Expression Of N-terminal Domain Of TET Oncogene Family Member 2 And Preparation Of Its Polyclonal And Monoclonal Antibodies

Malignant tumor has been a major killer of human's health all the time, and cancer mortality has been increasing to a rate of 1% per year. International Agency for Research on Cancer(IARC) of The World Health Organization(WTO) speculated that, by 2030, the global number of cancer patients will be double than that in 2000, meaning that, by 2030, there will be 2,700 million people diagnosed with cancer 1,700 people died of cancer, 7,500 million people with cancer worldwide. Currently, WTO and national governments have considered overcoming cancer as a priority.Since ancient Greece, human beings had been searching for reasons to cause cancer. About 150 years ago, the pathologist Virchow thought that cancer was derived from some underlying embryonic remnants during the growing process, and the speculation was based on the observation of the similarity of embryonic development and certain tumors, such as teratomas. In 1977, Hamburger found that in the 1000 to 5000 only one solid tumor cell formed to clone formation in soft agar. As these studies emerged continuously, many cancer theories is proposed: chemical carcinogenesis, cancer virus, cell cycle and apoptosis theory and so on. However, these have been related with cancer, but not the nature of cancer. In recent decades, through the analysis of tumor genetic family, epidemiology and a large number of animal studies, it proved that tumor effects by genetic factors. especially in the past two decades, tumor has further been proved that tumor was caused by interaction of environmental and genetic factors, and gradually found some relationship of genetic variation and cancer. The genes was difined as cancer genes, because they transformed the normal cells to tumor cell.In March 2009, Tefferi A found that there was TET2 mutations in about 14% of patients with JAK2V617F-positive MPNs, and these mutations exist in the early hematopoietic stem cells (CD34 + CD38-), which made TET2 to a study hotspot in blood tumor area. In subsequent studies, it found that TET2 mutations existed in other myeloid malignancies (Myeloid cancers), such as CMML, MDS, AML and M7 AML, whose ratio was about 15%. Another study showed that, there were 68 kinds of TET2 mutations in myeloid malignancies patients, which didn't have heredity, but would reduce the survival rate of AML patients.As a newly discovered cancer genes, the deletion or mutation of TET2 can cause malignant proliferation of bone marrow cells. Currently, the study of TET2 is limited in the gene level, and the tumor-suppressing function of TET2 is unknown for us. Recent studies have comfirmed that TET1 can catalyze the 5'-hydroxymethyl cytosine deoxynucleotide into deoxynucleotide 5'-methyl-cytosine which plays important role in the formation of bone marrow cells. Therefore, we speculate that TET2 may also be a DNA modifying enzymes as the same family of TET1, which may form a complex together with one or some proteins to perform the transcriptional regulation. According to this hypothesis, the present study plays a important role in intracellular localization, physiological function and detecting TET2 mutation in protein level.TET2 genes are located on chromosome 4p24, and the full-length of its cDNA is 9677 bp (coding region is 387-6395), including 13 exons, consisting of 2002 amino acids, with the molecular weight of 223.87 KD. Because of difficulty of cloning and expressing full-length TET2 and unnormal mutation locus in the central and C terminal TET2 protein, we use a N terminal peptide of TET2 protein (cDNA sequence is 381-974 with the expressed product of molecular weight 21.8KD, containing 196 amino acid residues) to prepare the Anti-TET2N polyclonal and monoclonal antibodies.In this study, we cloned and expressed two kinds of TET2N fusion proteins of GST-TET2N and MBP-TET2N. As the antigen GST-TET2N, experiment animals were immunized New Zealand white rabbits and BALB / c mice to prepare the monoclonal and polyclonal antibody. And another fusion protein MBP-TET2N was used for antibody screening and purification. Using GST-TET2N to immunize animals as the immunogen, it was inevitable to cause large antibodies against GST-tagged proteins. And the antibodies can be purified by the fusion MBP-TET2N in the antibody purification period, which can reduce the number of false positive protein antibodies to improve antibody specificity. Polyclonal antibodies can be combined with different epitopes, but only with a single monoclonal antibody epitope binding. In order to obtain more reasonable experiment design and more reliable results, Anti-TET2N polyclonal and monoclonal antibodies was prepared in this study, which can make the anti-TET2N polyclonal and monoclonal antibodies to verify the same results in the same time, and prepare for studying TET2N physiological and biochemical functions in the protein level.