【作者】 史伟；

【导师】 马春红；

【作者基本信息】 山东大学 ， 医学免疫学， 2009， 硕士

【摘要】 ZHX2（zinc finger homodomain box 2）是2003年发现的新型转录抑制因子,其目前已知的三个靶基因均在肝癌中特异性高表达。我们的前期研究结果显示ZHX2参与人肝癌细胞AFP表达调控,并抑制肝癌细胞生长。另有研究显示ZHX2参与多种疾病发生,其表达与多发性骨髓瘤病人预后负相关。因而,ZHX2可能在肿瘤诊断和治疗中具有重要研究价值。研究目的在原核细胞中表达人ZHX2的融合蛋白GST-ZHX2作为抗原,制备多克隆抗体,并利用该抗体检测肝癌组织及不同肿瘤细胞系中ZHX2的表达。研究方法1 GST-ZHX2融合蛋白的表达ZHX2融合蛋白表达载体pGEX-ZHX2（1-837AA）、pGEX-ZHX2（263-497AA）与空载体pGEX-5X-1分别经CaCl₂法转化E.coli BL21（DE3）菌株,抽提质粒并经酶切、测序鉴定。以终浓度为1mM的IPTG诱导工程菌表达目的蛋白,6h后收集菌体和上清液,分别进行SDS-PAGE电泳,考马斯亮蓝染色后,凝胶成像分析电泳图像,通过Quantity One分析融合蛋白表达率。2多克隆抗体制备GST-ZHX2（1-837AA）、GST-ZHX2（263AA-497AA）融合蛋白分子量约118KDa和52KDa,GST蛋白分子量26KDa。将上述菌体蛋白经SDS-PAGE电泳,考马斯亮蓝染色后,对照分子量Marker,切取目的蛋白条带的胶条（约含200gg融合蛋白）作为抗原,并添加2mlPBS后充分研磨,获得“胶体-蛋白”乳浊液。将其与弗氏完全佐剂按照体积比2:1比例混合,多点注射入新西兰大耳兔背部皮下,每2周免疫一次,共计3次（后两次加强免疫时将含有200μg融合蛋白的胶条按照2:1比例与弗氏不完全佐剂混匀）。最后一次免疫后14天,心脏采血法采血,利用饱和硫酸铵法对兔血清中的免疫球蛋白进行粗提,分装后-80℃保存,两种ZHX2-GST融合蛋白免疫后血清分别标记为ZHX2（1-837AA）pAb和ZHX2（263-497AA）pAb。3抗体特异性检测利用本实验室构建的ZHX2真核表达载体pcDNA3-ZHX2-HA及空载体pcDNA3分别转染HepG2。48h后收集细胞,并分别利用上述制备的两种多克隆抗体和商品化HA抗体（1:2000稀释）,进行western blot实验,检测转染后HepG2细胞内相应蛋白的表达。多克隆抗体按照1:100,1:500,1:1000,1:2000,1:4000比例稀释,用于验证该抗体对抗原识别的特异性,并确定抗体最适滴度。4抗体应用收集肝癌切除术术后组织标本和不同肿瘤细胞系细胞,SDS裂解液提取组织蛋白及细胞蛋白,利用自行制备的ZHX2多克隆抗体进行Western blot,检测ZHX2在肝癌组织及不同肿瘤细胞系中的表达水平。结果1 ZHX2融合蛋白在原核细胞中的表达1.1重组质粒pGEX-ZHX2（1-837AA）、pGEX-ZHX2（263-497AA）的鉴定重组质粒CaCl₂法转化大肠杆菌BL21（DE3）,抽提阳性重组子质粒DNA,经BamHI和NotI双酶切,酶切产物经1%琼脂糖凝胶电泳,可见分子量约为2500bp与700bp左右的特异性条带,与预期结果一致。DNA测序分析及BLAST对比分析证明,重组载体插入序列正确,pGEX-ZHX2（1-837AA）、pGEX-ZHX2（263-497AA）内分别含有编码ZHX2 1-837位氨基酸及263-497位氨基酸的相应编码序列。1.2 GST-ZHX2（1-837AA）、GST-ZHX2（263-497AA）融合蛋白在原核细胞中的表达工程菌经IPTG诱导6h后,分别收集菌体及上清,进行SDS-PAGE电泳,考马斯亮蓝染色显示:IPTG诱导的阳性工程菌菌体蛋白电泳后在118KDa、52KDa左右位置发现明显深染蛋白条带,而未经IPTG诱导的对照组未见该条带。上述结果提示:融合蛋白GST-ZHX2（1-837AA）、GST-ZHX2（263-497AA）可以在E.coli BL21（DE3）菌体中表达。收集等量菌体总蛋白,SDS-PAGE电泳及考马斯亮蓝染色后,扫描胶体,利用Quantity One分析各条带灰度,计算蛋白表达量。结果显示:融合蛋白表达率分别为GST-ZHX2（1-837AA）5.4%,GST-ZHX2（263-497AA）10.9%和GST11.7%。2 ZHX2多克隆抗体的制备2.1抗原制备大量培养工程菌,收集菌体蛋白,进行SDS-PAGE电泳,切取目的蛋白染色胶条[GST-ZHX2（1-837AA）,118KDa;ZHX2-GST（263-497AA）,52KDa;GST,26KDa)],根据蛋白表达率,按照每只兔子每次需要0.2mg抗原计算免疫动物所需胶体量,并秤取相应胶条。2.2 ZHX2多克隆抗体的制备及验证3次增强免疫结束后,采集并分离血清,硫酸铵沉淀法粗提血清免疫球蛋白IgG,用于Western blot检测pcDNA3-ZHX2-HA转染后HepG2细胞中的ZHX2蛋白。ECL显色结果显示,商品化HA抗体可检测出pcDNA3-ZHX2-HA转染组中ZHX2-HA融合蛋白的表达,蛋白条带位置在92KDa处;ZHX2（263-497AA）pAb、ZHX2（1-837AA）pAb亦可在相同位置检测出的目的条带,但ZHX2（1-837AA）pAb显色后出现较多的非特异性杂带。未转染组和pcDNA3转染组内,各抗体均未检测到ZHX2表达。上述结果提示,ZHX2（263-497AA）pAb可以与ZHX2抗原进行特异性结合,用于后续实验。利用不同稀释度的ZHX2多克隆抗体和相应的HRP羊抗兔酶标二抗,重复上述实验。结果证实一抗最佳抗体稀释度为1:1000,二抗稀释度1:500时实验结果最佳。3抗体的应用（1） ZHX2多克隆抗体检测不同肿瘤细胞系中ZHX2表达培养不同肿瘤细胞,并于对数生长期收集细胞,裂解后进行western blot。结果显示,ZHX2（263-497AA）pAb多克隆抗体作用后,肝癌细胞系Bel-7402,SMMC-7721细胞中92Kda处可见特异性条带,而HepG2和HepG2.2.15细胞中未见该条带,与本室原有结果相符。提示,自行制备的ZHX2多克隆抗体具有良好的特异性和灵敏度,可以用于检测培养细胞系中的ZHX2的表达。而在肺腺癌细胞系A549,子宫颈癌细胞系Hela,胃癌细胞系MGC-803和SGC-7901以及腺鳞癌细胞系的SKOV-3细胞内均查出ZHX2的表达,但表达强弱不同。（2） ZHX2多克隆抗体检测不同肝癌组织标本中的ZHX2表达随机选取10名肝癌患者手术后肝癌组织标本,抽提组织蛋白后,用自行制备的ZHX2（263-497AA）pAb进行western blot检测。结果显示不同患者肝癌组织中均有ZHX2表达,但表达水平不一致。提示自行制备的多克隆抗体可以用于组织内蛋白检测,为进一步研究ZHX2与肝癌关系提供了有利生物材料。结论成功制备并鉴定ZHX2多克隆抗体,并用该抗体进行western blot检测肿瘤细胞系和肝癌标本内源性ZHX2的表达。结果显示,制备的ZHX2多克隆抗体可以用于体内外检测,为进一步研究ZHX2与肿瘤关系及ZHX2功能研究提供了必要的条件。更多还原

