肠三叶因子在毕赤酵母中的表达、纯化及肠三叶因子受体的初步研究

发布时间：2018-04-03 12:44

本文选题：肠三叶因子　切入点：表皮生长因子　出处：《第三军医大学》2007年硕士论文

【摘要】： 背景: 肠三叶因子(intestinal trefoil factor,ITF)是由肠道杯状细胞特异分泌的具有特殊空间结构的小分子多肽,ITF不仅能促进细胞增殖和移行,还由于其空间结构中有和粘蛋白的寡聚糖或多糖侧链结合的位点,使之形成稳定的凝胶复合物,稳定肠粘液层。此外,ITF具有蛋白酶水解抗性和酸碱稳定性,可减轻多种致伤因子造成的消化道损害,这些特性决定了ITF在肠道的自我保护机制中占重要地位。目前对ITF的研究已很深入,但有关ITF受体的研究相对滞后,ITF是否具有独特的受体存在争议,一些学者认为ITF没有特异受体,它依赖表皮生长因子受体(EGFR)发挥生物学效应;另一些学者则持不同观点,他们发现了一类能和ITF特异结合的蛋白,将其称之为ITF结合蛋白,并对蛋白的基本特性进行了初步分析,但这些蛋白是否就是ITF受体还缺乏具有说服力的直接证据。目的: 在成功获取重组表达ITF的基础上,通过配体受体动力学研究和受体定位研究,探索在肠上皮细胞上是否存在ITF特异受体,以及受体在细胞上的分布特点。方法: 1.ITF在酵母中的表达和纯化:将成功转化的含ITF成熟肽序列的酵母X-33菌株进行Zeocin筛选,将阳性转化子接种于YPD液体培养基中摇瓶表达,表达产物上清经硫酸铵分级沉淀、Ni-NTA亲和层析、超滤离心三步纯化以获取重组表达的ITF。Tricine SDS-PAGE及Western bloting鉴定重组蛋白。 2.肠三叶因子受体放射性配基结合实验(Radioligand binding assay,RLBA):摸索ITF受体放射性配基结合分析的最佳反应温度、时间、细胞浓度和非标记ITF的浓度。在此条件下在IEC-6细胞上进行ITF受体放射性配基结合实验,得到亲和常数(K_D)及最大结合容量(B_(max))。在相同的条件下对HT-29、5E6L、HaCat细胞上ITF受体进行分析,比较不同细胞上ITF受体的差异。通过大量EGF、ITF及EGFR阻断肽对ITF受体和EGF受体竞争抑制,证实ITF特异受体的存在。 3.肠三叶因子受体在肠上皮细胞上的定位:将传代培养的IEC-6细胞用0.5%的胰酶消化后,细胞浓度调为约2.5×10~5个/ml,加入放有8×8mm盖玻片的24孔板于37℃,5%CO_2培养箱孵育,待IEC-6肠上皮细胞贴壁,细胞爬片用PBS清洗3遍后与B-藻红蛋白(B-PE)标记的ITF(BPE-ITF)在4℃下分别共同孵育0.5,1.0,2.0,4.0h,孵育结束后PBS清洗3遍×3min,细胞片置于载玻片上并用90%缓冲甘油封片,激光共聚焦显微镜下观察ITF受体在IEC-6细胞上的分布规律。并使用过量的EGF、ITF和EGFR阻断肽进行竞争抑制实验,观察荧光的变化情况。结果: 1.酵母表达获得ITF,产物经Tricine SDS-PAGE、Western blot鉴定具有良好的抗原性和特异性,三步纯化后重组蛋白的纯度超过95%,表达量达到约50mg/L。 2.放射性配基结合实验最佳反应条件为4℃,孵育30min,细胞浓度为5.0×10~5个/ml,非特异结合配基浓度为标记配基的10000倍。在此条件下,对IEC-6、HT-29、5E6L和HaCat细胞上ITF受体进行放射性结合分析,并通过Scatchard线性回归分析得到IEC-6细胞最大结合容量B_(max)=62.713×10~6结合位点/细胞,亲和常数K_D=4.3478×10~(-10)mol/L;HT-29细胞最大结合容量B_(max)=61.1583×10~6结合位点/细胞,亲和常数KD=0.8849×10~(-10)mol/L;5E6L细胞最大结合容量B_(max)=184.89×10~6结合位点/细胞,亲和常数KD=3.448×10~(-10)mol/L。根据所得到的K_D值和B_(max)分析,ITF与IEC-6、HT-29、5E6L细胞结合呈高亲和力,但受体密度有所差异。表皮细胞HaCat与ITF结合呈低亲和力。EGF对ITF受体及ITF对EGF受体均无竞争抑制作用,EGFR阻断肽对ITF与IEC-6细胞的结合并未受影响。 3.激光共聚焦观察发现,荧光标记的ITF首先聚集在细胞膜上,随时间和浓度的增加,荧光标记的ITF逐渐向胞浆与胞核转移。过量的ITF可明显抑制荧光标记的ITF与细胞结合,荧光强度明显降低。EGF和EGFR阻断肽对ITF在细胞膜上的荧光强度和向胞浆、胞核的转移没有影响。结论: 1.重组表达的ITF产量约50mg/L,纯度＞95%,符合受体研究中对配体的要求。 2.确定了放射性配基结合实验最佳反应条件,并证明肠上皮细胞IEC-6、结肠癌细胞HT-29、小肠上皮细胞5E6L上存在能与ITF结合的受体,受体的亲和力和受体密度存在一定差异,表皮细胞HaCat上没有与ITF结合的受体。 3.EGF和ITF与各自的受体结合,无相互竞争抑制作用,证明ITF不是通过EGF受体发挥生物学效应。 4.ITF受体存在于IEC-6细胞膜上,ITF与其受体结合后呈现向胞浆和胞核转移的特性。
