【作者】 韩艳玲；

【导师】 韩昱晨；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 目的金属硫蛋白家族(metallothieonein,MT)富含巯基的低分子量(6kD)蛋白质,MT由61-62个氨基酸组成。MT家族可分为MT1、MT2、MT3和MT4四种异构体。MT1(包括MT1A、MT1B、MT1E、MT1G、MT1F、MT1X、MT1H 7个亚型)。MT有调节重金属(锌等)与蛋白结合,调节基因表达、细胞生长、清除自由基,解毒等作用。以往对MTlH的研究主要集中在其与一些重金属(如锌、镉、铜、银等)代谢方面的研究,但有关MT1H相互作用蛋白所知甚少,未见相关报道。有关MT与肿瘤间关系报道不一,提示其在不同肿瘤中作用也各不相同。MT1G在甲状腺乳头状肿瘤中表达下调,可能是甲状腺瘤的抑癌基因；在浸润性乳腺导管癌中MTⅠ和Ⅱ过表达；在消化道癌、睾丸生殖腺肿瘤、涎腺肿瘤、卵巢癌、肺癌、黑色素瘤、结直肠癌组织中观察到MT过表达,以上对MT在肿瘤学中的研究并没有对MT的各个亚型进行划分,可能MT各个的亚型在不同的肿瘤中发挥不同的作用。有一些报道认为致癌过程中MT的产生与致癌物质的刺激,癌基因激活有关。如：化学致癌物质乙基甲硝脲可激活体外培养的S49细胞MT的产生,那么肿瘤中尤其是肺癌中有哪样一些蛋白与其作用呢?我们利用酵母双杂交技术初步筛选出与MT1H作用的相关蛋白,并为进一步依据其相互作用蛋白研究其功能提供依据。材料和方法1、文库、菌株、质粒和试剂第三代Matchmaker Ga14双杂交系统(MATCHMAKER GAI4 yeast two hybrid system3, Clontech):BD克隆质粒pGBKT7,AD克隆质粒pGADT7,BD质粒阳性对照pGBKT7-T, BD质粒阴性对照pGBKT7-Lam。酵母菌株AH109、酵母培养基YPDA、人工合成营养缺陷型培养基(synthetic dropout minimal base, SD) SD/-Trp、SD/-Leu、SD/-Trp/-Leu、SD/-Trp/-Leu/-His、SD/-Trp/-Leu/-His/-Ade、X-a-半乳糖苷酶(X-a-gal)均购自Clontech公司。肺癌cDNA文库购自Clontech公司。大肠杆菌Topo10C为本教研室保存。所需的限制性内切酶,LA TaqDNA聚合酶、T4 DNA连接酶均购自TaKaRa公司。酸洗玻璃珠购自Sigma公司。2、实验方法(1)诱饵BD质粒pGBKT7-MT1H的构建及鉴定获得的人全长MT1H基因利用经PCR引入的NdeI/BamHI双酶切位点定向亚克隆至pGBKT7,转化大肠杆菌Topo10,卡那霉素筛选相应阳性克隆,双酶切提取的质粒鉴定插入片断大小,为BD质粒pGBKT7-MT1H,测序结果与GeneBankMT1H序列比较同源性。(2)酵母双杂交筛选①采用醋酸锂法将酵母双杂交系统中的重组诱饵载体pGBKT7-MT1H与肺癌cDNA文库质粒DNA共同转化至酵母菌株AH109,涂布于SD/-Leu/-Trp/-His/-Ade/X-a-gal平板上,30℃培养至菌落长出,筛选蓝色的阳性克隆。挑取所有单菌落的阳性克隆,在SD/-Leu/-Trp/-His/-Ade/X-a-gal平板上反复传代3次,每次均以X-a-gal筛选,排除假阳性。②提取的大肠杆菌质粒DNA以pACT 5’、3’AD扩增引物PCR,产物经HaeⅢ酶切,琼脂糖凝胶电泳,去除重复的阳性克隆。对阳性质粒DNA测序分析鉴定。经GeneBank生物信息学分析确定基因。③回复杂交验证：将阳性克隆接种于SD/-Leu/-Trp/-His/-Ade液体培养基培养,玻璃珠法提取酵母。阳性克隆的质粒,转化至大肠杆菌Topol0C,卡那霉素筛选克隆,提取质粒DNA,与重组质粒pBGKT7-MT1H共同转化酵母菌株AH109,转化产物涂布于SD/-Leu/-Trp/-His/-Ade/X-a-gal,不能生长或生长菌落为白斑者作为假阳性排除。结果1、诱饵质粒pGBKT7-MT1H的构建及鉴定pGBKT7-MT1H经EcoRI/BamHI双酶切后,产生242bp左右大小DNA片段,与理论扩增大小一致。测序结果表明融合区域的读码框正确并有正确的方向。经测序证实为人MTlH序列且无碱基突变。2、诱饵质粒pGBKT7-MT1H的毒性及自激活效应检测将直径相同的AH109 (pGBKT7-MT1H)和AH 109 (pGBKT7)分别在3ml的YPDA液体培养基过夜培养后,600nm的吸光度分别为1.20和1.22,表明构建的诱饵质粒对酵母没有毒性,不能影响酵母正常生长。AH109 (pGBKT7-MT1H)在SD/-Trp/X-α-gal生长出白色菌落,在SD/-His/-Trp/X-α-gal不生长,排除了pGBKT7-MT1H激活自身报告基因的能力。3、基因序列的测定与分析(1)阳性克隆的分类在SD/-Leu/-Trp/-His/-Ade/X-agal平板上筛选蓝色的阳性克隆。将排除假阳性后的质粒用HaeⅢ内切酶进行酶切,以10g/L琼脂糖凝胶电泳,可以看到不同大小的cDNA片段,根据片段大小共分11类。(2)序列测定结果根据上述片段大小分类结果,我们共筛选出8种相关蛋白。结论1、筛选出8种与MT1H相互作用的蛋白。更多还原

【Abstract】 Metallothioneins (MT) are a group of low-molecular weight, cysteine-rich intracellular protein, which are encoded by a family of genes containing at least 10 functional isoforms (including MT1A, MT1B, MT1E, MT1Q MT1F, MT1X, MT1H). MT includes 61 to 62 amino acids, MT family can be divided into MT1, MT2, MT3 and MT4 four kinds of isomers. MTs regulate the binding of heavy metals (zinc, etc.)and protein,they also regulate gene expression,cell growth, free radical scavenging and detoxification.Formerly worke mainly concentrated on the MT1H between it and some heavy metals (for example zinc, cadmium, copper, silver and so on) metabolism aspect, but