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>

Expression of the recombinant fusion protein CP15-23 of Cryptosporidium parvum and its protective test.

The CP15 and CP23 surface proteins on the sporozoite of Cryptosporidium parvum are major protective antigens. The recombinant plasmid pET28-15-23 was constructed based on the plasmids pMD18-T-15 and pMD18-T-23 with two pairs of specific primers using DNA recombinant technique. In the primers, a synthetic linker sequence encoding a peptide (G-G-S) was designed. After identification, the recombinant plasmids were transformed to component cells of Escherichia coli BL21 (DE3). The positive strain containing the recombinant plasmid could express a specific fusion protein (CP15-23, MW approximately 25 kDa) induced by IPTG. The fusion protein could be recognized by the positive serum of mice infected with Cryptosporidium parvum oocysts specifically. The BALB/c mice were immunized with 80 microg of CP15-23 protein 4 times at 2 week intervals. The mice produced specific antibodies that responded to the lysate of Cryptosporidium parvum oocysts and could prevent Cryptosporidium parvum infection. The results indicated that the recombinant fusion protein CP15-23 would be used as a candidate antigen to prevent cryptosporidiosis.