Comparison of three methods in construction fusion gene of influenza A virus Nucleoprotein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a best method of constructing influenza NP fusion gene containing enhanced green fluorescent protein (EGFP).</p><p><b>METHODS</b>The full-length NP gene of influenza A was amplified by RT-PCR and was inserted into an eukaryotic expression vector pEGFP-N1 in order to construct a fusion gene of pEGFP-N1-NP using three different methods. Method one, NP gene containing restriction endonucleases and pEGFP-N1 were both digested using the same restrict enzymes and ligated, yielding the fusion gene of pEGFP-N1-NP. Method two, NP gene was cloned into pMD19-T Vector to construct a plasmid of pMD19-T-NP. The pMD19-T-NP cloned into pEGFP-N1 to construct the fusion gene of pEGFP-N1-NP; Method three, NP gene containing restriction endonucleases was cloned into pMD19-T Simple Vector to construct a plasmid of pMD19-T-NP. The pMD19-T-NP cloned into pEGFP-N1 to construct the fusion gene of pEGFP-N1-NP.</p><p><b>RESULTS</b>The fusion gene of recombinant eukaryotic expression vector pEGFP-N1-NP was successfully constructed by using method three.</p><p><b>CONCLUSIONS</b>The full-length NP gene is obtained and its fusion gene of recombinant eukaryotic expression plasmid is successfully constructed. This study provides foundation for further understanding the biological function of NP protein and the mechanism of diseases induced by influenza A virus.</p>

Screening of differentially expressed genes in the hypothalamus of a rat neuropathic pain model following sciatic nerve injury / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Neuropathic pain is induced by injury or disease of the nervous system. Most studies have so far focused only on a few known molecules and signaling pathways among neurons. However, all signal transmissions involved in neuropathic pain appear to be an integral system at different molecular levels. This study was designed to screen the differentially expressed genes of the hypothalamus in chronic constriction injury (CCI) rats and analyze their functions in developing neuropathic pain.</p><p><b>METHODS</b>Ten adult female Sprague-Dawley rats ((200 +/- 10) g) were used in experimental group and sham group (n = 5 in each group). Mechanical allodynia tests were performed to ensure that the CCI rat model was constructed successfully. Total hypothalamus RNAs were isolated from each group. Forward suppression subtractive hybridization (SSH) library of rat hypothalamus was constructed and up-regulated cDNA clones at neuropathic pain states were obtained via suppressed subtractive hybridization technique and the functions of these genes were analyzed bioinformatically.</p><p><b>RESULTS</b>Mechanical allodynia tests showed that the experimental rats had a significantly reduced mechanical allodynia threshold 3 to 13 days after CCI vs sham surgery rats (P < 0.01), indicating that the model was successful. Forward SSH library of the rat hypothalamus was constructed successfully and 26 over-expressed expression sequence tags (ESTs) were obtained from these up-regulated cDNA clones.</p><p><b>CONCLUSION</b>Twenty-six up-regulated genes, involved in the regulation of cell cycle and apoptosis, signal transduction, and neuroprotection, may play key roles in decreasing mechanical withdraw thresholds in CCI rats, which implicates a multidimensional and integrated molecular mechanism at gene level in developing neuropathic pain with the supraspinal contributions.</p>