摘要
目的构建类风湿关节炎(RA)滑膜组织T7噬菌体展示cDNA文库,为筛选和鉴定RA特异性基因的研究奠定基础。方法取确诊RA患者的滑膜组织,用Trizol试剂提取总RNA,Oligotex离心柱分离mRNA,电泳检测其质量,逆转录合成双链cDNA,经末端修平、接头连接、酶切后去除过多接头,收集>300bp的cDNA片段,与T7Select10-3载体连接,体外包装并扩增得到类风湿关节炎滑膜组织T7噬菌体展示cDNA文库。最后,通过铺平板滴度测定及PCR技术鉴定文库质量。结果所构建原始文库重组克隆数为2×107pfu,扩增滴度8.9×1010pfu/ml。PCR法检测片段插入率为90%,插入片段在300～2000bp之间。文库噬菌体裂解液PCR扩增得到BiP基因片段。结论成功构建了高质量的RA滑膜T7噬菌体展示cDNA文库,为下一步RA自身抗原的筛选奠定了基础。
Objective：To construct and evaluate T7 phage display cDNA library from synovium of rheumatoid arthritis patients.Methods：Total RNA was extracted from pooled RA synovium by Trizol reagents. Messenger RNA was isolated from total RNA by oligo （dT）-conjugated Oligotex particles,and then, the agarose gel electrophoresis showed the range of mRNA size. After mRNA was reverse-transcribed into double-stranded eDNA,end modification,adaptors ligation and EcoR Ⅰ and Hind Ⅲ digestion were performed.After eliminating excess adaptors and small fragments（less than 300 bp）, the eDNA was ligated into TTSelect 10-3 vector.The RA synovium phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library. Results： We showed that complexity of the library was about 2 × 10^7 ,and the phage titer of the amplified library was 8.9 × 10^10 pfu/ml.Insert ratio was proved to be 90% with the range of 300-2 000 bp inserts. Meanwhile, a target cDNA gene of BiPwas probed with PCR amplification. Conclusion：The phage display cDNA library from synovium was construted with high quality and can be used as a valuable source in screening of RA self-antigens.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2005年第12期936-939,共4页
Chinese Journal of Immunology
基金
国家自然科学基金重点项目(30430290)
