核受体共激活因子NCOA5单克隆抗体的制备及其在乳腺癌中的表达研究

发布时间：2018-01-16 07:10

本文关键词：核受体共激活因子NCOA5单克隆抗体的制备及其在乳腺癌中的表达研究　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

更多相关文章： NCOA5 单克隆抗体 特异性 乳腺癌

【摘要】：目的:应用小鼠杂交瘤技术制备能够识别NCOA5蛋白的单克隆抗体;检测乳腺癌标本中NCOA5的表达,并分析NCOA5与相关临床病理指标的相关性;观察沉默NCOA5对于乳腺癌细胞增殖、迁移的影响。方法:利用PCR技术体外扩增NCOA5羧基C端基因片段,将该片段插入原核表达载体pET-41a中;使用IPTG诱导NCOA5重组蛋白的表达;采用SDS-PAGE分析表达产物的可溶性并进行规模化表达,采用Ni-NTA柱亲和纯化NCOA5重组蛋白,采用紫外分光光度计、SDS-PAGE电泳及考马斯亮蓝染色分析蛋白浓度和纯度;利用小鼠杂交瘤技术制备抗NCOA5的单克隆抗体,通过酶联免疫吸附试验(ELISA)、蛋白质免疫印迹(Western Blot)、免疫荧光(Immunoflousece)和免疫组化(Immunohistochemistry)方法验证单克隆抗体识别NCOA5的敏感性及特异性;利用PCR技术分段扩增NCOA5 C端基因片段,将其插入pMAL-c2X原核表达载体并进行原核蛋白的表达,通过Western Blot初步分析单克隆抗体所识别表位氨基酸序列;利用所制备的单抗行免疫组化检测NCOA5在乳腺癌标本中的表达,统计其与相关临床病理指标的关系;应用慢病毒技术干扰NCOA5在乳腺癌细胞系MDA-MB-231中的表达,细胞增殖实验(CCK-8 assay)检测其对细胞增殖的影响,细胞划痕(wound healing)及Transwell实验检测其对细胞迁移的影响。结果:成功构建了pET-41a/NCOA5-C-ter重组质粒用于原核蛋白的表达,考马斯亮蓝染色显示诱导细菌裂解后上清蛋白大小约69 kDa,与预期一致,说明成功表达NCOA5重组蛋白,纯化后考马斯亮蓝染色显示蛋白纯度高、可用于免疫小鼠;杂交瘤细胞经间接ELISA检测成功筛选出一株抗体8B11,抗体效价为1:32,000。Western Blot、免疫荧光及免疫组化方法提示单克隆抗体8B11能够特异性识别细胞内NCOA5,NCOA5定位于细胞核并在多个肿瘤细胞系中存在表达;正确构建了pMAL-c2X/NCOA5-C-ter-N’及pMAL-c2X/NCOA5-C-ter-C’重组质粒用于分段表达NCOA5 C端原核蛋白,考马斯亮蓝染色显示诱导细菌裂解后上清蛋白大小与预期一致,说明重组蛋白表达成功,Western Blot显示8B11能够识别NCOA5蛋白第291-434个氨基酸组成的蛋白;乳腺癌标本的免疫组化结果显示,随着肿瘤的增大,NCOA5表达的阳性表达率升高(P=0.008);在有淋巴结转移组中,NCOA5表达的阳性表达率升高(P=0.042);随着肿瘤TNM分期的增加,NCOA5的阳性表达率逐渐上升(P=0.028);NCOA5在ER阴性标本中阳性表达率显著升高(P=0.006);NCOA5在PR阴性标本中阳性表达率升高(P=0.025);NCOA5表达与年龄、组织学分级、HER-2无明显统计学意义(P0.05)。慢病毒感染乳腺癌细胞株MDA-MB-231后,Western Blot显示沉默NCOA5表达有效,CCK-8法表明沉默NCOA5后细胞增殖能力下降(P0.05),细胞划痕(wound healing)及Transwell实验表明沉默NCOA5后细胞迁移能力下降(P0.01)。结论:(1)利用pET-41a原核表达载体,成功表达并纯化了NCOA5 C端重组蛋白;(2)利用杂交瘤技术成功制备了一株效价高、特异性好单克隆抗体8B11,可用于Western Blot、免疫荧光和免疫组化检测NCOA5的表达情况,其抗原表位范围为NCOA5蛋白第384-434个氨基酸;(3)NCOA5在肝癌、乳腺癌、结肠癌、胰腺癌细胞系中均存在表达,NCOA5在乳腺癌中阳性表达与肿瘤大小、淋巴结转移、TNM分期及ER、PR的表达水平相关,具有作为肿瘤标志物的潜在价值;(4)沉默乳腺癌细胞MDA-MB-231中NCOA5的表达导致细胞增殖及迁移能力下降,提示NCOA5能够通过ERα以外的途径影响细胞的增殖和迁移能力,为进一步探讨NCOA5参与乳腺癌发生发展过程中的具体机制提供基础。
[Abstract]:Objective: the application of mouse hybridoma technology for preparation of monoclonal antibodies to NCOA5 protein; the expression of NCOA5 in breast cancer tissues, and to analyze the correlation between NCOA5 and clinical pathological indicators; observe the silencing of NCOA5 for breast cancer cell proliferation, migration effect. Methods: using in vitro PCR amplification NCOA5 carboxyl end gene fragment of C. The fragment was inserted into the prokaryotic expression vector pET-41a; inducible expression of recombinant NCOA5 protein using IPTG; SDS-PAGE was used to analyze the expression of soluble and scale expression, using Ni-NTA affinity purified recombinant NCOA5 protein, using UV spectrophotometer, analysis of protein concentration and purity of SDS-PAGE electrophoresis and Coomassie blue staining; monoclonal the anti NCOA5 antibody in mice using hybridoma technique, by enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence (Western Blot) (Immuno Flousece) and immunohistochemistry (Immunohistochemistry) method to verify the sensitivity and specificity of NCOA5 monoclonal antibodies; C gene fragment amplified NCOA5 terminal using PCR technology, the expression of inserted into prokaryotic expression vector pMAL-c2X and prokaryotic protein by Western Blot, a preliminary analysis of monoclonal antibody epitopes using the amino acid sequence; preparation of monoclonal antibodies for immunohistochemical detection of NCOA5 expression in breast cancer specimens, the relationship between statistics and related clinical pathological factors; expression of lentiviral NCOA5 interference