the related MT1H interaction protein knows really few, we has not seen the related reports. There are few reports on the expression of the different isoforms of MT which have been analyzed by specific gene probes. They reveal that certain isoforms are expressed in specific cell types.Recently, downregulation of MT gene expression in PTC(papillary thyroid carcinoma)has been reported, suggesting a possible oncosuppressor role of this gene family in the pathogenesis of thyroid tumors., In wetting quality mammary gland drive pipe cancer MTⅠANDⅡare upregulation; At the digestive tract cancer, the testicle gonad tumor, the salivary gland tumor, the ovarian cancer, the lung cancer, the melanin lump, in the colon cancer organization observes the MT expression, the above has not carried on the division to MT in the oncology research to MT each hypotype, possible MT each hypotype to play the different role in the different tumor. A number of studies have shown an increased expression of MT in various human tumors of the breast, colon, kidney, liver, lung, nasopharynx, ovary, prostate, salivary gland, testes and urinary bladder. The above has not carried on the division to MT in the oncology research to MT each hypotype, each hypotype of MT possibly plays the different role in the different tumor.some reports reveal MTs production is related with the carcinogen stimulation and the cancer gene activation concerns,in the carcinogenic process.such as chemistry carcinogen ethyl armor nitrourea may activate in vitro raise S49 the cell and lead to MT incrensing. Then, which type of proteins has interaction with MT1H in the tumor,particularly in the lung cancer? We use the yeast double hybrid technology to screen initially related protein with the MT1H for further studying its function to provide the basis.Materials and Methods1、Library, strain, material particle and reagentMATCHMAKER GAI4 yeast two hybrid system3, Clontech:BD clone material particle pGBKT7, AD the clone material particle pGADT7, BD material particle masculine gender compares pGBKT7-T, the BD material particle negative compares pGBKT7-Lam. Yeast strain AH 109, yeast culture medium YPDA, the synthesized nutrition gap culture medium (synthetic dropout minimal base, SD) SD/-Trp, SD/-Leu, SD/-Trp/-Leu, SD/-Trp/-Leu/-His, SD/-Trp/-Leu/-His/-Ade, the X-a-glycosidase (X-a-gal) buys from Clontech Corporation. The lung cancer cDNA library buys from Clontech Corporation. Backwoods coli TopolOC is our working office preservation. Needs the restrictive interior contact enzyme, LA the TaqDNA polymerase, T4 the DNA ligase buys from TaKaRa Corporation. The acid pickling beaded glass buys from Sigma Corporation.2、Methods(1) Bait BD material particle’s(pGBKT7-MT1H) construction and identification Obtain the MT1H gene of the human.use the double enzyme cuts the position after PCR the insert NdeI/BamHI.that clones to pGBKT7, transformation backwoods coli Topo10, Kanamycin select Positive clone, the double enzyme cuts extract the particle that identifies the insertion piece size, the insertion piece size is BD particle pGBKT7-MT1H, compare the result with the MT1H of GeneBank sequence about homology.