Establishment Of GFAP Transgenic Mice Expressing The P24 And P40 Protein Of Borna Disease Virus In Astrocytes Specificly

Background and Objective:Depression is a kind of mental disease manifested by continuing depressive, lacking of interest and fun. Its has become the world's fourth largest burden disease and with high incidence, recurrence rate, morbidity and rate of suicide. Research on the etiology of depression involved biological factors, psychological factors, social factors, and they have varying degrees of impact on the incidence and development of the disease . The role of Viruse as a common pathogenic factor in the pathogenesis of depression and its mechanism are worth concerning and researching. BDV is a highly neurotropic, single negative-stranded RNA virus, it's can infectes many species of animals in the the central nervous system, especially in the hippocampus of the limbic system, causing the host behavioral disorders such as anxiety, attacks, over-excited, abnormal Game behavior and cognitive deficits, these symptoms similar to human schizophrenia, affective disorder and autism and other diseases. The epidemiological study in many countries and regions suggested that BDV infection is related to mental illness such as depression and schizophrenia.P24 and p40 protein are two virus structural and major pathogenic proteins encoded by BDV ORFâ… and ORFâ…¡and they both highly expressed in infected cells and tissues. The loss and dysfunction of glial cells especially the astrocytes are found in major depression and bipolar disorder instead of neuronal degeneration in neurodegenerative diseases, suggesting that depression disease are very closely related with glial cells.Therefore, this research is aimed to establish GFAP transgenic mice expressing both the p24 and p40 protein of BDV in astrocytes specificly.The following work is assessment of depression behavior and finally establish a new model of depression-BDV infection induced depression model. Then it will facilitate further research on the cellular and molecular mechanism in depressoin and provide evidence for the hypothesis of BDV infectious agent of depression.Methods:1.P24 and p40 were amplified and cloned from the plasmid of pQE30-p24 and pQE30-p40 .2.P24 and p40 were connected to the pMD18-T vector3.PMD18T-p24, pMD18T-p40 and pCMVie-GFAP-Venus were digested by EcoRI and NheI respectively,The gel extraction kit was used to recovery the p24, p40 gene and pCMVie-GFAP vector fragment for the ligation to prepared pCMVie-GFAP-p24 and pCMVie-GFAP-p40 plas- mid.4.The AseI, NotI, and ClaI was used for the third digestion,the largest fragment obtained by electrophoresis is the injection fragment (including CMVie, GFAP, p24 or p40 gene, BGH PolyA four components), cutting the strip with the Whatman DNA recovery systems Purification.5.The fertilized pronuclei of fertilized eggs was transplanted into transgenic mice to leave tubal pregnancy, birth and development mice.6.Detection of p24/p40 in G1 G0 generation mice.Results:The p24 and p40 gene were cloned and amplified in correct size from the pQE30-p24 and pQE30-p40 plasmid and connected to the pMD18-T vector to form pMD18-T-p24 and pMD18-T-p40 vector and Sequenced correctly.The pCMVie-GFAP-p24 and pCMVie-GFAP-p40 plasmidsequencing showed no abnormalities were prepared by using double-digested and connections. The largest fragment is the fragment for injection after purification from three digestion products of the three bands seen in electrophoresis. The purity of p24 is 55ng/ul and p40 is 26ng/ul; The final product was used to establish 144 mouse,PCR test results were positive in 31 mouse,two-gene positive Rate was 18.8%. In G1 mice ,there were 48 mice show gene-positive and the two-gene positive rate is 20.60%.Therefore, this research is aimed to establish GFAP transgenic mice expressing both the p24 and p40 protein of BDV in astrocytes specificly.The following work is assessment of depression behavior and finally establish a new model of depression-BDV infection induced depression model.Then it will facilitate further research on the cellular and molecular mechanism in depressoin and provide evidence for the hypothesis of BDV infectious agent of depression.Conclusion:This research successfully established a GFAP transgenic mice expressing both the p24 and p40 protein of Borna disease virus in astrocytes specificly.It supplied a usefull tool to the following assessment of depression, further research for cellular and molecular mechanisms of depression and proteomics research.