Cloning And Expression Of The X Gene, 11kd Gene And Preliminary Research Their Function Of The Human Parvovirus B19

Human parvovirus B19 (B19) is one member of the parvoviridae family (the other one is human bocarvirus) known to cause disease in human, and this virus is widely distributed all over the world. The report from our lab has shown that the rate of infection of B19 is about 4.50% in the normal population in Wuhan, but the rate of the pregnant women infected by the B19 virus is 8.50%. B19 infection shows a very strong erythroid tropism and destroys erythroid progenitor cells drastically, then leads to most of the disease outcomes associated with B19 infection. The disease resulting from B19 is called as fifth disease, which is very popular in children, arthropathy, particularly in women, transient aplastic crisis in individuals with a high red cell turnover rate, pure red cell aplasia in immunocompromised patients, and hydrops fetails following infection of pregnant women. Although B19 infection shows a very strong erythroid tropism, some studies show that with the help of the adenovirus, B19 can replicate in nonpermissive cells, such as 293T, and produce the infectious virus. Recently, Wang reported that B19 in the patients infected by the HCV sustained in the lung tissue for a long time, and it is unclear that whether it has effects on HCV replication.In the thesis, we finished two parts of experiments:First, we constructed the prokaryotic expression vector pET-28a-x which can express the fused His-X protein after it was induced with IPTG. On the other hand, we cloned the x gene into the pEGFP-Nlin order to observe the location of the fused x-GFP protein in the host cells. The results demonstrated that the fused protein was located in both nucleus and cytoplasm of the transfected cells.Second, we constructed the pET-49b-11 and pcDNA3.1-Grb2, and the expressin of proteins was assayed by western blot. This is helpful for GST PULL DOWN experiment. In order to study the function of the riched-proline region of the llkd protein during the B19 replication, the key amino acids of the llkd protein were mutanted according to the method of site-specific mutagenesis, and the the mutant gene was expressed successfully in E. coli.