肝癌相关抗原的扩增及肝癌相关蛋白的抗体制备

【作者】 霍景瑞；

【导师】 王清明；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 原发性肝癌(hepatocellular carcinoma,HCC)是我国第二大恶性致死肿瘤,其发病率和死亡率有逐年上升的趋势。肝癌的发病十分隐匿,导致发现晚,预后差。提高肝癌患者存活率的重要措施就是“早发现、早诊断、早治疗”。目前,国内外大量文献中,均涌现出很多肝癌诊断标志物。为了开展肝癌的免疫诊断和治疗,我们根据现有的文献调研,选择了11中肝癌相关标志物,有目的的从不同的癌细胞中成功克隆了11种各种基因的cDNA,他们分别是MAGEA1、MAGEA2、MAGEA6、MAGEA10、GPC3、GP73、HCA519、HCA661、CTp11、TPTE和PTEN。其中MAGEA1和MAGEA10在我国肝癌患者中的有较高的表达比率,分别为66%和36.7%,是最有可能的肝癌疫苗候选分子。GPC3和GP73是目前最有希望的肝癌诊断候选标志物,并且与AFP的表达并无相关性,可与APF联合应用以提高肝癌的诊断率。HCA519和HCA661是wang等人利用重组cDNA表达文库血清学分析(SEREX)技术筛选到的两种肝癌相关抗原,其中HCA519高表达于肝癌细胞,在前列腺癌、肺癌和胰腺中也有表达,但在正常组织中不表达,是一种核增殖相关的蛋白,仅存在于处于细胞周期的细胞中;HCA661主要表达于睾丸组织,在正常的胰腺中有弱表达,但在其他正常组织中无表达,应属于癌睾抗原,有可能成为候选疫苗,但由于在胰腺中有弱表达,因此,基于HCA661设计的治疗性疫苗应首先考察其对胰腺的毒性。CTp11首先被发现表达于黑色素瘤细胞中,该抗原在肝癌中的表达率高达62.9%,存在潜在的HLA-A2限制性CTL表位,而HLA-A2在亚洲人中的表达率可能超过50%,因此,CTp11也是一种可用于肝癌免疫治疗的候选分子。TPTE1在肝癌患者中的表达率为39%,而肝癌患者血清中存在TPTE1抗体的阳性率达到25%,因此,TPTE1有可能用于肝癌的筛查。PTEN是一种肿瘤抑制基因,在肝癌中表达降低。PTEN正常表达可能对于抑制肿瘤的浸润和迁移具有重要意义,因此,PTEN可用于基因治疗肝癌及其他肿瘤。GPC3是一种新的肝癌标志物,其表达与AFP无相关性。为开展肝癌症的免疫诊断研究,我们在克隆了GPC3 cDNA的基础上,表达并纯化了人血清中可溶性GPC3。可溶性GPC3是GPC3蛋白在358-359位氨基酸残基之间断裂后产生的N端部分释放到血液中的。用纯化的可溶性GPC3免疫小鼠,在血清抗体效价达到1:106后,通过细胞融合技术,将免疫小鼠的脾细胞与小鼠骨髓瘤细胞融合,经间接ELISA法筛选阳性克隆,并采用有限稀释法对阳性克隆进行传代,最终得到了两株能稳定分泌鼠抗人可溶性GPC3蛋白的单克隆抗体的杂交瘤细胞D1和G6。经鉴定,D1和G6均为IgG1亚类抗体,两株抗体均具有较好的特异性和亲和力,但D1的亲和力更高,可以检测到10 ng/mL的可溶性GPC3,而G6仅能检测到10μg/mL的可溶性GPC3。通过ELISA叠加实验证明,D1和G6识别GPC3不同的抗原表位,可以作为配对抗体用于夹心ELISA以检测病人血清中的可溶性GPC3。另外,通过免疫荧光实验证明,D1和G6能够识别细胞表面表达的GPC3,这表明D1和G6抗体可以用于免疫荧光实验研究。HCCR是另外一种较理想的肝癌诊断标志物,是韩国Ko等通过差异显示RT-PCR从人宫颈癌组织中克隆的一种新的肿瘤相关基因。在肝癌患者血清中,HCCR蛋白含量升高,而在正常人和良性肝病患者的血清中HCCR含量很低。由于前期我们并未能够克隆出HCCR的cDNA,所以,我们采用了另外一种策略制备HCCR的抗体。一般蛋白质的C末端肽具有较好的可及性,常常成为蛋白质的抗原表位。HCCR的C末端八肽(TNYLGTRR)具有较好的亲水性,并且经过比对发现HCCR的C末端八肽在人蛋白质库中具有唯一性,可以作为HCCR蛋白的特异性标签。基于此,我们将HCCR的C末端八肽展示于T7噬菌体表面,每个噬菌体表面展示有415个小肽,用这种重组噬菌体作为HCCR抗原的替代抗原免疫兔,经过四次免疫后,血清抗体效价均达1:105。其中1号兔免疫血清特异性很好,能够特异性识别HCCR的C末端小肽和HCCR167-360蛋白,但2号兔免疫血清的特异性很差,除能够结合HCCR的C末端小肽外,与其他无关蛋白和小肽也有反应,这可能与免疫动物的个体差异有关。Hepatopoietin Cn (HPPCn)是一种新的肝增殖刺激因子,是从小牛肝刺激物(hepatic stimulatory substance,HSS)中分离出来的,属于LANP(leucine-rich acidic nuclear protein, LANP)家族。HPPCn在肝再生过程中发挥着重要作用,它不仅能够激活肝细胞增殖相关的信号通路,刺激肝细胞DNA合成,而且能够缓解CCl4引起的肝损伤。为进一步了解HPPCn的生物学功能,分析其在肝炎、肝硬化及肝癌等肝病发生过程中的动态变化及其与疾病的相关性,我们研制了抗HPPCn的单克隆抗体(mAb)。用纯化好的HPPCn蛋白免疫雌性BALB/c小鼠,在小鼠抗血清效价达到105后,采用经典的杂交瘤细胞融合技术进行细胞融合,采用间接ELISA检测阳性克隆,经有限稀释法进行亚克隆,最终得到了一株能稳定分泌抗人HPPCn单克隆抗体的杂交瘤细胞W2-D5,该抗体具有很好的特异性,与其他无关蛋白无反应。该抗体还具有较高亲和力,能够检测到低至1ng/ml的HPPCn蛋白;采用噬菌体肽库技术分析了W2-D5抗体所识别的抗原表位,其所识别的抗原表位为HPPCn7-13(IHLELRN)。综上所述,通过本课题研究,我们成功克隆了11种肝癌相关基因,并成功表达、纯化了一种肝癌标志物GPC3,并研制了特异性好和亲和力高的GPC3配对抗体D1和G6;利用替代抗原策略研制了另一种肝癌标志物HCCR的抗血清,该抗血清特异性好,可与鼠源抗体作为配对抗体用于夹心ELISA检测血清中的HCCR。另外,我们还研制了一种肝增殖刺激因子HPPCn的单克隆抗体W2-D5,该抗体能够特异性识别HPPCn蛋白,并具有较高的亲和力,通过噬菌体肽库技术最终鉴定了该抗体所识别的抗原表位。