【Abstract】 Zinc-fingers and homeoboxes 2(ZHX2) is a transcriptional repressor,which was cloned in 2003.AFP,GPC3 and H19,known as targets of ZHX2,were highly expressed in HCC.Our lab have provided direct evidence that ZHX2 repressed AFP in human liver tumors,and suppressed hepatoma cells growth.Clinical study showed that ZHX2 expression was negatively correlated to the risk of multiple myeloma. Therefore,ZHX2 might be used as a diagnostic marker and an antibody against ZHX2 with good specificity will be very helpful for the diagnosis of tumors.AimTo prepare ZHX2 polyclonal antibody and detect ZHX2 expression in cell cultures and tumor samples.Methods1 Expression of GST-ZHX2 fusion proteinThe plasmids,pGEX-ZHX2(1-837AA),pGEX-ZHX2(263AA-497AA) and pGEX-5X-1 were transformed into E.Coli BL21(DE3).The positive clones were identified by restrictive enzymes assays and automatic DNA sequencing respectively. Expression of GST and recombinant protein fused with GST tag,GST-ZHX2 (1-837AA),GST-ZHX2(263-497AA),were induced by IPTG(1mM) for about 6 hours.The cell lysate were loaded on 12%SDS-PAGE gels,followed by coomassie brilliant blue staining.Protein expression level was analyzed by Quantity One.2 Immunization of rabbits and immunoglobulin purificationThe target bands(GST,26KDa;GST-ZHX2(1-837AA),118KDa;GST-ZHX2(263-497AA),52KDa) were cut off from stained gels and triturated for more than 10 min, then stored at -20℃. The triturated suspensions of target antigen in 0.9%NaCl was used for immunization.The homogenate was used to immunize male New Zealand white rabbits.Four rabbits were inoculated at multiple sites in the back,with the target protein(200μg/rabbit) mixed with equal volume of Complete Freund’s Adjuvant.And the booster immunizations with equal volume of the expressed protein mixed with Incomplete Freund’s Adjuvant were carried out every two weeks for three times.Two weeks after the final boost,the serum was separated from blood by storing the blood.The serum immunoglobulin obtained was skimpily purified by saturated ammonium sulfate(SAS) precipitation method(purified three times using 50%,33% and 33%(v/v) of SAS,respectively),and sodium azide(NaN3) was added as a preservative at a final concentration of 0.02%(w/w).The purified serum was then subpackaged and stored at -80℃.3 Specificity and Sensitivity assay for ZHX2 polyclonal antibodyIn order to assay the specifiity and sensitivity of self-made antibody,HepG cells was transiently transfected with 1.0μg of pcDNA3-ZHX2-HA or pcDNA3 using Lipofectamine TM2000.Forty-eight hours after transfection,the cells were collected to detect the expression of the protein ZHX2-HA by ZHX2 pAb and HA mAb.4 Application of ZHX2-pAb(1) ZHX2 expression in different tumor cell lines.Human hepatoma cell line HepG2(ATCC HB-8065),HepG2.215 and Bel-7402, epithelial human Caucasian lung carcinoma cell line(A549),cervical cancer cell line(Hela),human gastric cancer cell lines(MGC-803 and SGC-7901),human adenocarcinoma cell line(SKOV-3) were cultured and collected for western blot. Endogenous ZHX2 protein was probed using ZHX2 pAb.