Molecular cloning of a novel retroviral NP9 gene of human endogenous retrovirus and its viral protein expression / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the relationship between retroviruses and autoimmune diseases, to clone the novel retroviral NP9 gene from human endogenous retrovirus (HERV), and to construct its expression vector.</p><p><b>METHODS</b>The viral NP9 gene was amplified and cloned by RT-PCR and T-A clone techniques, and its sequence was determined with Perkin-Elmer 377 DNA Sequencer. The amplified viral NP9 gene was subcloned into the prokaryotic express vector pQE30. The recombinant plasmids were identified by restriction endonuclease digestion and sequencing. The recombinant pQE30-NP9 protein was expressed in M15 host cells under the IPTG induction and showed with SDS-PAGE,and the corresponding NP9 viral protein was identified with Western blot analysis.</p><p><b>RESULT</b>A specific band of 250 bp was amplified using RT-PCR from total RNA of peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) and confirmed as the NP9 gene via T-A clone and DNA sequencing analyses. SDS-PAGE profile showed a clear protein band with a relative molecular weight 9 kD in the IPTG-induced samples, which was confirmed as viral NP9 protein by Western blot analysis.</p><p><b>CONCLUSION</b>The NP9 gene has been successfully isolated and cloned from PBMCs of SLE patients and the corresponding NP9 viral protein expressed in prokaryotic expression vector.</p>