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors and is the second most common cause of cancer death in China. The incidence and mortality of HCC is found increasing year by year. Because of late diagnosis, the prognosis of HCC remains poor. The important measures to improve the survival rate of HCC are early detection, early diagnosis and early treatment.At present, there has the research on immune diagnosis and immune therapy of HCC, 11 kinds of cDNA encoding HCC-related antigens were successfully cloned from several different cancer cell lines, including MAGEA1, MAGEA2, MAGEA6, MAGEA10, GPC3, GP73, HCA519, HCA661, CTp11, TPTE and PTEN. The positive detection of MAGEA1 and MAGEA10 in HCC was 66% and 36.7% respectively, they are the promising candidate molecules for therapeutic vaccine against HCC. GPC3 and GP73 are the most promising candidate biomarkers for HCC diagnosis. Because their expression has no relation with AFP, combined detection of GPC3, GP73 and AFP could improve the diagnosis of HCC. HCA519 and HCA661 are two HCC associated antigen cloned by recombinant cDNA expression library serological analysis (SEREX) technique. HCA519 is a nuclear proliferation-related protein, which only exists in cells during cell cycle. HCA519 is highly expressed in hepatoma cells, and also expressed in prostate, lung and pancreas, but not expressed in normal tissues; HCA661 is a cancer-testis antigen, it is mainly expressed in testis, weakly expressed in normal pancreas, but not expressed in other normal tissues, it may be a vaccine candidate molecule. Because of the weak expression of HCA661 in normal pancreas, the pancreatic toxicity should be focused on when the therapeutic vaccine is designed based on HCA661. CTp11 was first discovered in melanoma cells, it was also found expressed in 62.9% of HCC. CTp11 has a potential HLA-A2 restricted CTL epitope and more than 50% of Asians express HLA-A2, therefore, CTp11 is candidate molecule for immune therapy for HCC. TPTE1 was found in 39% of HCC patients, while the anti-TPTE1 antibody in serum was found in more than 25% of HCC patients. TPTE1 might be used for HCC screening. As a tumor suppressor gene, the expression of PTEN was found decreased in HCC patients. PTEN may play an important role in preventing tumor invasion and the migration. PTEN can be used for gene therapy for HCC and other tumors.GPC3 is a new marker for HCC diagnosis and its expression has no correlation with AFP. For carrying out the study of immune diagnosis of HCC, the human serum soluble GPC3 (sGPC3) was expressed and purified on the basis of cloning of GPC3 cDNA. sGPC3 is the NH2-terminal portion of GPC3 cleaved between Arg358 and Ser359, and could be detected in the sera of patients with HCC. In order to make monoclonal antibody(McAb) to sGPC3, mice were immunized with purified sGPC3. When the serum antibody titers reached 1:106, the splenocytes from the immunized mice were fused with SP2/0 cells by PEG. The positive clones were selected by indirect ELISA. Two positive hybidoma cell lines (D1 and G6) stably secreting antibodies against sGPC3 were obtained after successive limiting dilutions. The McAb D1 and G6 were found to be of immunoglobulin IgG1 subclass, and had high specificity for sGPC3. The McAb D1 had higher affinity than G6, it could detect 10 ng/mL of sGPC3 while G6 could only detected 10μg/mL of sGPC3. The results of the competition ELISA indicated that D1 and G6 