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Objective@#To construct a Tat-dependent MazF expression vector pcDNA3.1-GFP-HA-MazF-U3TAR.@*Methods@#HIV-1 U3TAR and MazF gene were amplified from pCR2.1-U3TAR and pET28a-MazF, respectively. Two gene fragments were linked together to form U3TAR-MazF by an overlapping PCR, and then cloned into pMD18T. An N-terminal HA tag was added to MazF to form U3TAR-MazF-HA. After double enzyme digestion using EcoR I and Hind Ⅲ, U3TAR-MazF-HA was reversely inserted into pcDNA 3.1 to form pcDNA3.1-HA-MazF-U3TAR. Then, a fluorescent reporter gene GFP was inserted into the downstream of U3TAR to form pcDNA3.1-GFP-HA-MazF-U3TAR.@*Results@#The co-transfection experiments with pcDNA3.1-tat-flag showed that pcDNA3.1-GFP-HA-MazF-U3TAR is Tat dependent. MazF was expressed only under the presence of Tat. In addition, compared with the cells transfected with pcDNA3.1-GFP-HA-MazF-U3TAR, less green fluorescent signal was observed in the cells co-transfected with pcDNA3.1-GFP-HA-MazF-U3TAR and pcDNA3.1-tat-flag, indicating that expressed MazF down-regulated the expression of GFP.@*Conclusions@#The expression vector pcDNA3.1-GFP-HA-MazF-U3TAR will provide an important tool for the development of MazF-based AIDS gene therapy strategies.
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Objective To investigate the antimicrobial susceptibility of Pseudomonas aeruginosa (P. aeruginosa) isolated from Zhenjiang area to 13 routinely used antibiotics and identify the structure and dissemination of class Ⅰ integron. Methods K-B test was used to determine the resistant rate of 71 strains of P. aeruginosa. DNA template was extracted by boiling method, PCR method was utilized to detect class Ⅰintegron, and subsequently gene cassettes were analyzed by sequencing. Results The resistant rates to 13 routinely used antibiotics were quite different from 18. 3 to 77.5% among 71 strains of P. aeruginosa. The prevalence of class Ⅰ integron was 38%. These integrons include 5 gene cassettes ( aadB, aac (6) - Ⅱ , PSE-Ⅰ , dfrA17 and aadAS), in which dfrA17 and aadA5 gene cassette were frequently found. Comparing with the negative strains of integron, the positive strains of integron has obviously higher resistance to ten the antibiotics including piporacillin, piperacillin-tazobactam, ceftriaxone, cefepime, ceftazidime, gentamicin,amikacin, tobmmycin, levofloxacin, and ciprofloxacin. Conclusions The resistant rates of P. aeruginosa to 13 drugs were different, and the resistant rates of integron positive strains were obviously higher than integron negative strains, which indicates that integron may play an important role in multidrug reisistance of P. aeruginoosa.
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Objective:To investigate if a plant-derived polysaccharide sulfate(M33A) binding to HIV1 gp120 may induce the exposure of neutralization epitopes of gp120,and if M33A-bound HIV-1ⅢB antigens may be used as a AIDS vaccine for inducing neutralizing antibodies against HIV-1.Methods:Whole-inactivated M33A-bound HIV-1ⅢB antigens were prepared and used to immunize mice after mixing with FCA or FIA.The titers of anti-HIV-1 IgG antibodies in immunized mouse plasma were detected by ELISA,and the HIV-1 neutralization by those plasma was detected by the improved microtiter neutralization assay.Results:M33A-bound HIV-1 antigens induced higher titers of anti-HIV-1 IgG antibodies(group C:1.5?10~6;group D:1.5?10~6) than HIV-1 antigens alone(4.9?10~5),and female mice produced 3 times higher titers of anti-HIV-1 IgG antibodies than male mice after immunized with various HIV-1 antigens.All three immunization schemes did not induce the production of anti-HIV-1 neutralizing antibodies.Conclusion:M33A binding does not induce gp120 to expose neutralization epitopes.However,M33A may improve the level of mice immune responses to HIV-1 antigens,suggesting M33A may enhance immune response to HIV-1 antigens.