Construction Of Prokaryotic Expression Plasmid With Two Subgroups Of Human Ndrg2 Gene And Expression And Purification And Anti-tumor

Our group discovered the ndrg2 gene(N-myc downstream regulated gene 2)and cloned it from normal human whole brain cDNA library in 1999,which has been adopted in Genbank with the number of AF159092.This gene locates on human chromosome 14q1.11-11.2.There are two subtypes about ndrg2,encoding about 43kDa protein with 357 amino acid and 371 amino acid respectively.The ndrg2 gene is one member of ndrg gene family,which consists of 4 members:ndrg1,ndrg2,ndrg3 and ndrg4.This gene family has high similarity in nucleotide or amino acid sequence from inferior creature to advanced creature.Those indicate that the ndrg gene family plays an important role in life of cells.Our preliminary studies showed that there were higher expression of ndrg2 mRNA in brain,salivary gland,skeletal muscle,lower expression of ndrg2 in bone marrow,testis,peripherial blood and placenta and none expression in bone marrow cells(BMC)of leukaemia patients,colorectal cancer tissue,lung cancer tissue,liver cancer tissue and some tumor cell lines.In addition,the ndrg2 gene could inhibit the proliferation of many tumor cells,such as glioma cell line BT325.All above suggested that gene might be a new candidate tumor suppressor gene(TSG).However,the mechanism on suppressive effect of ndrg2 isn't clear.First, we constructed the prokaryotic recombinant vectors for two subgroups of human ndrg2 gene, and identified their expression and purification. The coding sequence of two subgroups of human ndrg2 were amplified with specific primers and cloned into the 6×Histidine (6×His)fusion expression vectors pPROEX-HTb. Then the recombinant plasmids were transformed into E.coli BL21 and the expressions of 6×His fusion protein were induced by adding isopropylthiogalactoside (IPTG). Then fusion proteins were purified by Protino Ni-TED Resin. Restriction digestion of the constructed recombinant plasmid revealed the existence of gene segments about 1100bp in length, which were in accordance with what had been expected and the results of sequencing showed that the coding sequence of two subgroup of human ndrg2 gene were correct. Two protein bands with the molecular weight of about 45kd were induced by IPTG in the recombinant plasmids. Western blot assay revealed that the 6×His fusion protein could be specifically recognized by ndrg2 antibody.And by Protino Ni-TED Resin,the fusion proteins were purified successfully.By the test, the coding sequence of two subgroups of human ndrg2 gene have been successfully cloned into the 6×his fusion expression vectors pPROEX-HTb and efficiently expressed and purified, which lay the foundation for study on biological activity and suppressing tumor.The second, we detected the inhibition action of ndrg2-357 protein and ndrg2-371 protein on tumor cell. The fusion protein of ndrg2-357 protein and ndrg2-371 protein were induced to express and purified by Protino Ni-TED Resin.The protein purified were concentrated by ultrafiltration centrifuge tube and the balanced solution were replaced. The inhibition action of ndrg2-357 protein and ndrg2-371 protein on tumor cell of U251,BT325 and A549 were observed by in vitro tests.And the inhibition action of ndrg2-357 protein and ndrg2-371 protein on tumor cell were compared.The tests showed us that the inhibition action of ndrg2-357 protein and ndrg2-371 protein on tumor cells were obvious, but there were no significant difference between the ndrg2-357 protein and ndrg2-371 protein.