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
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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。
Background:Intestinal trefoil factor(ITF),a small,promoting mucosa proliferation polypeptide,is contained by one characteristic trefoil domain.Expression of ITF is closely related to that of mucin glycoproteins in diverse biological sources.ITF play an important role in both maintaining the barrier function of mucosal surfaces and facilitating healing after injury.Up to now,the research of ITF is extensive and profound,including tissue distribution,gene localization,gene recombination,amino acid sequence and spatial structure analysis as well as mass of function research.But the research progression of ITF receptor is slow-moving,there is or not ITF unique receptor in intestinal epithelial cell has no accepted argument.Some researchers deem ITF has no its own special receptor,it educes biological effects depend on epidermal growth factor receptor(EGFR),others presume there is ITF unique receptor in intestinal epithelial cell.But there is no credible direct evidence.
Objective:To explore if ITF special receptor exists on the intestinal epithelial cell and it's disposition by radioligand binding assay and receptor mapping.
Methods:
1.Expression and purification of ITF:Successfully transformed Pichia pastoris strain X-33 which contained ITF gene encoding mature peptide were screened with Zeocin. Subsequently,ITF was constitutively expressed and purified by ammonium sulfate precipitation,Ni-NTA affinity chromatography and ultrafiltration,determined by SDS-PAGE and Western blot analysis.
2.Radioligand binding assay of ITF receptor:Purified ITF was labeled by ~(125)I, determined the optimal temperature,reaction time,quantity of intestinal epithelial cell and concentration of unlabelled ITF.The binding assay was performed under this condition. Data from binding experiments were calculated by linear regression analysis of Scatchard plots,dissociation constant(K_D) value and maximum density of binding sites(B_(max)) of the cell was gained.Subsequently,analysis HT-29、5E6L、Hacat cell by the same method. Compare the K_D and B_(max) of different cells with addition of excess unlabeled EGF、ITF in the reaction,EGFR blocking peptide screened the binding sites of EGF,to prove ITF receptor presence on the intestinal epithelial cells.
3.ITF receptor localization in intestinal epithelial cells:subcultured intestinal epithelial cells were digested by 0.5%protease at 37℃for 5 minutes,adjust the cells concentration of 5.0×10~5 cells/ml and inoculate to 8×8mm coverslip.After cells adherence, washed the coverslip by PBS buffer 3 times for 3 minutes,and incubated with B-Phycoerythrin labeled ITF for various time at 4℃,At the end of incubations,cells were washed by PBS buffer 3 times for 3 minutes,put the cell coverslip on a slide and mounting by 90%glycerine,observed the disposition of ITF receptor on intestinal epithelial cells by laser confocal microscopy.
Results:
1.ITF was successfully expressed in Pichia pastoris,Tricine SDS-PAGE and Western blot analysis showed that ITF had much good antigenicity and specificity.The purity after purification was above 95%,the yield of ITF was about 50mg/L.
2.Established optimal reaction conditions of radioligand binding assay:standard incubation temperature was 4℃,an incubation time of 30 min and cell concentration of 5.0×10~5 cell/ml were used in radioligand binding assay experiments.A 4000pmol/ml unlabelled ITF concentration was used in competition experiments.Analysed IEC-6 cell, HT-29 cell,5E6L cell and Hacat cell by radioligand binding assay.According to comparison of each cells' K_D and B_(max),ITF bind with IEC,HT-29 and 5E6L showed high affinity,and Hacat cells showed low affinity.
3.Discovered that the B-Phycoerythrin labeled ITF gathered at membrane in the intestinal epithelial cells by laser confocal microscopy,as increased incubation time and concentration of BPE-ITF,B-Phycoerythrin labeled ITF transfered into cytoplasm and nucleus.
Conclusion:
1.ITF was successfully produced in Pichia pastoris expression system.The purity after purification above 95%and fit for radioligand binding analysis experiment.
2.Established optimal reaction conditions of radioligand binding assay,and demonstrated presence of ITF receptors on intestinal epithelial cells(IEC-6),colon carcinoma cells(HT-29) and small intestine epithelial cells(5E6L).Epidermic cells(Hacat) have no ITF specific receptor.
3.EGF and ITF specific band with each receptor,competitive inhibition between EGF receptor and ITF receptor did not exist.It is indicated that there is ITF specific receptor on the intestinal epithelial cells.
4.Successfully label ITF with B-Phycoerythrin,and certificate that there is ITF receptor on membrane of intestinal epithelial cells and labeled ITF could transfer into cytoplasm and nucleus.
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。
Background:Intestinal trefoil factor(ITF),a small,promoting mucosa proliferation polypeptide,is contained by one characteristic trefoil domain.Expression of ITF is closely related to that of mucin glycoproteins in diverse biological sources.ITF play an important role in both maintaining the barrier function of mucosal surfaces and facilitating healing after injury.Up to now,the research of ITF is extensive and profound,including tissue distribution,gene localization,gene recombination,amino acid sequence and spatial structure analysis as well as mass of function research.But the research progression of ITF receptor is slow-moving,there is or not ITF unique receptor in intestinal epithelial cell has no accepted argument.Some researchers deem ITF has no its own special receptor,it educes biological effects depend on epidermal growth factor receptor(EGFR),others presume there is ITF unique receptor in intestinal epithelial cell.But there is no credible direct evidence.
