Establishment Of Transgenic Mouse Model Of Human Cytomegalovirus Ie2 Gene And Its Mechanism Research In Immunosuppression

ObjectInfection with human cytomegalovirus causes severe diseases in congenitally infected neonates and immunocompromised individuals.It is not completely clear that whether the occurrence of these diseases is caused by the effect of viral protein molecules on the immune system.There are no vaccines and drugs that can effectively prevent and treat HCMV.The human cytomegalovirus IE86 protein is ecoded by the ie2 gene,which not only regulates the viral own genes but also regulates the transactivation of host genes.It plays an important role in the replication and pathogenicity of human cytomegalovirus.Because human cytomegalovirus has a strict species specificity and infects humans as the only host,no animal model can be used to study the effect of HCMV IE86 protein on organisms.Therefore,we broke the species specificity of HCMV and constructed ie2transgenic mice to study the effects of IE86 protein on the immune system of mice.Methods1.By constructing a pAV.Ex1d-CMV-IE86-IRES/eGFP vector containing a cDNA fragment of the ie2 gene,C57 mouse fertilized eggs and embryos were transplanted to obtain the first generation ie2 mouse.The PCR method was used to identify the transgenic mice.Using the fluorescent quantitative PCR method,we established the standard curve to detect the copy number of the founder mice.Some mice of F1 were sacrificed and their organs such as kidney,heart,muscle,brain,spleen and fat were used for further study.RNA was extracted to detect the transcription level of ie2 gene.Immunohistochemistry was used to identify the expression of IE86 protein.2.Assess the immune status of ie2 transgenic mice:The proportion of peripheral blood CD4~+,CD25~+and CD4~+CD25~+T cell to lymphocytes were measured.The mouse spleen was taken and the expression of cytokines such as spleen index and IL-2,IL-6,IFN-γ,Foxp3,TGF-β1 in positive transgenic mice and negative mice was detected.Results1.The pAV.ExBi-CMV-IE2-IRES-eGFP expression vector was successfully constructed and three positive transgenic mice were obtained by microinjection and other techniques.A PCR identification method for transgenic mice was established.At the same time,the copy number of F0 were 30.77±0.10,6.89±0.98 and 39.53±1.4,respectively.Quantitative statistics were performed on mice:59 F0 and 3 positive mice were obtained,with a positive rate of 5.1%.57 mice were obtained from the first generation(F1)and there were 21 positive mice and the positive rate was 36.8%.51 mice were obtained from the second generation(F2),22 positive mice,the positive rate was 43.1%;29 offspring of the third generation(F3),18 positive mice,the positive rate of 62.1%.The expression level of ie2 mRNA in muscle,heart muscle and kidney tissue of F1 transgenic mice was higher,and the expression levels in brain,spleen and adipose tissue were relatively low.Immunohistochemistry showed that IE86 protein was expressed in kidney,heart,muscle,brain and spleen of positive transgenic mice and located in the nucleus.2.The results of flow cytometry showed that the CD4~+CD25~+Treg cells in peripheral blood of positive transgenic mice were significantly increased,(5.58±0.40)%and(4.82±0.59)%,respectively.The proportion of CD4~+and CD25~+in the positive mice was also significantly increased,with CD4~+being(38.83±4.73)%and(27.58±5.14)%,and CD25~+being(5.99±0.38)%and(5.25±0.26)%,respectively.Positive transgenic mice had less spleen weight and spleen index than the negative mice.Detection of cytokines in the spleen showed that immunosuppressive cytokines Foxp3,TGF-β1 were higher in positive mice than in negative mice,and IFN-γwas lower than negative mice.There was no significant difference between IL-2 and IL-6.Conclusions1.The ie2 transgenic mouse model expressing IE86 protein was successfully constructed.2.The detection of copy number of F0 show that we obtained different copy number transgenic lines.3.The preliminary study on the immune status of mice showed that the immunosuppressive index was highly expressed in the transgenic mice,and the mice may be in an immunosuppressive state.