[Abstract]:Background:
Intestinal trefoil factor (intestinal trefoil, factor, ITF) is a small molecule with special structure of polypeptide secreted by intestinal goblet cell specific, ITF can not only promote cell proliferation and migration, but also because of its spatial structure and mucin oligosaccharides or polysaccharides of side chain binding sites, so as to form a composite gel a stable, stable intestinal mucus layer. In addition, ITF with protease hydrolysis resistance and acid-base stability, can reduce the injury caused by a variety of factors of digestive tract damage, these characteristics determine that ITF occupies an important position in the self - protection mechanism of intestine. At present the study on ITF has been very thorough, but the Research on ITF receptor the relative lag, whether ITF has a unique receptor is controversial, some scholars believe that there is no specific ITF receptor, it is dependent on the epidermal growth factor receptor (EGFR) exert biological effects; others have different opinions, they A class of proteins that can specifically bind to ITF have been discovered. They are called ITF binding proteins. The basic characteristics of proteins have been preliminarily analyzed. But whether these proteins are ITF receptors still lack convincing direct evidence.
Objective:
Based on successful acquisition of recombinant expression of ITF, we investigated the existence of ITF specific receptors on intestinal epithelial cells and the distribution of receptors on cells by ligand receptor kinetic studies and receptor localization studies.
Method:
Expression and purification of 1.ITF in yeast: successfully transformed with the mature peptide sequence of ITF strain X-33 was screened by Zeocin, the positive transformants were inoculated in YPD liquid culture medium in shake flask expression, expression supernatant by ammonium sulfate precipitation, Ni-NTA affinity chromatography, ultrafiltration from three steps of purification to obtain recombinant expression ITF.Tricine SDS-PAGE and Western bloting identification of recombinant protein.
