文中拟通过采用基因重组技术获得ALT1和ALT2同工酶重组蛋白，分别制备筛选出高特异性、高活性的ALT1和ALT2单克隆抗体 (ALT1单克隆抗体已成功制备并发表)，初步探讨ALT1和ALT2同工酶在人体组织中的定位、分布及表达情况。采用RT-PCR方法从人肝癌细胞 (HepG2) 中扩增ALT2基因，将成熟的ALT2基因亚克隆至pET32a-ALT2原核表达载体中，并将其连接产物转化至BL21(DE3) 感受态细胞中经IPTG诱导表达ALT2蛋白，镍柱 (Ni+) 亲和层析法纯化ALT2重组蛋白。ALT2重组蛋白免疫Balb/c小鼠，选取阳性血清小鼠脾细胞和骨髓瘤细胞SP2/0进行细胞融合，间接ELISA法挑选阳性细胞株，有限稀释法进行亚克隆，采用亲和层析柱法纯化ALT2抗体。通过RT-PCR和Western blotting方法检测ALT1和ALT2在人体正常组织中的表达和分布，研究结果显示组织中的ALT同工酶基因在mRNA水平和蛋白水平上的表达几乎一致。ALT1在肝脏、肾脏、骨骼肌高表达，胃肠道平滑肌中等表达；ALT2在脂肪、骨骼肌、心肌中高表达，胃肠道平滑肌低表达。免疫组化研究表明，ALT1在肝细胞、肾髓质小管和肌纤维中高表达，ALT2在脂肪细胞、心肌细胞中高表达，胃肠道组织ALT1和ALT2主要表达于肠壁上部区域黏膜，上述结果显示同工酶ALT1和ALT2在组织中分布广泛，为理解不同病理条件下ALT活性升高的变化机制提供理论依据。
This study intends to obtain recombinant proteins of ALT1 and ALT2 isozymes by using genetic recombination technology. Monoclonal antibodies ALT1 and ALT2 with high specificity and high activity were prepared and screened (ALT1 monoclonal antibody has been successfully prepared and published). The localization, distribution and expression of ALT1 and ALT2 isozymes in human tissues were discussed. The ALT2 genes were amplified from human liver cancer cell (HepG2) by RT-PCR method. The mature ALT2 gene was subcloned into the pET32a-ALT2 prokaryotic expression vector. Its ligation product was transformed into BL21(DE3) competent cells, and transformed into competent cells to express ALT2 proteins induced by IPTG. The recombinant proteins of ALT2 were purified by nickel column (Ni+) affinity chromatography. Balb/c mice were immunized with recombinant proteins of ALT2. Positive serum mouse spleen cells and myeloma cells SP2/0 were selected for cell fusion. The positive cell lines were selected by indirect ELISA and subcloned by limited dilution method. Affinity chromatography was used to purify ALT2 antibodies. The expression and distribution of ALT2 in human normal tissues were detected by RT-PCR and Western blotting. Results show that the expression of ALT isoenzyme in tissues was almost the same at gene mRNA level and protein level. ALT1 is highly expressed in liver, kidney and skeletal muscle, and moderately expressed in gastrointestinal smooth muscle. ALT2 is highly expressed in fat, skeletal muscle and myocardium, and is poorly expressed in gastrointestinal smooth muscle. Immunohistochemical studies show that ALT1 is highly expressed in hepatocytes, renal medullary tubules and muscle fibers, ALT2 is highly expressed in adipocytes and myocardial cells, and ALT1 and ALT2 in gastrointestinal tissues are mainly expressed in mucosa of upper intestinal wall region. The results showed that the isoenzymes ALT1 and ALT2 were mainly expressed in the mucosa of the upper part of the intestinal wall. It is widely distributed in the tissues, providing theoretical basis for understanding the mechanism of ALT activity increase under different pathological conditions.
国家自然科学基金 (No. 81101318) 资助。