Construction of a recombinant lentiviral vector of p38 MAPK and establishment of a human prostatic carcinoma cell line stably expressing p38 MAPK / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant lentiviral vector for p38 MAPK and establish a human prostatic carcinoma cell line that stably expresses p38 MAPK.</p><p><b>METHODS</b>EGFP/p38 fusion gene was subcloned into the lentiviral vector pTYF- EF1α-IRES-EGFP. The recombinant lentiviral vector pTYF-EF1α-EGFP/p38 was indentified by restriction enzyme digestion, and packaged in HEK 293T cells using lipofectamintm2000 with the packaging plasmid psPAX2 and envelope plasmid pMD2.G. The viral titer was tested according to the expression level of GFP. The resulting recombinant lentiviral vector was transduced into human prostatic carcinoma DU145 cells, and stably transduced cells were selected by limiting dilution analysis. The intracellular expression level of total p38 was detected by Western blotting and the cell growth curve was drawn.</p><p><b>RESULTS</b>DNA restriction enzyme digestion demonstrated that the recombinant lentiviral vector of the fusion gene EGFP/p38 (pTYF-EF1α-EGFP/p38) was constructed successfully. The recombinant lentiviral vector was packaged in 293T with a viral titer of 4.7×10(6) TU/ml. A stable cell line, EGFP/p38-DU145, was established, which stably expressed exogenous EGFP/p38 MAPK fusion protein as detected by Western blotting and showed a lowered growth rate compared to the control cells.</p><p><b>CONCLUSION</b>We have successfully constructed a recombinant lentiviral vector of the fusion gene EGFP/p38 and established a stable cell line EGFP/p38-DU145. Overexpression of p38 has a significant inhibitory effect on the proliferation of DU145 cells in vitro.</p>

A comparative analysis of the methods for titering adenoviruses / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare different methods commonly used for titering adenovirus and analyze the advantages and limitations of each method.</p><p><b>METHODS</b>Four recombined adenoviruses (Ad-G-AT2R-EGFP, Ad-CMV-EGFP, Ad-mif-shRNA-EGFP and Ad-CBA-GFP) were amplified and purified, and each was titered by optical absorbance, real-time PCR, green fluorescent protein (GFP)-labeled method, immunoassay, and cytopathic effect (CPE). The results were then comparatively analyzed.</p><p><b>RESULTS</b>No significant difference was found in the titer amounts derived from GFP-labeled method, immunoassay, and cytopathic effect method (P>0.1). A positive correlation was noted in the titer amounts determined by real-time PCR and immunoassay (r=0.965), even though the value (vg/ml) obtained by real-time PCR was 10 times higher than that by immunoassay (ifu/ml).</p><p><b>CONCLUSION</b>GFP-labeled method and immunoassay allow rapid determination of the adenoviral titer. Real-time PCR can not directly determine the real infectious titer of the adenovirus, but the result is well correlated to that of immunoassay and reflects, though indirectly, the actual infectious titer of adenovirus. Considering the procedural convenience and shorter time consumption, real-time PCR is still a practical method for adenoviral titration.</p>

Development of the recombinant SAG1 antigen of Toxoplasma gondii by high-density fermentation and identification of its immunoreactivity / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a technology for production of recombinant SAG1 of Toxoplasma gondii (T.g) in batches.</p><p><b>METHODS</b>The rSAG1 of T.g was expressed in E.coli by high-density fermentation and purified by Sephadex G-75 column chromatography after Ni-NTA agarose at native condition. The activity of rSAG1 and its efficacy in T.g diagnosis were identified by Western blotting and ELISA, respectively.</p><p><b>RESULTS</b>The optical density (OD) of the bacteria reached 20.21 after induction, and 300 g bacteria were harvested from 11.5 L broth. The rSAG1 was highly expressed in E.coli as a fusion protein, accounting for about 25.82% of the total bacterial protein. The purity of rSAG1 reached 98.54% after purification by Ni-NTA combined with Sephadex G-75 column chromatography. Western blotting revealed a distinct band reacting with the sera of rabbits vaccinated by T.g. Twenty-four of the 25 sera of mice infected with T.g and 36 of the 38 sera of human subjects with IgG antibody against T.g were detected by rSAG1-ELISA.</p><p><b>CONCLUSION</b>A large-scale production of immunoreactive SAG1 of T.g is developed by high-density fermentation and purification with Ni-NTA combined with Sephadex G-75 column chromatography.</p>

The Procaryotic Expression, Purification and Activity Analysis of VIP-sTNFRII / 中国生物工程杂志

A prokaryotic expression plasmid containing VIP (vasoactive intestinal peptide) and sTNFRII(soluble tumor necrosis factor receptor II ) genes was constructed. The sTNFRII was cloned by PCR by using special primers which contained VIP gene ORF and a linker in its forward primer. The amplified fragment was inserted into the expression vector pET32a between BamHI and Hind III restriction sites. Transformed E.coli DH5 by pET32a-VIP- sTNFRIIexpressed the fusion protein. After being identified, the protein was purified by ion exchange chromatography and by hydrophobic interaction chromatography. The reconstructed protein showed high bio-activity and could be applied for further use.