technology in breast cancer cell line MDA-MB-231, cell proliferation assay (CCK-8 assay) to detect its effect on cell proliferation, cell scratch (wound healing) and Transwell test to detect its effect on cell migration. Results: successfully constructed pET-41a/NCOA5-C-ter recombinant plasmid for prokaryotic expression, Coomassie brilliant blue Staining induced bacterial lysis after the supernatant protein size is about 69 kDa, consistent with expectations, indicating the successful expression of NCOA5 recombinant protein, purified by Coomassie brilliant blue staining showed that the protein of high purity, can be used for immunization of mice; hybridoma cells were screened by indirect ELISA detection of a 8B11 antibody, the antibody titer was 1:32000.Western Blot, immunofluorescence immunohistochemistry and prompt 8B11 monoclonal antibody could specifically recognize NCOA5 cells, NCOA5 localized in the nucleus and in the presence of multiple tumor cell lines expression; the correct construction of the recombinant plasmid pMAL-c2X/NCOA5-C-ter-C pMAL-c2X/NCOA5-C-ter-N 'and' NCOA5 C end for segmented expression of prokaryotic protein, Kaumas shows Coomassie brilliant blue staining is consistent with the expected size of the supernatant protein induced by bacteria after cracking, that the recombinant protein was successfully expressed, Western Blot shows that 8B11 can recognize NCOA5 protein 291-434 amino Acid protein; immunohistochemical results of breast cancer were showed as the tumor increased, the expression of NCOA5 positive expression rate increased (P=0.008); in the group with lymph node metastasis, the expression of NCOA5 positive expression rate increased (P=0.042); with the increase of TNM staging, the positive expression rate of NCOA5 increased gradually (P=0.028); NCOA5 in ER negative samples positive expression rate was significantly increased (P=0.006); NCOA5 in PR negative samples positive expression rate increased (P=0.025); the expression of NCOA5 with age, histological grade, HER-2 had no statistical significance (P0.05). The lentiviral infection of breast cancer cell line MDA-MB-231, Western Blot silence the expression of NCOA5, CCK-8 showed a decrease in cell proliferation ability after silencing NCOA5 (P0.05), cell scratch (wound healing) and Transwell experiments showed that the decreased cell migration ability after silencing NCOA5 (P0.01). Conclusion: (1) using pET-41a prokaryotic expression As the carrier, NCOA5 C successfully expressed and purified the recombinant protein; (2) by hybridoma technique was successfully prepared a high titer and specificity of 8B11 monoclonal antibody can be used for Western Blot expression by immunofluorescence and immunohistochemical detection of NCOA5, the epitope of NCOA5 protein in the range of 384-434 amino acid; (3) NCOA5 in hepatocellular carcinoma, breast cancer, colon cancer, expressed in pancreatic cancer cell lines, NCOA5 in breast cancer expression and tumor size, lymph node metastasis, TNM staging and ER, related to the expression level of PR, which has potential value as a tumor marker; (4 the silence of NCOA5 expression in breast cancer cells) in MDA-MB-231 resulted in decreased proliferation and migration of cells, suggesting that NCOA5 can approach by ER alpha outside affect the ability of cell proliferation and migration, to further explore the NCOA5 specific mechanisms involved in breast cancer development process Basics.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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