【作者】 栾好飞；

【导师】 宋海峰； 范礼斌；

【作者基本信息】 安徽医科大学 ， 细胞生物学， 2010， 硕士

【摘要】 卵巢癌是严重威胁广大妇女的恶疾,由于发病部位隐匿,早期症状不明显,卵巢癌死亡率居妇科恶性肿瘤之首,70%~80%卵巢癌患者发现时已为晚期,五年生存率仅20%,而早期患者5年生存率可达70%~90%,所以提高卵巢癌早期诊断水平对提高其治愈率有重要意义,这也是目前妇科肿瘤研究关注的热点。肿瘤标志物的检测简便且无创,在肿瘤的筛查、诊断、指导治疗、预后评价等方面有着非常广泛的应用前景。目前只有癌抗原125(cancer antigen 125, CA125)检测被广泛应用于临床卵巢癌的诊断,大量研究证实CA125敏感性和特异性不高,对卵巢癌早期诊断率低。人附睾蛋白4(human epididymis protein 4, HE4)是一种新的卵巢癌肿瘤标志物,最初是在人附睾上皮中发现的,可能有蛋白酶抑制剂的作用,但具体功能不详。HE4在卵巢癌组织中高表达,而在正常组织中不表达或低表达,在卵巢癌诊断方面其灵敏度要明显高于CA125,且能更好区分良性与恶性肿瘤,大量研究证实HE4是检测早期卵巢癌的最佳标志物之一。因此制备针对HE4的卵巢癌早期诊断试剂,对于提高卵巢癌患者的生存率有重要意义。传统的抗体制备方法通常采用纯化的蛋白质来免疫动物,但分离纯化蛋白费时费力,难以获得理想产品,有时即使获得了目的蛋白,其免疫原性也很低(如某些原核表达蛋白),难以获得理想的免疫效果。电穿孔辅助DNA免疫能克服上述缺点。瞬时电场可使细胞生物膜产生可恢复的微孔,外源基因在电场力的作用下通过微孔进入目标组织或器官,并在该组织或器官中直接表达目的抗原,引发免疫反应。DNA被细胞摄取后,基因半衰期延长,可持续表达目的蛋白,增强了淋巴细胞记忆持久性;另外,电穿孔引起局部组织炎症反应,使大量炎症细胞浸润,提高了抗原递呈效率,因此,电穿孔辅助DNA免疫能获得高效价的抗体。本研究利用RT-PCR从患者卵巢癌组织中获取HE4基因编码序列,通过T-A克隆将HE4基因克隆到pMD19T载体中,经测序鉴定后将其亚克隆至pPICZαA和pcDNA3.1(+)表达载体中,并通过PCR、双酶切及测序对其进行鉴定。将构建好的pPICZαA-HE4表达质粒通过电穿孔转染毕赤酵母GS115,在选择培养基上筛选阳性表达菌株,诱导其表达HE4重组蛋白,进一步通过ELISA、Western-blot鉴定表达的HE4重组蛋白。通过考察不同甲醇浓度、pH、诱导时间、生物量及诱导温度对HE4表达水平的影响,优化了HE4诱导表达的条件。对含HE4重组蛋白的酵母培养上清采用镍亲和层析进行纯化。另一方面,我们采用活体电穿孔法辅助pPICZαA-HE4和pcDNA3.1-HE4分别免疫BALB/c小鼠,通过B淋巴细胞杂交瘤技术进行细胞融合来制备单抗,经多次克隆化培养,筛选出特异分泌鼠抗人HE4 mAb的杂交瘤细胞株。采用Western-blot,Ig亚型分析和mAb表位分析等对mAb的生物学特性进行鉴定。研究结果显示,成功从卵巢癌组织中获取了HE4基因,并构建了pcDNA3.1-HE4和pPICZαA-HE4两个真核表达载体;构建的毕赤酵母能分泌表达HE4蛋白,表达的重组蛋白已被糖基化修饰并具有一定的空间构象;HE4重组蛋白的最佳诱导条件为:pH4.0,2%甲醇,2倍菌量,温度26℃,诱导12h;从1L培养上清中,可纯化出约10mg的HE4重组蛋白;单抗制备方面,获得了2株持续、稳定分泌鼠抗人HE4 mAb的杂交瘤细胞株,命名为1-7-C和3-12-C,两株单抗培养上清抗体效价分别为1:160和1:320,ELISA和Western-blot结果均显示:本实验获得的两株mAb能够特异识别有天然构象的HE4蛋白,针对HE4蛋白的相同表位,两株mAb的Ig亚类均为IgM,轻链均为λ链。上述结果表明,HE4基因被成功克隆至pcDNA3.1-HE4和pPICZαA-HE4两个真核表达载体中;成功构建了表达HE4蛋白的酵母工程菌,获得了有空间构象的HE4重组蛋白,并获得2株分泌鼠抗人HE4 mAb的杂交瘤细胞株,为卵巢癌早期诊断和HE4蛋白功能研究奠定了基础。更多还原

