The Eukaryotic Expression Of PD-L1 And PD-L2 And The Construction Of Their Transgenic Mice

PD-Ll and PD-L2 are two co-stimulate molecule ligands found recent years .which belong to B7 family. They share the same receptor PD-1 .By the interaction with PD-1 they affect activated T cells and diminish their proliferation and cytokine production. The engagement of two ligands to the receptor PD-1 result in a negative regulatory effect in T cell response. On the other hand they can also regulate B cell response though the effect on cytokine production. The PD-Ll and PD-L2/PD-1 pathway is not only take an important role in peripheral self tolerance but also found act on the process of tumor cells evading immune systems.In this research we cloned encoding sequence of PD-L1 and PD-L2 by RT-PCR from the total RNA isolated from PHA stimulated mouse peripheral blood mononuclear cells and the total RNA isolated from mouse thymus. After sequencing we found that the sequence of cloned PD-Ll is exactly the same as Genebanks while the sequence of cloned PD-L2 has one mutation. We consult reference and consider it not important.We successfully construct two recombinant eukaryotic expression vectors pEF6/-PD-Ll-V5 His and pEF6/-PD-Ll-V5 His which contain the target genes. Then the recombinant vectors were transfected into CHO cells separately for transient expression and the expression was identified by immunofluorescence technology. We also obtained the stable expression cell lines through the selection of the transfected cells and was identified using western-blot. These suggest that these cell lines, which stably express PD-Ll or PD-L2, can be strongly useful tools to study the immune response regulated by PD-L2 in vitro.We got the DNA fragments for microinjection by restriction endonuclease digestion. Using microinjection into male nuclei two founder transgenic mice of PD-L1 and one of PD-L2 was established after PCR detection. But only PD-L2 founder mouse can passage the transgene to its offspring and was identified by southern-blot.All these results suggest that the construction of stable cell lines and transgenic mouse are successful and will be strongly useful tools to study the function of PD-Ll and PD-L2 in vitro and vivo.