Expression Of C-terminal Domain Of TET2and Preparation Of Antibodies Against TET2C-terminal Domain

Myeloproliferative neoplasms (MPNs) are a class of hematopoietic tumordiseases, mainly caused by the myelodysplastic hematopoietic disorders.Several recurrent chromosomal aberrations have been associated with MPNs,so prevalent that the changes in gene resulted in the production of MPNs,but the specific impact of genetic changes in how diseases are stillunclear.In2009, Tefferi A found that there was TET2mutations in about13%ofpatients with BCR-ABL-negative MPNs. TET2mutations are present in avariety of myeloid malignancies, such as MPNs, MDS, CMML. The mutationrate of TET2in MPNs is about15%, approximately20%in DMS, up to50%in CMML.TET2gene is located in the long arm of chromosome4, locus24(4q24).The full-length of the cDNA is9766bp, including13exons, encoding2002amino acids which the molecular weight is223.87kd. TET2is a putativetumor suppressor gene, and the deletion or mutation of the gene favoursmyeloid tumorigenesis. TET2-/-mice were found to show symptoms of myeloidmalignancies in2010, and it indicates that Tet2functions as a tumorsuppressor to maintain hematopoietic cell homeostasis.TET2, together with TET1and TET3, belongs to a family of oxygenase thatis dependent on Fe2+and α-oxoglutarate.TET2protein is shown to be ableto demethylate DNA by converting5-methylcytosine (5mC) into5-hydroxymethylcytosine (5hmC).5-Hydroxymethylcytosine can be furtheroxidized into5-carboxylcytosine (5caC), which can subsequently berecognized by thymine-DNA glycosylase base-excision repair enzymes and replaced by unmethylated cytosines. This study may give us a betterunderstanding of the tumorigenesis mechanisms.TET2takes an important part in DNA demethylation that can affect geneexpression, cell differ and renew, etc. The mutation of TET2gene can leadto myeloid malignancies. So it is must be meaningful for diagnosis andprognosis of myeloid malignancies to detect the mutation of TET2generapidly. In the studies of TET2gene mutation, researchers all detect themutation at DNA level. It can not exactly describe the quantity of mutatedprotein of TET2in human body and in which way the mutated protein affectthe human body. And how the mutation results of the myeloid malignanciesis still unclear. In order to further research the function of TET2protein,we clone and express the TET2C terminal in this study, and use it toproduce the polyclonal and monoclonal antibodies. We can use theantibodies to study the proteins interacted with TET2and detected themutated protein of TET2by Western blot in the future experiments.We clone and express the fusion protein GST-TET2C and purify it bypreparative electrophoresis. As the antigen GST-TET2C, experimentanimals are immunized New Zealand white rabbits and BALB/c mice to preparethe polyclonal and monoclonal antibodies. Use two kinds of antibodies inthe experiment can reduce the error and make the results more credible.