Intestinal trefoil factor 2. receptor radioligand binding assay (Radioligand binding, assay, RLBA): exploring ITF receptor radioligand binding analysis of the optimum reaction temperature, time, concentration of cell concentration and non labeled ITF. Under these conditions, ITF receptor radioactive ligand binding experiments were performed on IEC-6 cells by affinity constants (. The maximum binding capacity (K_D) and B_ (max)). In the same conditions of HT-29,5E6L, analyzes on the HaCat cell receptor ITF, ITF receptor in different cell differences. Through a large number of EGF, ITF and EGFR blocking peptide inhibition and EGF receptor competition on ITF, confirmed that the ITF specific receptor.
3. localization of intestinal trefoil factor receptor in intestinal epithelial cells: IEC-6 cells with 0.5% trypsin digestion, the cell concentration to about 2.5 * 10~5 /ml, adding 8 * 8mm were placed in 24 holes plate at 37 DEG C, box were incubated for 5%CO_2, IEC-6 for intestinal epithelial cell paste the wall, cell body cleaning 3 times after B- and phycoerythrin (B-PE) labeled with PBS ITF (BPE-ITF) at 4 C were incubated for 0.5,1.0,2.0,4.0h, after incubation of PBS cleaning 3 times * 3min, slides and 90% glycerol buffer mounting piece is placed on the distribution of load cell, confocal laser under the microscope of ITF receptor in IEC-6 cells. And the excessive use of EGF, ITF and EGFR were blocking peptide competitive inhibition experiment, observe the fluorescence changes.
Result:
1. ITF was obtained from yeast expression. The product was identified by Tricine SDS-PAGE and Western blot. It had good antigenicity and specificity. After three steps, the purity of recombinant protein was over 95%, and its expression reached about 50mg/L..
2. radioligand binding experiments the optimum reaction conditions of 4 DEG C, incubated for 30min, cell concentration of 5 * 10~5 /ml, non-specific binding ligand concentrations 10000 times labeled ligands. Under this condition, the IEC-6, HT-29,5E6L and HaCat on ITF cell receptor binding analysis and analysis of radioactive, IEC-6 cells the maximum binding capacity of B_ by Scatchard linear regression (max) =62.713 * 10~6 binding sites per cell, the affinity constant of K_D=4.3478 * 10~ (-10) mol/L; HT-29 cell maximum binding capacity of B_ (max) =61.1583 * 10~6 binding sites per cell, the affinity constant of KD= 0.8849 * 10~ (-10) mol/L; 5E6L (maximum binding capacity of B_ cells max) =184.89 * 10~6 binding sites per cell, the affinity constant of KD=3.448 * 10~ (-10) mol/L. according to the values of K_D and B_ (max) analysis, ITF and IEC-6, HT-29,5E6L cells with a high affinity receptor, but the density difference. Epidermal cells of HaCat combined with ITF The low affinity.EGF has no competitive inhibition effect on ITF receptor and ITF to EGF receptor, and the binding of EGFR blocking peptide to ITF and IEC-6 cells is not affected.
3. laser scanning confocal microscope, fluorescence labeled ITF first gather in the cell membrane, increase the time and concentration of fluorescent labeled ITF to cytosolic and nuclear transfer. The combination of ITF and ITF can inhibit the cell excessive fluorescent marker, the fluorescence intensity decreased.EGF and EGFR blocking peptide on fluorescence intensity ITF on the cell membrane and the cytoplasm to the nucleus, did not affect the transfer.
Conclusion:
1. the yield of recombinant ITF was about 50mg/L, and the purity was more than 95%, which was in line with the requirement for ligand in the receptor study.
2. determine the combination of experimental optimum reaction conditions of radioactive ligand, and that intestinal epithelial cell IEC-6, colon cancer cell HT-29, are capable of binding ITF receptor 5E6L in intestinal epithelial cells, there are some differences in receptor affinity and receptor density, epidermal cells HaCat no binding to ITF receptor.
3.EGF and ITF are combined with their respective receptors, and there is no mutual competitive inhibition, which proves that ITF does not play biological effects through the EGF receptor.
The 4.ITF receptor exists on the IEC-6 cell membrane, and the ITF and its receptors bind to the cytoplasm and nucleus.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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