小鼠免疫共刺激信号B7-1和B7-2联合表达载体的构建及包装

The construction and packaging of double expressing vector of mouse immune-costimulating signal B7-1 and B7-2

目的构建含小鼠B7-1和B7-2基因的双表达载体pLXSN-mB7-1-IRES-mB7-2,并包装到PA317细胞中。方法1将EcoRI和BamHI酶切下来的pMD18-mB7-2质粒上的mB7-2基因片段克隆到含有IRES片段的MINV质粒上,得到MINV-IRES-mB7-2载体;2MunI酶切载体MINV-IRES-mB7-2,末端钝化,然后BamHI再酶切,得到含钝末端和BamHI粘末端的IRES-mB7-2基因片段;3XhoI酶切质粒pLXSN-mB7-1,末端钝化,然后BamHI再酶切,形成含钝末端和BamHI粘末端的开环pLXSN-mB7-1质粒,将IRES-mB7-2基因片段插入其中,得到pLXSN-mB7-1-IRES-mB7-2双表达载体;并PA317细胞包装,PCR和电镜鉴定。结果经酶切、PCR、测序等鉴定载体构建成功,并包装到PA317细胞内。结论成功得到了双表达载体pLXSN-mB7-1-IRES-mB7-2和包装后的PA317/mB7-1-mB7-2细胞。

[Objective]To construct double expressing vector pLXSN-mB7-1-IRES-mB7-2 with murine B7-1 and B7-2 gene and package them into PA317 cell. [Methods]（1）The mB7-2 fragment obtained from plasmid pMD18-mB7-2 which was cutted by EcoRⅠ and BamHⅠ was inserted into vector MINV to gain vector MINV- IRES-mB7-2, which include IRES gene fragment. （2）Digesting vector MINV-IRES-mB7-2 by enzyme MuntI and BamHⅠ to obtain IRES-mB7-2 gene fragment with ambly end and BamHⅠ sticking end. （3）Digesting pLXSN-mB7- 1 by enzyme XhoI and BamHⅠ to gain the opened plasmid pLXSN-mB7-1 with ambly end and BamHI sticking end; Inserting IRES-mB7-2 gene fragment into it to gain the double expressing vector pLXSN-mB7-1-IRES- mB7-2;Then, pLXSN-mB7-1-IRES-mB7-2 was packaged into PA317 cells, which were detected by PCR and electron microscope. [Results] Vector was constructed successfully which was proved by cutting, PCR and sequencing, and packaged into PA317. [Conclusion] Double expressing vector pLXSN-mB7-1-IRES-mB7-2 and PA317/mB7-1-mBT-2 cells were obtained successfully.