(2) ZHX2 expression in HCC samples.Liver samples from 10 patients with HCC,who underwent hepatectomy in the Shandong provincial hospital,Jinan,China were obtained.Clinical data backuped from medical records.These fresh samples were washed by Rnase free PBS in order to remove the humor fluid as much as possible and then frozen immediately in liquid nitrogen and stored at -80℃for use.Protein were extrzctd from all HCC samples for western blot.Endogenous ZHX2 protein was probed using ZHX2 pAb.Results1 Expression of GST-ZHX2 fusion proteinRestrictive enzymes assay and automatic DNA sequencing confirmed the successful construction of plasmid pGEX-ZHX2(1-837AA) and pGEX-ZHX2 (263-497AA).12%SDS-PAGE showed the specific band of GST-ZHX2(1-837AA) at 118KDa and GST-ZHX2(263-497AA) at 52KDa.Protein expression level was analyzed by Quantity One.The expression of the recombinant GST-ZHX2(263-497aa) was estimated up to 10.86%of the total expressed bacterial protein,while the expression of GST-ZHX2(1-837AA)is about 5.4%.These data would be used in evaluating dosage of antigen for immunization.2 Purification and characterization of ZHX2 pAbSerum against human ZHX2,named ZHX2-pAb,was concentrated and simply purified by SAS precipitation method.To further study the specificity and sensitivity of our self-made ZHX2-pAb, transfected pcDNA3-ZHX2-HA was transfected into HepG2 cells which have less endogenous expression of ZHX2.Western blot results showed that the recombinant ZHX2-HA protein in HepG2 transfected with pcDNA3-ZHX2-HA could be detected by both ZHX2-pAb and anti-HA,but not showed in both pcDNA3-transfected HepG2 and non-transfected HepG2.All these evidence proved that our self-made ZHX2-pAb had good specificity and sensitivity for human ZHX2 detection.3 Application of ZHX2-pAb(1) ZHX2 expression in different tumour cell linesNine tumour cell lines were detected for ZHX2 protein expression.Very interestingly,ZHX2 could be detected using self-made ZHX2-pAb in all cell lines, including liver cancer,lung cancer,cervical cancer and gastric cancer cell lines. ZHX2 expression varied greatly in four HCC cell lines which is consistent with our previous results.These indicated the potential use of our self-made ZHX2-pAb in further study for ZHX2. (2) Detection of ZHX2 in HCC samplesZHX2 protein expression were also detected in 10 HCC samples,which obtained from Shandong provincial hospital,Jinan,China.The results showed that 2 samples have high expression levels of ZHX2,5 samples have a middle level,while the others have much lower levels.This indicated that our self-made pAb can be used in clinical detection.ConclusionWe succesufuly prepared polyclonal antibody against human ZHX2 and developed a approach for preparing polyclonal antibody:cutting off fusion proteins from SDS-PAGE gel,injecting protein-mixed gel into rabbits,and then concentrating antibody by SAS,which can be accomplished in all laboratory.Western blot results of transfected HepG2 cells show the good quality of the antibody.We established the western blot assay with our self-made pAb to detect ZHX2 expression in both tumor cell lines and clinical tissues.Further study will focus on the correlation ship between ZHX2 expression and disease prognosis.更多还原