(2) yeast double hybrid screening①Apply the lithium acetate method to make yeast two-hybrid system bait vector pGBKT7-MT1H reorganization and lung cDNA library plasmid DNA co-transformed into yeast strain AH 109, coated in SD/-Leu/-Trp/-His/-Ade/Xa-gal plates,30℃cultured colony grew, the blue screen positive clones. All single colony picked positive clones in the SD /-Leu /-Trp /-His /-Ade /Xa-gal plates, passaged three times repeatedly, Use Xa-gal each time filter to the exclude false positive.②Extracte E.coli plasmid DNA with pACT 5’,3’AD primers PCR, products are HaeⅢdigestion, agarose gel electrophoresis, remove the duplicate clones. Plasmid DNA sequencing of the positive identification. The GeneBank bioinformatics analysis to determine gene.③Back cross validation:the positive clones are inoculated in SD/-Leu/-Trp/-His /-Ade liquid culture medium, the glass bead extraction of yeast. Positive clone plasmid was transformed into E. coli TopolOC, kanamycin select clones. plasmid DNA and the recombinant plasmid pBGKT7-MT1H yeast strains co-transformed into AH109. Inoculated the products on the SD/-Leu/-Trp/-His/-Ade/Xa-gal.If not growth or the growth of white colonies were ruled out as a false positive.Results1、the bait plasmid pGBKT7-MT1H Construction and identificationpGBKT7-MT1H by EcoRI/BamHI double-digested, resulting in about the size of DNA fragments of 242bp, and the theory of the same size was amplified. Sequencing results showed that the integration of the region and have the correct reading frame in the right direction. Confirmed by DNA sequencing,the result is no mutation.2、The bait plasmid pGBKT7-MT1H toxicity testing and self-activating effectThe diameter of the same AH 109 (pGBKT7-MT1H) and AH 109 (pGBKT7) were in 3ml of YPDA liquid medium overnight incubation,600nm absorbance of 1.20 and 1.22 respectively, showed that the constructed yeast bait plasmid hasn’t toxicity and don’t affect yeast normal growth. AH109 (pGBKT7-MT1H) in SD/-Trp/X-α-gal white colonies grown in SD/-His/-Trp/X-α-gal showed no growth, excluding the pGBKT7-MT1H ability to activate the reporter gene itself.3、Gene sequence and analysis(1) the classification of positive clonesIn SD/-Leu/-Trp/-His/-Ade/X-agal plate blue screen positive clones. False positive will be excluded after the plasmids were digested with HaeⅢendonuclease to 10 g/L agarose gel electrophoresis, can see the different sizes of cDNA fragments is divided into 11 categories according to fragment size.(2) sequencing resultsAccording to the fragment size of the classification results, we have selected eight kinds of proteins.ConclusionWe select eight kinds of proteins that interact with MT1H.更多还原