Biological characterisation and application of human MTH1 and monoclonal antibody preparation
Biological characterisation and application of human MTH1 and monoclonal antibody preparation
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人MTH1及单克隆抗体制剂的生物学特性及应用
DOI：
10.3892/or.2018.6942
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发表时间：
2019-03-01
期刊：
影响因子：
4.2
通讯作者：
Ji, Xuemei
中科院分区：
文献类型：
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作者：
Chen, Chuyuan;Li, Xiaoping;Ji, Xuemei
人类突变同源基因1(MTH1)可以分解氧化的核苷酸三磷酸，从而阻止它们被结合到DNA中；MTH1已被发现在许多类型的癌症中都升高，包括肺癌、胃癌、黑色素瘤和乳腺癌。因此，肿瘤靶向的hMTH1可能对开发新的抗癌治疗方法有价值。在本研究中，我们制备了人MTH1蛋白及其单抗。将hMTH1基因克隆到原核表达载体pET28a中，并在大肠杆菌Transetta(DE3)中进行了优化表达。采用Ni-NTA色谱柱和G-50凝胶过滤色谱柱，从1000ml细菌培养物中分离得到20.1 mg的活性hMTH1，经高效液相色谱检测，其纯度可达98%以上。用重组hMTH1蛋白测定hMTH1抑制剂TH287的半数抑制浓度(IC50)为3.53+/-0.47 nM。酶活测定表明，该蛋白的米氏常数(K-m)为106.13+/-48.83u M，催化常数(k(CAT))为3.64+/-0.58s(-1)。用杂交瘤技术获得了抗hMTH1的单抗，并用免疫印迹方法进行了验证。此外，免疫荧光分析(IFA)和酶联免疫吸附试验(ELISA)表明，该mAb能有效结合人乳腺癌细胞株MCF-7上表达的天然hMTH1。综上所述，结果表明，hMTH1是一种具有活性的蛋白质，在抑制物选择方面具有实际应用价值，我们制备的hMTH1单抗将为hMTH1的进一步鉴定和抗肿瘤药物的开发提供有价值的工具。
Human MutT homolog 1 (MTH1) hydrolyses oxidised nucleotide triphosphates, thereby preventing them from being incorporated into DNA; MTH1 has been found to be elevated in many types of cancers, including lung, stomach cancer, melanoma and breast cancer. Thus, tumour-targeted hMTH1 may be valuable for developing novel anticancer therapies. In the present study, we prepared human MTH1 protein and its monoclonal antibody (mAb). The hMTH1 gene was cloned into the prokaryotic expression vector pET28a and optimally expressed in the E. coli Transetta (DE3) strain. Using an Ni-NTA column and a G-50 gel filtration column, 20.1 mg of active hMTH1 was obtained from 1,000 ml of bacterial culture, and the purity was over 98%, as detected by high-performance liquid chromatography (HPLC). The half maximal inhibitory concentration (IC50) of TH287 (hMTH1 inhibitor) was determined to be 3.53 +/- 0.47 nM using the recombinant hMTH1 protein (rhMTH1). The enzyme activity assay showed the Michaelis constant (K-m) and the catalytic constant (k(cat)) of the protein were 106.13 +/- 48.83 mu M and 3.64 +/- 0.58 sec(-1), respectively. The anti-hMTH1 mAb was obtained via the hybridoma technique and validated by western blot analysis. In addition, an immunofluorescence assay (IFA) and ELISA determined that the mAb could efficiently bind to natural hMTH1 expressed on the human breast cancer cell line MCF-7. Taken together, the results showed the rhMTH1 is an active protein and has practical applications for inhibitor selection, and our prepared hMTH1 mAb will provide a valuable tool for the further characterisation of hMTH1 and antitumour medicinal development in future.