JC病毒VP2蛋白的抗原抗体制备及核定位信号与入核转运受体的鉴定

发布时间：2018-09-14 20:13

【摘要】： 目的: JC病毒属于人类多瘤病毒属,是一种重要的机会感染性疾病病原体。JC病毒广泛分布于人群中,有80㳠的成人血清学检测为阳性[1]。当免疫力低下时,JC病毒会引发多灶性脑白质病(PML)[2]。然而,JC病毒的临床检测,以及它的包膜蛋白VP2入核的机制和入核转运受体并不清楚。本研究拟通过原核表达、抗体制备、核定位信号鉴定和入核转运受体的鉴定几个方面初步阐释JC病毒的小包膜蛋白VP2基因在PML发生发展过程中的生物学功能。方法: (1)构建原核表达载体pET-32a(+)-VP2,转化BL21宿主菌后经IPTG诱导,获得了带有组氨酸标签的重组融合蛋白的优化表达。 (2)利用Ni+亲和柱纯化表达蛋白并免疫BALB /c小白鼠,获得抗VP2多克隆抗体。以纯化VP2为抗原,利用Western blot和ELISA法对多克隆抗体进行特异性和效价检测。 (3)构建重组绿色荧光表达载体pEGFP-C1-VP2,转染7402细胞,24hr后观察各截短体的亚细胞定位情况。 (4)表达纯化pGEX-2T-importin(importinα1、3、5、7和imporinβ)的GST融合蛋白。 (5)通过GST-pull down方法,寻找并确定VP2的入核转运受体。结果: (1)通过PCR方法从病人的脑脊液中获得VP2的目的片段,并成功构建了pET-32a(+)-VP2原核表达载体。pET-32a(+)-VP2原核表达载体在IPTG的诱导作用下,成功表达融合蛋白通过Ni+亲和层析法获得了融合蛋白纯品。 (2)用纯化的VP2融合蛋白免疫BALB/c小鼠,成功制备了高效价,高特异性的鼠抗VP2多克隆抗体。通过ELISA法表明多克隆抗体效价1:160,000, Western blot检测证明多克隆抗体的特异性良好。 (3)通过PCR方法获得了VP2截短体的目的片段,成功构建了VP2各截短体的绿色荧光表达载体;将构建好的VP2各截短体绿色荧光表达载体转染7402人肝癌细胞,通过各截短体的亚细胞定位分析出了VP2蛋白的核定位信号为946-972段。 (4)成功表达纯化了五个pGEX-2Timportin融合蛋白。 (5)通过GST-pull down实验,分析发现VP2能够与入核转运受体蛋白importinα1、importinα3结合,从而被转运入细胞核,发挥它的生理和生化作用。结论: 1.我们成功构建了pET-32a(+)- Vp2原核表达载体,在IPTG的诱导下,成功表达纯化了VP2融合蛋白。这就为抗体的制备以及研究VP2蛋白与其它蛋白的相互作用做了物质上的准备。 2.将VP2蛋白免疫BALB/c小鼠,制备了高效价、高特异性的鼠抗VP2多克隆抗体。这对于VP2抗原的检测,以及免疫组化奠定了基础。 3.成功的鉴定出VP2的核定位信号,通过构建四个pEGFP-C1-Vp2绿色荧光载体,脂质体转染7402细胞株,在荧光显微镜下观察pEGFP-C1-Vp2各截短体的亚细胞定位。这是第一次鉴定出VP2的核定位信号,为进一步的研究提供了信息。 4.通过GST-pull down方法,我们成功的鉴定出VP2的入核转运受体为importinα1、importinα3。初步阐明了VP2入核过程中参与的入核转运受体。
[Abstract]:Objective: JC virus belongs to human polytumour virus, which is an important opportunistic infectious disease pathogen. JC virus is widely distributed in the population, has 80? The adult serological test was positive [1]. When immunity is low, JC virus can cause multiple focal white matter disease (PML) [2]. However, the clinical detection of JC virus, the mechanism of its envelope protein VP2 entry and its nuclear transporter receptor are not clear. The purpose of this study was to elucidate the biological function of the small envelope protein VP2 gene of JC virus during the development of PML through prokaryotic expression, antibody preparation, nuclear localization signal identification and nuclear transport receptor identification. Methods: (1) the prokaryotic expression vector pET-32a () -VP2was constructed and transformed into BL21 host strain and induced by IPTG. The optimized expression of recombinant fusion protein with histidine label was obtained. (2) Ni affinity column was used to purify the expressed protein and immunize BALB / c mice to obtain anti-VP2 polyclonal antibody. Using purified VP2 as antigen, Western blot and ELISA were used to detect the specificity and titer of polyclonal antibody. (3) Construction of recombinant green fluorescent expression vector pEGFP-C1-VP2, for 24 hours after transfection to 7402 cells to observe the subcellular localization of each truncated body. (4) expression and purification of the GST fusion protein of pGEX-2T-importin (importin 伪 _ 1H _ 3H _ 5N _ 7 and imporin 尾). (5) the fusion protein was purified by GST-pull down. To identify the nuclear transporter receptor of VP2. Results: (1) the target fragment of VP2 was obtained from cerebrospinal fluid of patients by PCR method, and pET-32a () -VP2 prokaryotic expression vector. PET-32a () -VP2 prokaryotic expression vector was successfully constructed under the induction of IPTG. The fusion protein was successfully expressed by Ni affinity chromatography. (2) the purified VP2 fusion protein was used to immunize BALB/c mice, and a high titer and high specificity mouse anti-VP2 polyclonal antibody was successfully prepared. The polyclonal antibody titer was 1: 160 ~ (000) by ELISA, and the specificity of the polyclonal antibody was proved by Western blot. (3) the target fragment of VP2 truncated body was obtained by PCR method, and the green fluorescent expression vector of VP2 truncated body was constructed successfully. The constructed VP2 truncated green fluorescent expression vectors were transfected into 7402 human hepatoma cells. The nuclear localization signal of VP2 protein was 946-972. (4) five pGEX-2Timportin fusion proteins were successfully expressed and purified. (5) five pGEX-2Timportin fusion proteins were successfully expressed and purified by GST-pull down assay. It was found that VP2 could bind to importin 伪 1 importin 伪 3 and be transported into the nucleus to play its physiological and biochemical role. Conclusion: 1. The prokaryotic expression vector of pET-32a ()-Vp2 was successfully constructed, and VP2 fusion protein was successfully expressed and purified under the induction of IPTG. This made a material preparation for the preparation of antibodies and the study of the interaction between VP2 protein and other proteins. 2. 2. BALB/c mice were immunized with VP2 protein, and high titer and specificity of anti VP2 polyclonal antibodies were prepared. This laid a foundation for the detection of VP2 antigen and immunohistochemistry. The nuclear localization signal of VP2 was successfully identified. Four pEGFP-C1-Vp2 green fluorescent vectors were constructed and transfected into 7402 cell line by liposome. The subcellular localization of pEGFP-C1-Vp2 truncated bodies was observed under fluorescence microscope. This is the first time to identify the nuclear localization signal of VP2, which provides information for further research. By GST-pull down method, we successfully identified the nuclear transport receptor of VP2 as importin 伪 1 importin 伪 3. The translocation receptors involved in the process of VP2 nucleation were preliminarily elucidated.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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