斑筛选后，再用PcR方法筛选阳性克隆，从而构建胰腺癌抑制消减cDNA文库。结果文库扩增后得到257个白色克隆，随机挑取50个阳性克隆进行PCR扩增分析，其中47个克隆有插入片段．克隆阳性率为94％，片段大小主要集中在300～600bp之间。结论成功构建了人胰腺癌抑制消减cDNA文库，为进一步筛选、克隆胰腺癌特异性表达基因奠定了基础。

Construction of suppressive subtractive cDNA library of human pancreatic cancer

Objective To construct a suppressive subtractive cDNA library of differentially expressed genes of human pancreatic cancer and normal pancreas tissues by suppressive subtractive hybridization (SSH). Methods Total RNA and mRNA were isolated from human pancreatic cancer (as tester) and normal paracancerous pancreas tissues (as driver), respectively. Then double-strand cDNA were synthesized. After Rsa I digestion, the tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2R respectively. After tester cDNA was hybridized with driver cDNA twice and underwent nested PCR twice, the cDNA fragments of differentially expressed genes of human pancreatic cancer were isolated. Then the fragments were cloned into T/A vector and transformed E. coli TOP 10F', screened through the blue-white screening system, and then positive clones were confirmed by PCR method to set up the suppressive subtractive cDNA library. Results The amplified library contained approximately 257 white bacteria clones. Random analysis of 50 white clones with PCR amplification showed that 47 clones contained inserted fragments. The positive rate was 94%. The size of inserted fragments was between 300 - 600 bp. Conclusions A suppressive subtractive cDNA library of human pancreatic cancer is constructed successfully. The library would lay the foundation for further screening and cloning differentially expressed genes in human pancreatic cancer.