Construction and identification of recombinant adeno-associated virus expressing siRNA / 病毒学报

Adeno-associated virus (AAV) mediated RNA interference can be used to inhibit the expression of homologous genes in different mammalian cells. In this study, a transfer plasmid (pAAV-EGFP-H1) containing the H1 promoter and EGFP-expressing cassette was constructed based on the backbone of pAAV-MCS. Using calcium phosphate precipitation method, pAAV-EGFP-H1 was co-transfected into AAV-293 cells with helper plasmids and infective recombinant AAV was generated. EGFP gene was selected as the interfering target. EGFP gene was removed from pAAV-EGFP-H1 and a new transfer plasmid pAAV-H1 was constructed. Recombinant AAV-H1-shEGFP then infected 293 cells which was pre-transfected with plasmid pEGFP-N1. After 72 hours, the interference effect on EGFP expression was investigated by fluorescence microscope, fluorescence quantitative PCR and fluorescence activated cell sorting (FACS). All results showed that rAAV-H1-shEGFP could effectively reduce more than 60 percent of EGFP expression in 293 cells. The study demonstrates that a recombinant AAV transfer plasmid for RNAi is constructed, and the generated recombinant AAV can be used for further investigation on RNAi research.

The effect of HPV16E7 DNA vaccine transdermal delivery with microneedle array / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the effects of DNA vaccine transdermal delivery with microneedle array.</p><p><b>METHODS</b>The pcDNA3.1-HPV16E7 recombinant vector acting as gene vaccine was established. The infiltration quantity of pcDNA3.1-HPV16E7 getting across the microchannels generated by microneedle arrays in vitro was observed. 30 BALB/c mice were divided into 3 groups (experimental group, in vain plasmid group, negative control). Each group had 10 mice. Then immunized BALB/c mice with a dose of 200 microg with microneedle array every two weeks. The control groups did the same as that as the study groups. Two weeks after the third immunization, the serum and lymphocytes were separated to detect the functions of humoral immunity with indirect immunofluorescence test, while, the functions of cellular immunity with lymphocyte transformation test was also detected.</p><p><b>RESULTS</b>The DNA vaccine could easily get across the microchannels generated by microneedle arrays in vitro. Moreover, the course was permanent and the whole infiltration quantity was comparatively high, reaching 0.73819 mg/cm2 at the 30th hour. And among immunized BALB/c mouse, DNA vaccine transdermal delivery with microneedle array could induce specific antibodies. Lymphocyte transformation test showed that there was significant difference for the lymphocyte transformation rate between experiment (the average of lymphocyte transformation rate was 47.25%) and control group (the average of lymphocyte transformation rate was 30.00%) (chi2 = 12.903, P < 0.001). Also, the difference was found between in vain plasmid group (the average of lymphocyte transformation rate was 43.00%) and negative control(chi2 = 7.292, P = 0.007). While, no difference was observed in the experimental group and in vain plasmid group (chi2 = 0.817, P = 0.366).</p><p><b>CONCLUSION</b>The DNA vaccine combined administering with microneedle array might get across the microchannels generated by microneedle arrays in vitro and induce humoral and cellular immune response in vivo.</p>