Gene Clone And Expression Of Human Parvovirus B19 NS1 Region Of Chinese Strain

Objective Parvovirus B19 is the only parvovirus having pathogenicing in humans. The virus exists widely and the infection rate of B19 is high in china. Transmission of infection occurs via the respiratory route,through blood-derived products administered parenterally, and from mother to fetus vertically. Manifestation of infection varies with the immunologic and hematologic status of the host, including asymptomatic infection and severe aplastic crisis. In healthy irnmunocornpetent children, B19 is the cause of erythema infectiosum, an innocuous rash illness. B19 infection is associated with an acute symmetric polyarthropathy that may mimic rheumatoid arthritis in adults.Infection in individuals with an underlying hemolytic disorder causes transient aplastic crisis. In the immunocopromised host, persistent B19 infection is manifested as pure red cell aplasia and chronic anemia. Likewise, the immature immune response of the fetus render it susceptible to infection, leading to fetal death in utero, hydropsfetalis, or development of congenital anemia. Pavovirus B19 encodes two viral protein (VP1 and VP2) and one major nonstructural protein (NS). NSI gene contains a well-conserved nucleoside triphosphate-binding motif, which is essential for a variety of biological functions. Cytotoxicity is abolished when single amino acid mutations are introduce in this domain. Ns is thought to possess site-specific DNA-binding, DNA-nicking, ATPase, transcriptional, and helicase activities, which may explain its cytotoxicity. It s variability is associate with it s pathogenicity. It was demonstrated that NSI cytotoxicity is closely related to apopotosis by a pathway involving caspase 3, whose activation may be a key event during NSI-induced cell death. The NS-specific antibody is suggested to be associated with an altered course of disease. Von Poblotzki et al cloned and expressed the full-length NS-1 protein in escherichia coli in 1995. Hicks established a Hela cell line to express NS by an Epstein-Barr virus vector, which have provided an opportunity to study the functional and immunologic properties of NSI. The study is designed to clone human parvovirus B19 NSI gene, construct the recombinant vector and express NSI protein.Methods NS 1 gene was amplified by PCR from xi'an-20 strain kept in our laborary and cloned into pGEM-T-easy vecor.lt was analysised and identified by sequencing. Then the correct NSI gene was subcloned into pQE30 vector. The plasmid pQE30-NSl was transformated into M15 competent.cells. After induced by IPTG for 4h, the expression of Mr 77kd fusion protein was confirmed by SDS-PAGE.Results (l)Compared with the sequence of Au strain, a base-pairs were found to be changed in XA20, C was substituted for T at nt2439. Accordingly the amino acid was changed from arginine to methionine. (2) Recombinant clonevector NS1-pGEM-T-easy and expression vector NS1-pQE30 were constructed and they were proved by restriction digestion. (3) The moleculor weight of the expressed fussion protein which was confirmed by 12% SDS-PAGE is 77kD.Conclusion (1) NSI were cloned and expressed in prokaryotic cell. NS1 can be expressed steadily in escherichia coli. For the production of full-size NS1, it was necessary to lower the growth temperature to 25 C to obtain high level of expression. Expression was induced by ImM-IPTG for at least 3h of culture. (2) The fusion protein of pavovirus B19 NS1 was firstly gotten in china. It can be used to study the biological character of B19 NS1 further and produce monoclonal antibody of NS1.