recognized different epitopes of GPC3, this meant that D1 and G6 could be paired for the use in sandwich ELISA for detection of sGPC3 in serum. In addition, D1 and G6 could also recognize the GPC3 proteins on cell surface.HCCR is another good marker for HCC diagnosis, which was first cloned from human cervical cancer by South Korea’s Ko using differential display RT-PCR. Compared to normal people or patients with benign liver diseases, the level of HCCR protein was found increased significantly in the serum of HCC patients. Because HCCR cDNA was not successfully cloned, another strategy for generation antibody directed to HCCR was adopted. In general, the C-terminal peptide of protein has good accessibility, it is often good B cell epitope candidate for the corresponding protein antigen. The C-terminal peptide of HCCR (TNYLGTRR, 37c) also has good accessibility and hydrophilicity, and its sequence is unique analyzed by blasting in human protein library. This means that the C-terminal peptide of HCCR can be used as a specific tag for HCCR. Based on above analysis, the peptide was displayed on the surface of T7 phage, each T7 phage displayed 415 copies of the peptide. This recombinant T7 phage was used as the alternative antigen of HCCR to immunize two rabbits to make antiserum. After four times of immunization, the serum antibody titer reached 1:105. Serum from rabbit 1 had good specificity, it not only specifically recognized the C-terminal peptide of HCCR, but also specifically recognized HCCR167-360 protein, whereas the serum from rabbit 2 showed poor specificity. This may be related to the individual differences of animals.Hepatopoietin Cn (HPPCn) is a novel liver stimulating factor isolated from hepatic stimulatory substance (HSS), it belongs to LANP (leucine-rich acidic nuclear protein) family. HPPCn plays an important role in liver regeneration. It could activate the hepatocyte proliferation related signal pathway, and could alleviate CCl4–induced liver damage. For further study of the biological functions of HPPCn and analysis of its dynamic changes in liver diseases, it necessary to make McAb against HPPCn. The recombinant HPPCn was used to immunize female BALB/c mice. The hybridoma cell lines were established by cell fusion. After screening, a stable positive cell line W2-D5 was obtained. McAb W2-D5 possessed good specificity and high affinity. Its detection limit of HPPCn is 1 ng/mL. Using phage peptide library technology, the binding epitope of McAb W2-D5 was mapped to HPPCn7-13(IHLELRN).In summary, 11 kinds of cDNA encoding HCC-related antigen were successfully cloned. Of them, GPC3 was expressed and purified, and two specific, high affinity McAbs to GPC3 were generated, they could be paired for sandwich ELISA. Using the alternative strategy, the specific antiserum against HCCR was produced, it could be paired with mouse McAbs for sandwich ELISA for detection of serum HCCR. In addition, McAb W2-D5 to HPPCn was developed, it could specifically recognize HPPCn protein, its binding epitope was also confirmed.更多还原