Construction Of Recombinant Adenoviruses Expressing Two Subtypes Of Ndrg2 And The Suppressive Effect Of Ndrg2 On The Glioma Cell Lines

Glioma is the most malignant tumor in the nervous system.It has high incidence, mutilation and mortality.The therapeutic efficacy is not satisfactory now. The activation of the oncogene and(or) the inactivation of the anti-oncogene are the most primary molecule event.Then the gene therapy has a good future in glioma.It is the first choice to transfer the anti-oncogene into patient.Ndrg2 gene was discovered and cloned from normal brain tissure of human with the differential hybridization by professor Deng Yanchun,which has been adopted in Genbank with the number of AF159092.Ndrg2 is the differential expression gene in the tissue of different tumors and corresponding normal tissue.Ndrg2 has the high expression in the brain,skeletal muscle and salivary;ndrg2 has the moderate expression in the liver, epithelium tissure of the gastrointestinal and epithelium tissure of the lung.;ndrg2 has the low expression in the thymus gland,bone marrow and testicle.We discovered that the expression of ndrg2 step down in the tissue and cell line of glioma. Simultaneously, ndrg2 is suppressor of the cell proliferation when it has overexpression in the glioma .The G1 phrase of the cell in the glioma is appeared block.Ndrg2 has the two subtype (ndrg2S and ndrg2L). ndrg2S is 2024bp in length, encoding about 357 amino acid;ndrg2L is 2066bp in length, encoding about 371 amino acid.The preliminary studies were concentrate in the ndrg2S with the immunohistochemisty,RT-PCR and gene transfection.Since the difference is 42bp of nucleotide (14 amino acid) and the high concordance of the nucleotide sequence and the similarity of the protein antigen ,it is very difficult to distinguish the function of ndrg2L an ndrg2S.The function of antitumor by ndrg2S was proved in the gene transfection.No person to prove the function of antitumor in the ndrg2L.Adenovirus is the nonencapsulated DNA virus having double strands and linearity.It is generally distributed in the nature. its pathogenicity is low .It is safe since it can not induce the cancer and unconformity in the cell geneome.The adenvirus has low genetoxic ,extensive hostrange,high transfection efficient,esay preparation and purification.The adenovirus vector was used in the gene expression ,transfusion and therapy.Now,there is recombinant adenovirus expressing ndrg2S having been constructed.For further investigation on the function of ndrg2 and the mechanism of antitumor,to identify the role and mechanism of the ndrg2L in the glioma.,to explore the pathogenesy and new therapy strategy of the glioma,we are going to construct the recombinant adenoviruses expressing the two subtypes of the ndrg2 .The recombinant adenoviruses are going to be used to detect the index of cytobiology in the human glioma cell lines for the difference in the two subtypes of the ndrg2.First,Invitrogen's ViraPowerTM Adenoviral Expression System was used to construct recombinant adenovirus vectors. The two subtypes of ndrg2 gene was cloned into the entry vector pENTR2B to obtain the entry clones, pENTR2B-ndrg2L and pENTR2B-ndrg2S. After they were identified by double enzymes digestion and PCR analysis, the entry clones were recombinanted with the expression vector pAd-CMV/V5-DEST using Invitrogen's LR Clonase? II Enzyme Mix to obtain the expression clones, pAd-CMV/V5-DEST-ndrg2L and pAd-CMV/V5-DEST- ndrg2S identified by PCR method. After linearized by PacI, the expression clones were transfected into HEK293A cells for adenovirus packaging and amplification. Two adenoviruses were designated as Ad-ndrg2L and Ad-ndrg2S. Adenovirus titers were determined by TCID50 method and ndrg2 protein expression was detected by western blotting.The result is that enzyme digestion and PCR analysis proved the pAd-CMV/V5-DEST-ndrg2L and pAd-CMV/V5-DEST-ndrg2S expression clones were successfully constructed .High-titer recombinant adenoviruses were acquired after being packaged in HEK293A.The human glioma cell lines infected by the recombinant adenoviruses of the two subtypes of the ndrg2 are detected using the flow cytometry, MTT assay and plate clone formation assay.It was observed that the cell percentages of G1 phrase infected by the recombinant adenoviruses of the two subtypes of the ndrg2 are more than the cell percentages of G1 phrase infected by the recombinant adenoviruses of the recombinant adenoviruses of the EGFP and the normal glioma cells.It was observed that the curve of cells of the glioma infected ndrg2 increased relatively slowly in MTT assay.But there are not significant difference in the ndrg2L and ndrg2S.The plate clone formation assay indicated that plate clone formation efficiency of the cell infected adenoviruses of ndrg2 was lower than the control groups. But there are not significant difference in the ndrg2L and ndrg2S in the plate clone formation assay.