Analysis of interaction among proteins expressed in retinal pigment epithelium

为了阐明MFRP与CTRP5之间的相互作用，我们制备了针对这些蛋白的抗体。我们已经制造了两种不同的MFRP抗体。我们在今年制作了另一种识别MFRP不同抗原位点的抗体和针对CTRP5的抗体，以便更准确地分析它们的相互作用。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。我们利用高保真DNA聚合酶扩增这些cDNA。cDNA序列经测序证实，无突变。我们还克隆了导致rd6小鼠表型的截断突变的Mfrp的cDNA。我们将cDNA亚克隆到pET-17b载体上。我们一直在尝试使用PURE SYSTEM classic mini体外蛋白表达系统来表达这些蛋白。然而，到目前为止，这个系统还不是很好用。我们一直在尝试几种排除故障的方法。

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.