Objective:To explore if ITF special receptor exists on the intestinal epithelial cell and it's disposition by radioligand binding assay and receptor mapping.
Methods:
1.Expression and purification of ITF:Successfully transformed Pichia pastoris strain X-33 which contained ITF gene encoding mature peptide were screened with Zeocin. Subsequently,ITF was constitutively expressed and purified by ammonium sulfate precipitation,Ni-NTA affinity chromatography and ultrafiltration,determined by SDS-PAGE and Western blot analysis.
2.Radioligand binding assay of ITF receptor:Purified ITF was labeled by ~(125)I, determined the optimal temperature,reaction time,quantity of intestinal epithelial cell and concentration of unlabelled ITF.The binding assay was performed under this condition. Data from binding experiments were calculated by linear regression analysis of Scatchard plots,dissociation constant(K_D) value and maximum density of binding sites(B_(max)) of the cell was gained.Subsequently,analysis HT-29、5E6L、Hacat cell by the same method. Compare the K_D and B_(max) of different cells with addition of excess unlabeled EGF、ITF in the reaction,EGFR blocking peptide screened the binding sites of EGF,to prove ITF receptor presence on the intestinal epithelial cells.
3.ITF receptor localization in intestinal epithelial cells:subcultured intestinal epithelial cells were digested by 0.5%protease at 37℃for 5 minutes,adjust the cells concentration of 5.0×10~5 cells/ml and inoculate to 8×8mm coverslip.After cells adherence, washed the coverslip by PBS buffer 3 times for 3 minutes,and incubated with B-Phycoerythrin labeled ITF for various time at 4℃,At the end of incubations,cells were washed by PBS buffer 3 times for 3 minutes,put the cell coverslip on a slide and mounting by 90%glycerine,observed the disposition of ITF receptor on intestinal epithelial cells by laser confocal microscopy.
Results:
1.ITF was successfully expressed in Pichia pastoris,Tricine SDS-PAGE and Western blot analysis showed that ITF had much good antigenicity and specificity.The purity after purification was above 95%,the yield of ITF was about 50mg/L.
2.Established optimal reaction conditions of radioligand binding assay:standard incubation temperature was 4℃,an incubation time of 30 min and cell concentration of 5.0×10~5 cell/ml were used in radioligand binding assay experiments.A 4000pmol/ml unlabelled ITF concentration was used in competition experiments.Analysed IEC-6 cell, HT-29 cell,5E6L cell and Hacat cell by radioligand binding assay.According to comparison of each cells' K_D and B_(max),ITF bind with IEC,HT-29 and 5E6L showed high affinity,and Hacat cells showed low affinity.
3.Discovered that the B-Phycoerythrin labeled ITF gathered at membrane in the intestinal epithelial cells by laser confocal microscopy,as increased incubation time and concentration of BPE-ITF,B-Phycoerythrin labeled ITF transfered into cytoplasm and nucleus.
Conclusion:
1.ITF was successfully produced in Pichia pastoris expression system.The purity after purification above 95%and fit for radioligand binding analysis experiment.
2.Established optimal reaction conditions of radioligand binding assay,and demonstrated presence of ITF receptors on intestinal epithelial cells(IEC-6),colon carcinoma cells(HT-29) and small intestine epithelial cells(5E6L).Epidermic cells(Hacat) have no ITF specific receptor.
3.EGF and ITF specific band with each receptor,competitive inhibition between EGF receptor and ITF receptor did not exist.It is indicated that there is ITF specific receptor on the intestinal epithelial cells.
4.Successfully label ITF with B-Phycoerythrin,and certificate that there is ITF receptor on membrane of intestinal epithelial cells and labeled ITF could transfer into cytoplasm and nucleus.
引文
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1.沈玉美,张慧,刘志民.放射配体结合检测方法及注意事项[J].第二军医大学学报,2001,22(11):1098-1099.
2.班婷婷,吴强恩,周志俊.放射性配体受体结合实验方法介绍[J].毒理学杂志,2007,21(3):231-232.
3.Tayebati S K,Codini M,Gallai V,et al.Radioligand binding assay of M1-M5muscarinic cholinergic receptor subtypes in human peripheral blood lymphocytes.J Neuroimmunol,1999,99(2):224-229.
4.聂克,王汝俊,王建华.大鼠胃壁细胞胃泌素受体的放射配基结合法[J].中国药理学通报,2001,17(6):645-647.
5.吴健虹,谢秋玲,陈小佳,等.表皮生长因子受体EGFR及其信号传导.生命科学,2006,18(2):116-122.
6.詹臻,吴慧平.表皮生长因子受体(EGF-R)放射受体分析方法(RRA)的建立[J].南京中医药大学学报(自然科学版),2001,17(4):239-241.
7.Ricci A,Bronzetti E,Felici L,et al.Dopamine D4 receptor in human peripheral blood lymphocytes:a radioligand binding assay study.Neurosci Lett,1997,229(2):130-134.
8.Romain S,Dussert C,Martin P M.Determination of oestrogen receptors:application of the Passing-Bablock linear regression technique for comparison of enzyme immunoassay and radioligand binding assay in 1841 breast cancer tumours.Eur J Cancer,1991,27(6):715-720.
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