Analysis of interaction among proteins expressed in retinal pigment epithelium

为了阐明MFRP和CTRP5之间的相互作用，我们制作了针对这些蛋白的抗体。我们已经制作了两种不同的MFRP抗体。为了更准确地分析MFRP和CTRP5的相互作用，我们今年又制作了识别MFRP和CTRP5不同抗原位点的抗体。为了在体内表达MFRP和CTRP5，我们分别克隆了小鼠MFRP基因和Clqtnf5基因的cDNA。我们用高保真DNA聚合酶扩增了这些基因。经测序证实，该基因未发生突变。我们还克隆了与RD6小鼠表型相关的截短突变的MFRP的cDNA。我们已将其亚克隆到pET-17b载体上。我们一直在尝试使用纯系统、经典的MINI即体外蛋白表达系统来表达这些蛋白质。然而，到目前为止，这个系统并没有很好地发挥作用。我们一直在尝试几种故障排除方法。

To elucidate the interaction between MFRP and CTRP5, we have made antibodies against these proteins. We have already made two different antibodies against MFRP. We made another antibody that recognize different antigenic site of MFRP and antibody against CTRP5 this year, to analyze the interaction more precisely. To express MFRP and CTRP5 in vivo, we have cloned cDNA of Mfrp gene and Clqtnf5 gene of mice respectively. We amplified those cDNA by using high-fidelity DNA polymerase. cDNA sequence has confirmed by sequencing and there was no mutation. We have also cloned cDNA of Mfrp with truncated mutation that is responsible for the phenotype of rd6 mice. We have subcloned the cDNA to pET-17b vector. We have been trying to express those proteins by using PURE SYSTEM classic mini that is in vitro protein-expression system. However, this system does not work well so far. We keep trying several trouble shooting methods.