Shen Li-jia, Fang Wei-yi, Xie Si-ming, He Ying, Jiang Hui-yong, Zhao Tong
Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2006 Feb;26(2):144-9.
To detect and clone mCD99L2 gene from mouse B lymphoma cell line A20 and construct its eukaryotic expression vector pcDNA3.1-mCD99L2.
The expression of mCD99L2 mRNA 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(+).
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.
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.
从小鼠B淋巴瘤细胞系A20中检测并克隆mCD99L2基因，构建其真核表达载体pcDNA3.1-mCD99L2。
采用原位杂交法检测A20细胞系中mCD99L2 mRNA的表达。提取A20细胞的总RNA，通过逆转录聚合酶链反应（RT-PCR）获得mCD99L2基因编码区的全长cDNA，将其产物连接到pMD18-T载体中，并检测插入片段的DNA序列。使用含EcoR I和Xho I酶切位点的引物通过PCR从pMD-mCD99L2中扩增mCD99L2基因的编码区，并克隆到真核表达载体pcDNA3.1/MycHis(+)中。
原位杂交鉴定出A20细胞系中mCD99L2基因呈阳性表达。通过RT-PCR获得了A20细胞系mCD99L2编码区的全长cDNA，其产物大小为712 bp，与GenBank中报道的mCD99L2 cDNA序列完全同源。限制性内切酶酶切和DNA测序表明真核表达载体pcDNA3.1(+)-mCD99L2构建成功。
已从小鼠B淋巴瘤细胞系A20中克隆出mCD99L2 cDNA，并成功构建其真核表达载体pcDNA3.1(+)-mCD99L2，这为进一步研究mCD99L2基因在小鼠B淋巴瘤细胞系A20中的功能奠定了基础。
