小鼠B淋巴瘤A20细胞株mCD99L2基因表达检测及真核表达载体构建

目的 检测及克隆小鼠B淋巴瘤A20细胞株mCD99L2基因，构建真核表达载体。方法 设计寡核苷酸探针。应用原位分子杂交方法检测A20细胞中mCD99L2基因的mRNA表达，RT-PCR法从A20细胞总RNA中获取mCD99L2基因含编码区全长cDNA，克隆入T载体，筛选阳性克隆、酶切鉴定并经序列测定；设计含酶切位点的PCR引物，PCR获取含mCD99L2基因编码区片段，用限制性内切酶EcoRI和Xho I双酶切取所需目的片段．利用分子克隆技术与pcDNA3．1／MycHisC（＋）质粒重组，对产生的重组子进行PCR、双酶切鉴定，并经过测序证实。结果 原位杂交方法检测A20细胞中mCD99L2基因mRNA呈阳性表达；RT-PCR法成功获得mCD99L2基因编码区全长cDNA序列，DNA序列测序结果与GenBank提供的已知序列（NM—138309）比较，结果完全一致；重组质粒pcDNA3．1．mCD99L2中的插入片段经DNA测序后与GenBank中mD99L2基因相应序列比较，100％同源。结论 小鼠B淋巴瘤A20细胞株存在mCD99L2基因，采用RT-PCR和T．A技术克隆了A20细胞的mCD99L2基因，成功构建了pcDNA3．1．mCD99L2真核表达载体，为下一步探索mCD99L2基因在小鼠B淋巴瘤A20细胞株中的功能奠定了实验基础。

Expression and cloning of mCD99L2 gene from mouse B lymphoma cell line A20 and construction of its eukaryotic expression vector

Objective To detect and clone mCD99L2 gene from mouse B lymphoma cell line A20 and construct its eukaryotic expression vector pcDNA3.1-mCD99L2. Methods The expression of mCD99L2 mKNA in A20 cell line was detected by in situ hybridization. The total RNA of A20 cells was extracted to obtain the full-length cDNA of the coding region of mCD99L2 gene by RT-PCR, the product of which was ligated into pMD18-T vector and the DNA sequence of the insert was detected. The coding regions of mCD99L2 gene was amplified from pMD-mCD99L2 by PCR using primers containing EcoR I and Xho I sites and cloned into the eukaryotic expression vector pcDNA3. 1/MycHis( ). Results In situ hybridization identified positive expression of mCD99L2 gene in the A20 cell line. The full-length cDNA of mCD99L2 coding region of A20 cell line was obtained by RT-PCR, which yielded a product of 712 bp as expected, and the DNA sequence was completely homologus to the mCD99L2 cDNA reported in GenBank. Restriction endonuclease digestion and DNA sequencing indicated that the eukaryotic expression vector pcDNA3.1( )- mCD99L2 had been constructed successfully. Conclusion mCD99L2 cDNA has been cloned from mouse B lymphoma cell line A20 and its eukaryotic expression vector pcDNA3. 1 ( )- mCD99L2 successfully constructed, which facilitates further functional study of mCD99L2 gene in mouse B lymphoma cell line A20.