Xue Tao, Xue Xiao-ping, Huang Qing-sheng, Wei Li, Sun Kai, Xue Tian
Department of Otorhinolaryngology, Head and Neck Surgery, Xijing Hospital, The Fourth Military Medical University, Road, Xi'an, China.
Hybridoma (Larchmt). 2009 Aug;28(4):251-7. doi: 10.1089/hyb.2009.0017.
We newly cloned the gene encoding the human aspartyl (asparaginyl) beta-hydroxylase (HAAH) from the surgical tissue of a patient with hepatocellular carcinoma. This study was designed to generate HAAH-specific monoclonal antibody (MAb) for further exploration of its structure and function. Mice were co-immunized with naked plasmid DNA containing N-terminal domain of encoding HAAH gene and recombinant HAAH polypeptide. Hybridomas were developed by the electrofusion of the splenocytes from mice immunized with plasmid DNA to Sp2/0 myeloma cells in vitro. Three hybridoma cell lines (designated G3, G9, and F11, respectively) stably secreting HAAH-specific MAbs were obtained. The specificity and sensitivity of MAbs were assessed by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Results showed that the three MAbs belong to IgG1 kappa isotype, the titer of MAbs reached was 5 x 10(4) - 1 x 10(5), and the affinity constant (k(aff)) of MAbs ranged between 2.5 x 10(8) - 1.1 x 10(9). MAb G3 was preliminarily applied to detection expression of HAAH for seven tumor tissues, including hepatocellular carcinoma, lung cancer, kidney cancer, cholangiocarcinoma, prostate cancer, breast cancer, and glioblastoma by immunohistochemical stain. Our studies demonstrated that co-immunization of naked DNA containing encoding gene of target antigen and recombinant target protein, and combined with in vitro electrofusion, is an effective and simple method to raise MAbs.
我们从一名肝细胞癌患者的手术组织中首次克隆了编码人天冬氨酰（天冬酰胺酰）β-羟化酶（HAAH）的基因。本研究旨在制备HAAH特异性单克隆抗体（MAb），以便进一步探索其结构和功能。将含有编码HAAH基因N端结构域的裸质粒DNA与重组HAAH多肽共同免疫小鼠。通过将用质粒DNA免疫的小鼠脾细胞与Sp2/0骨髓瘤细胞在体外进行电融合来制备杂交瘤。获得了三个稳定分泌HAAH特异性单克隆抗体的杂交瘤细胞系（分别命名为G3、G9和F11）。通过间接酶联免疫吸附测定（ELISA）和蛋白质印迹分析评估单克隆抗体的特异性和敏感性。结果表明，这三种单克隆抗体属于IgG1 κ同种型，单克隆抗体的效价达到5×10⁴ - 1×10⁵，单克隆抗体的亲和常数（k(aff)）在2.5×10⁸ - 1.1×10⁹之间。单克隆抗体G3初步应用于通过免疫组织化学染色检测包括肝细胞癌、肺癌、肾癌、胆管癌、前列腺癌、乳腺癌和胶质母细胞瘤在内的七种肿瘤组织中HAAH的表达。我们的研究表明，将含有靶抗原编码基因的裸DNA与重组靶蛋白共同免疫，并结合体外电融合，是一种有效且简单的制备单克隆抗体的方法。
