The Construction And Expression Of Plasmids PEGFP-N1-ApoE ?2

Objective In order to survey the expression of ApoeE2 protein in Spague-Dawley(SD)mouse brain at different time points,we constructed pEGFP-NI-APOE e2 plasmid models and injected non-toxic pEGFP-N1-APOE s2 into SD mouse tricorn.We studied whether pEGFP-N1—APOE ?2 could enter the living mouse brain tissue and express ApoeE2 protein successfully.By means of recording the time and trend of ApoE2 protein expression,we hoped to provide experimental basis for pathogenesis of alzheimer disease(AD)and gene therapy.Methods To make basic group C of the 526 base pair in ApoeE s3 mutations into T and the 3 ' end to TGAterminator(beginning with ATG).Then we cloned pEGFP-N1 into carrier and constructed pEGFP-N1-APOE ?2 plasmid models.Next we extracted plasmid pEGFP-N1-APOE ?2 and pEGFP-N1 without endotoxin.We respectively maked pEGFP-N1-APOE ?2 and pEGFP-N1 mixed into EntransterTM-in vivo solution in proportion.We selected 84 healthy male SD mice of aged 3 months and weighted 30-35 grams.According to random number table we divided all the mice into two groups,A as case group and B as control group.Each group was set up observation points at 24 h,48 h,72 h,five days,7 days,10 days,14 days after pEGFP-N1-APOE ?2 injection through lateral ventricle puncture.(1)In order to confirm whether the direction and sequence of plasmid pEGFP-N1-APOE e2 is correct and reading frame was errorless?we detected rebuilded plasmid pEGFP-N1-APOE ?2 and sequenced the inserted parts.Then we conducted plasmid agarose gel electrophoresis to check the size of plasmid DNA.(2)According to the different time points we took the brain tissues and observed on biopsy by using fluorescent microscope.(3)Immunohistochemical experiment method was used to convert brain tissue staining.Under the 40 x 10 times light microscopy,we observed on the regions of periventricular.In the positive regions,five non-overlapping views were randomly selected.We counted the classifications and scores of positive cells'number using the microscope and ApoE2 immune response positive cells by applicating of multifunctional true color cell image analysis system.Then we averaged as the classifications and scores of positive cells' number at each time point.(4)To measure ApoE2 protein by using western blot method.Results 1.We got pEGFP-N1-APOE s2 plasmid by means of adopts fixed point mutation technology and conducted plasmid pEGFP-N1-APOE ?2 and pEGFP-N1 agarose gel electrophoresis tests.The size of itself PEGFP-N1 carriers was 4.7 kb,but the gotten size of plasmid pEGFP-Nl-APOE s2 was about 5.7kb by cloning ApoE s2 gene to pEGFP-N1 carriers.In order to confirm whether the direction and sequence of plasmid pEGFP-N1-APOE ?2 is correct and reading frame was errorless,we detected rebuilded plasmid pEGFP-Nl-APOE ?2 and sequenced the inserted parts.2.In vivo transfection process we respectively injected pEGFP-N1-APOE s2 and pEGFP-N1 into the lateral ventricle of mice.In the first 24 hours,the mice of case group and control group periventricular could be detected the expression of green fluorescent protein.3.Comparing with the control group,the expression of ApoE2 positive cells was more than the case group at the same time point.The number of the case group's ApoE2 positive cells was more and more as time goes on,but there is no obvious change for the number of the control group.Contrasting with cell grading scores of the A and B groups at the same time point,the results of statistical analysis was different.The difference was statistically significant(P<0.01).The immunohistochemical scores of positive cells were compared between the A and B groups at the same time point.The difference was statistically significant(P<0.01).According to the results of the immunohistochemical method within A group,we maked the statistical analysis.The results showed that the classification and score of immunohistochemical positive cells number in the first 24 hour were different from the 72th hour,the 5th day,the 7th day,the 10th day,the 14th day.4?Only in the plasmid pEGFP-Nl-ApoE E2 transfection group we found the expression of ApoE2 protein,the control group did not.Conclusions 1?We obtained relatively rare anthropogenic ApoE2 gene fragment by means of making fragments of ApoE3 gene point mutation.Then we connected the gene fragments to pEGFP-N1 carrier and constructed the recombinant pEGFP-N1-ApoE ?2 plasmids.2.With the help of EntransterTM-in vivo transfection reagent,we transferred the restructured pEGFP-N1-ApoE ?2 plasmids into brain tissues by using the method of lateral ventricle injection.The target protein got expressed.As times went by,the quantity of protein expression was increased.