大鼠癌钙调蛋白_OM基因克

发布时间：2018-07-20 15:38

【摘要】： 视觉神经中成熟的神经纤维和中枢神经系统(central nervous system,CNS)的其他组成部分一样,一旦受损将非常难以修复。成熟的视网膜神经节细胞(retinal ganglion cell,RGC)经历了损伤或视神经眶内损伤而发生凋亡后,无法再生。因此,如何延缓RGC的变性,并重新激活轴突再生程序,使它们能够幸存下来并在受损的视神经中再生出长长的的轴突一直是眼科及CNS研究领域关注的重点。近年来研究发现,眼内活化的巨噬细胞对RGC具有显著的促进轴突再生效应。而其中,由活化的巨噬细胞表达和分泌的癌钙调蛋白(oncomodulin,OM)是这一效应的最重要的介导者。进一步研究发现,在活体内,OM对损伤的成熟视神经及周围感觉神经元也有促进轴突再生作用。而OM发挥再生促进效应的确切机制尚不清楚。目前,国内外均尚未有报道。对于OM的结构和神经再生功能方面的研究,目前国内尚无报道。因此,对其进行相关研究,可为神经再生新途径的研究提供一定的实验依据。本实验通过RT-PCR从活化的大鼠巨噬细胞中获得OM基因,构建了pET22b(+)-OM原核表达载体并成功表达了OM,为进一步完成对该蛋白的纯化及对其活性研究与探讨其促进轴突再生的作用机制奠定基础。目的: 1.对大鼠癌钙调蛋白(OM)进行基因克隆,构建原核表达质粒并加以鉴定。 2. OM的诱导表达及鉴定。方法: 实验一:大鼠癌钙调蛋白_OM基因原核表达载体的构建及鉴定 1.从SD大鼠腹腔中提取巨噬细胞培养活化,经巨噬细胞特异性抗体CD68鉴定; 2.从活化的巨噬细胞中提取总RNA,利用相应引物,通过RT-PCR的方法获得OM基因序列,1%琼脂糖凝胶电泳鉴定; 3.通过中间载体PUC57进行目的基因克隆,将阳性克隆质粒酶切,与原核表达载体PET-22b(+)连接,构建PET-22b(+)-OM原核表达质粒,经菌检PCR、DNA测序鉴定。实验二:大鼠癌钙调蛋白_OM基因原核表达载体在大肠杆菌中的表达及鉴定 PET-22b(+)-OM重组质粒转化入大肠杆菌BL21,IPTG诱导表达,SDS-PAGE观察表达产物,Western-blot鉴定。结果: 1. 1%琼脂糖凝胶电泳获得目的基因OM,大小为346bp左右,与设计的预期目的基因大小一致; 2.回收目的基因OM与中间载体PUC-57连接后转化入大肠杆菌感受态细胞DH5a,菌检PCR获得长度500bp左右的阳性克隆,与预期大小完全一致。抽提质粒PUC-57-OM,DNA测序鉴定证实与预期序列一致。 3.质粒PUC-57-OM酶切后与载体PET-22b(+)连接转化入大肠杆菌感受态细胞DH5a,抽提质粒PET-22b(+)-OM,经酶切鉴定及DNA测序后证实与预期序列一致。 4.重组表达质粒pET22b(+)-OM转化大肠杆菌BL21,IPTG诱导蛋白表达,经SDS-PAGE电泳分析表明目的蛋白OM主要以可溶形式存在于细菌裂解后的上清中,并经过western blot鉴定确定为目的蛋白OM。结论: 大鼠癌钙调蛋白在原核细胞中成功表达,为OM的获得提供了一条切实可行的途径。为后续实验中,蛋白的活性研究及进一步探讨其促进轴突再生的作用机制奠定了基础。
[Abstract]:In recent years , it has been found that in vivo and in vivo , OM plays an important role in promoting axonal regeneration .

Purpose :

1 . Cloning , constructing prokaryotic expression plasmid and identifying the gene of the rat ' s cancer calcineurin ( OM ) .

2.OM induced expression and identification .

Method :

Experiment 1 : Construction and Identification of Prokaryotic Expression Vector of Calcineurin _ OM Gene in Rats

1 . The activation of macrophages was extracted from the abdominal cavity of SD rats , and the macrophages - specific antibody CD68 was identified .

2 . extracting total RNA from activated macrophages , using corresponding primer , obtaining OM gene sequence through RT - PCR , 1 % agarose gel electrophoresis identification ;

3 . Using the intermediate vector PUC57 to clone the target gene , the positive cloning plasmid was digested and ligated with the prokaryotic expression vector PET - 22b ( + ) , and the prokaryotic expression plasmid of PET - 22b ( + ) - OM was constructed .

Experiment 2 : Expression and identification of prokaryotic expression vector of calcium - regulating protein _ OM gene of rat carcinoma in E . coli

The recombinant plasmid of PET - 22b ( + ) - OM was transformed into E . coli BL21 . The expression was induced by IPTG . The expression product was observed by SDS - PAGE and Western - blot .

Results :

1 . The target gene OM was obtained by 1 % agarose gel electrophoresis . The size of OM was about 346bp , which was consistent with the expected gene size of the designed gene .

2 . The recombinant plasmid PUC - 57 - OM was cloned into E . coli competent cell DH5a after being ligated with the intermediate vector PUC - 57 , and the positive clones with the length of 500 bp were obtained by PCR . The DNA sequencing and identification of PUC - 57 - OM were consistent with the expected sequence .

3 . The plasmid PUC - 57 - OM was digested with the vector PET - 22b ( + ) and transformed into E . coli competent cell DH5a . The plasmid PET - 22b ( + ) - OM was extracted and confirmed to be consistent with the expected sequence after digestion and DNA sequencing .

4 . The recombinant expression plasmid pET22b ( + ) - OM was transformed into BL21 and IPTG - induced protein expression .

Conclusion :

The successful expression of calcium - modulating protein in prokaryotic cells provided a feasible way for the acquisition of OM . For the subsequent experiments , the activity of protein was studied and the mechanism of promoting axonal regeneration was further explored .
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774

【共引文献】