大鼠心肌细胞钙调磷酸酶融合基因的克隆及其原核表达

Cloning and expression of gene coding for calcineurin of rats

目的:构建钙调磷酸酶(CaN)融合基因的原核表达载体,并使其获得表达,为研究该蛋白的功能,在病理生理过程中的变化及相关疾病的治疗奠定基础。方法:利用OLIGO6.0生物学软件设计一对特异性引物,并在引物中引入适当的酶切位点,采用逆转录聚合酶链反应(RT-PCR),扩增出CaN基因,经BamHI和XhoI双酶切后,将其克隆于上游融合了GST标签的pGEX-6P-1表达载体中,选取鉴定为阳性的重组表达载体,转化于受体菌BL21中,并利用1mMIPTG对其进行诱导,收获表达产物进行SDS-PAGE电泳分析和Western印迹(blot)鉴定,以确定融合表达的CaN基因的反应活性。结果:RT-PCR后扩增得到了大小约为700bp的目的片段,克隆于表达载体pGEX-6P-1,经测序分析后,证实所获得的与Genebank提供的序列相一致,CaN基因融合表达产物经SDS-PAGA电泳分析发现该重组蛋白大小约为45kD,利用CaNAβ抗体进行Western blot分析,结果证实该表达的融合蛋白具有较好的免疫反应性。结论:钙调磷酸酶Aβ以融合蛋白的形式获得了表达,而且融合表达的蛋白具有反应活性。

Objective： To construct the prokaryotic expression vector of the calcineurin gene and to guide expression. The results could be used for researching the function of the protein further and its changes in pathological and physiological processes of correlated diseases. Methods： To design a pair of specific primer by OLIGO 6.0 biological software and to draw into adequate cutting site of enzyme. The mRNA amounts of the calciveurin gene were acquaired by RT-PCR and amplify the calcineurin gene. The gene were cut by BamHI and XholI and then was cloned into a prokaryotic expression vector pGEX- 6P- 1 whose upstream has a GST taq. The recombinant vector which is positive was transformed into E. coil BL21. Samples were collected after induction with 1mM IPTG. The expression product was analysed by SDS-PAGE and appreciated the react activity of the calcineurin gene by western blot. Results： The objective fragment about 700bp was obtained by RT-PCR. The custom-crafted expression vector was confirmed by sequencing analysis that the sequence was coincided with the Genebank. A fusion-expressd protein was about 45 kD in size and was recognized by Aβ antibody in western blot. Conclusion： The calcineurin Aβ protein is expressed as fusion protein and the fusion protein has react activity.