关键词: DNA甲基转移酶3B, 异构体, 胚肾细胞293A, 增殖

Abstract: Objective To construct six kinds eukaryotic expression vectors of human DNA methyltransferase 3B isofroms and overexpress these recombinant DNMT3B isoforms, study its biological effects on human embryonic kidney (HEK) cell line 293A.Methods The human DNMT3B1 and DNMT3B2 were amplified from HCT116 using high fidelity Taq PCR. Others isoforms were amplified from DNMT3B1 or DNMT3B2. Then the PCR product were linked into eukaryotic expression vector pCMV-2B. These recombination plasmids were identified by double enzyme digestion with BamHⅠand ECORⅠand by sequence. These recombination plasmids were transfected into 293A cells when the sequencing results of recombination plasmids were correct. The G418 was used to get the stable cell line which express DNMT3B isofoms respectively. MTT assay was used to detect the proliferation of cells which stable express the DNMT3B isoform. Results six kinds of Human DNMT3B CDS sequence were cloned. The stable 293A cells overexpressing human DNMT3B isoforms were established. Overexpression DNMT3B3、DNMT3B4 and DNMT3B7 can inhibit the growth of 293A cells. Conclusion Eukaryotic expression vectors encoding human DNMT3B isoforms and corresponding stable overexpression cells were constructed. Different human DNMT3B isoforms protein may have different effects on 293A cell growth.

Key words: DNA methyltransferase 3B, isoform, human embryonic kindey cell line, proliferation