The Base Excision Repair Functional Investigation Of The Mutant Dna Polymeraseβ In Human Gastric Cancer Tissue

BackgroundGastric cancer is one of the most popular malignant tumors.The morbility of Gastric cancer has achieved(5～40)/10 ten thousand,therefore it is great important to research etiopathogenisis for it's prevention and cure.Up to now,it has been discovered excessively and/or anormaly expression of the DNA polymerase beta in many human tumor,for instance,rectal cancer,prostatic carcinoma,esophageal carcinoma,gastric cancer,nasopharyngeal carcinoma and so on.It has a lot of complicated etiological factors for gastric cancer,and it must be follow DNA damage in the process of the gastric cancer'development.It is starded that the mutation rate of the DNA polymerase beta is 23.7%(9/38) in gastric cancer,the mutation rate of the DNA polymerase beta is 57.1%in the poorly differentiated gastric cancer,which surpass obviously the midrange and completely differentiated gastric cancer that is 4.2%,it is implied that it has the mutation of the DNA polymerase beta in gastric cancer,and it is related to the extent of the differentiation in gastric cancer. The DNA polymerase beta is a very important DNA repair enzyme,which distribute generally in the mammalian cell,and it's major function is to participate the DNA repair and supply mononucleotide gap in the progress of BER(Base Excision Repair),which contain short-patch repair(single mononucleotide) and/or long-patch-repair(several nucleotide incorporated),also it is one of the most important polymerase in the progress of BER.The DNA polymerase beta play a great significant part in the progress of vital movement due to it's homology with mammal cell and extremely conservatism in the evolution of structure,therefore it is named "house-guard enzyme"in the progress of organic evolution,and it's defect is concemed with some tumors.At present,many researchers have investigated systematically the mutations and expression states of DNA polymerase beta, however,it hasn't reported the study of the DNA polymerase beta's functions in our country.For this reason,it may provide original theory evidences to reveal molecule mechanisms that induce tumors by studing the functions of the DNA polymerase beta.ObjectiveTo detect the metergasis of the mutant DNA polymerase beta detected in human gastric cancer at the process of DNA reparation,and to investigate etiological factors to provide foundations for the tumor.Methods1.The construction and identification of the recombinant expression vectors pET28a(+)-polβ.Wide-tipe DNA polβand mutant DNA polβwere amplified and cloned into pGEM-T-polβplasmid.After PCR selecing,they were subcloned into pET28a(+) plasmid.The recombinant expressive vectors of DNA polβgene(pET28a(+)-polβ) were constructed and identified by PCR.2.The expression of the recombinant expression vectors pET28a(+)-polβby IPTG induced and identification To inoculate the BL21(DE3) bacterium which containing the recombinant expression vectors pET28a(+)-polβcultivated overnight in 5mlLB liquid medium,and oscillate at 37℃to achieve saturated concentration,then put the culture into 10mlLB liquid medium which containing the kanamycin,and cultivate 2h at 37℃.12.5μl IPTG was put into 10mlLB liquid medium that experimental groups to achieve it's concentration to lmmol/L,to follow cultivating 5h at 37℃.To crush cells by hypersound and determine the content of proteinum by ultraviolet spectr0photometry. Then make electrophoresis with 15%SDS-PAGE,and stain by coomassie brilliant blue,decolorizate by methyl hydrate-glacial acetic acid and observe the consequence. Eventually,scann the result by gel analysis system to analyze staining straps in the gel after SDS-PAGE.3.The purification of DNA polβproteinum by Nickel affinity chromatographTo purify the DNA polβproteinum according to demonstrations of the kit-Hislink ^TMProtein Purification Resin.4.The synthesis of DNA substrateDesigne three oligonucleotide strands P1,P2 and P3,after annealing they were synthesis DNA substrate which contain a basic deletion and a site which can be cut open by the HindⅢenzyme.5.The experiment of BER repairAccording to proportion diluting polymerase beta proteinum in order,it is easily to observe the results of repairation for DNA substrate which contain a basic deletion,and identify them by HindⅢenzyme.Results1.The recombinant expression vectors pET28a(+)-polβwere constructed successfully by PCR identification.It was confirmed that the insertion element was concordant completely together with the DNA polβgene sequence publicated.2.After the recombinant expression vectors pET28a(+)-polβtransf- orming into the Escherichia coli BL21(DE3),inducing the expression of exogenous gene by IPTG.It was displayed that the recombinant bacterium induced bright obviously exceed the bacterium which there was no induced where the proteinum strap of relative molecular mass was about 39KD by analysis of SDS-PAGE,which was conformit with the magnitude of expectant fusion protein.3.It was discovered that the wide-tipe DNA polβand mutant DNA polβall can display the proteinum straps where the relative molecular mass was about 39KD after purifing the DNA polβproteinum according to demonstrations of the kit-Hislink ^TMProtein Purification Resin.4.After annealing,the three oligonucleotide strands P1,P2 and P3 were synthesis 56bp DNA substrate which contain a basic deletion,and the result was conformit with the magnitude of expectant fragment.5.In the BER experiment,wide-tipe DNA polβ1 and mutant DNA polβ2 can repair the DNA substrate and cut open the DNA substrate to two short fragments that were 27bp and 28bp by the HindⅢenzyme when diluting proteinum at the proportion 1:4, however mutant DNA po1133 cann't or can partly repair the DNA substrate,and the DNA substrate still or partly to remain 56bp.In addition,wide-tipe DNA polβ1 can repair the DNA substrate and cut open the DNA substrate to two short fragment that were 27bp and 28bp by the HindⅢenzyme,mutant DNA polβ2 and mutant DNA polβ3 cann't or can partly repair the DNA substrate,and the DNA substrate still or partly to remain 56bp when diluting proteinum at the proportion 1:8 and 1:16.ConclusionsTo contrast the Wide-tipe DNA polymerase beta,the base excision repair functional of the mutant DNA polymerase beta detected in human gastric cancer was weakened obviously.