【Abstract】 Ovarian cancer is the leading cause of death from gynecologic malignancy among women worldwide. Early stage ovarian cancer has an excellent prognosis if properly evaluated. Unfortunately, 70~80% of patients could not be found until the cancer progressed to the late stages, and their 5-year survival rate is lower than 20%. In contrast, if we can detect and treat the cancer at early stage, when tumor is found to be only in the ovary, the 5-year survival rate is up to 70~90%. Therefore, a sensitive and specific early detection method is no doubt appealing.In the present, the only clinically accepted serum marker for ovarian cancer is CA125. Numerous studies showed that the sensitivity and specificity of CA125 is not high enough for a screening test. HE4 (human epididymis protein 4) is one of the most promising new serum biomarkers for ovarian cancer, which is a glycoprotein and first detected in human epididymal epithelium. The biological function of HE4 is still unknown although some research suggested it may play a role of protease. HE4 is highly expressed by ovarian carcinomas and serum HE4 has been shown to elevate in both early and advanced stage endometrial cancers. Clinical studies also confirmed that the combination of serum HE4 and CA125 raised the sensitivity of detection compared to that of CA125 alone. Therefore, the objectives of this study were to obtain recombinant HE4 and its antibodies for developing serum HE4 assay methods, which would provide more powerful tool for early detection and prognosis supervision of ovarian cancer.During the antibodies preparation methods, animals were commonly immunized with purified protein. But sometimes it is difficult to obtain a quantity of purified proteins enough for immunization, and the recombinant proteins might be deficient in immunogenicity to initiate immune reaction, such as inclusion body proteins expressed by E.coli. DNA immunization via electroporation could make a way to produce antibodies without proteins preparation. As we known, the proteins expressed in vivo have structures more similar to its natural structure. Electroporation could help the DNA taken up by the cells, prolong its expression, and the continually expressed protein could provoke and boost the memory lymphocyte responses. In addition, electroporation causing local tissue inflammation and large numbers of inflammatory cell infiltration improved the antigen presentation. Therefore, we will try to obtain high titer antibodies by DNA immunization via electroporation in vivo.We obtained the cDNA encoding HE4 from ovarian cancer tissues by RT-PCR. The amplified product was inserted into PMD19T vector via T-A complement and then subcloned into pPICZαA and pcDNA3.1(+), pichia pastoris and mammalian cell expression vectors, respectively. Recombinant plasmids were identified by PCR, restriction enzyme digestion and sequencing. The indentified pPICZαA-HE4 plasmid was then transformed into pichia pastoris GS115 by electroporation and positive recombinant strains were screened by PCR and induced expression. The expressed recombinant protein was identified by ELISA and Western-blot with commercial anti-HE4 monoclonal antibody. The expression conditions were also optimized for obtaining a quantity of recombinant HE4 to develop a quantitative method.On the other hand, the plasmids constructed were also used to immune BALB/c mice via electroporation in vivo. The hybridomas were achieved by fusing the immunized spleen cells with Sp2/0 myeloma cell line and the positive clones secreting anti-human HE4 antibodies were screened by further subcloning. The secreted antibodies properties were determined by Western blot, Ig subtyping and epitope analysis.The results showed that we obtained the cDNA encoding HE4 from ovarian cancer tissues and successfully constructed the pcDNA3.1-HE4 and pPICZαA-HE4 eukaryotic expression vector. The secretory expression of HE4 protein could be detected from, the recombinant pichia strains transformed with pPICZαA-HE4 and the recombinant HE4 was glycosylated with special confirmations; The expression level of recombinant HE4 reached the highest after 12 hours induced by 2% concentration of methanol, at pH 4.0 and 26℃. After purification, 0.413 mg/mL recombinant HE4 protein was obtained from 1 liter culture medium. At the same time, we obtained two hybridoma cell lines, named 1-7-C and 3-12-C respectively, which could continuously and stably secrete specific anti-HE4 monoclonal antibodies. The antibodies titers were 1:160 and 1: 320 respectively. ELISA and Western-blot assay showed that the monoclonal antibodies recognized the HE4 protein with natural conformation. Subgroup assay showed that they are both IgM antibodies, and their light chains were typeλ.Therefore, the research successfully developed the engineered pichia pastoris strains, which could produce a quantity recombinant HE4 with special conformation. It is valuable in the future studies for its diagnostic and therapeutic application. In addition, two mouse anti-human HE4 hybridomas were obtained by DNA immunization and they secreted antibodies specifically recognize the recombinant HE4 which made it possible to develop a quantitative method for detection of serum or urinary HE4 level.更多还原