1. Prevalence And Molecular Typing Of Antiseptic Genes QACA/B Among Staphylococcus Aureus Strains 2. Direct Molecular Cloning To Construct Recombinant Adhu5-040-fsp27 And Its Amplification And Purification

PRAT ONE PREVALENCE AND MOLECULAR TYPING OF ANTISEPTIC GENES QACA/B AMONG STAPHYLOCOCCUS AUREUS STRAINSObective:To detect the distribution and epidemiological features of antisepitic gene qacA/B of staphylococcous aureus from Jan 2004 to Jun 2009 in our hospital, which will be helpful for rational administration on antisepitics.Methods: PCR was employed to detect the mecA gene and the qacA /B gene in the isolates. Also, pulse-field gel electrophoresis (PFGE) typing, Staphylococcus protein A (spa) typing, staphylococcal chromosomal cassette (SCC) mec typing and antimicrobial-resistant phenotypic typing were used to analyse on the homology of these qacA/B positive strains. Meanwhile, the clinical information of the patients was collected to understand the epidemic characteristics of qacA/B-carried staphylococcous aureus.Results: The frequency of carriage of the qacA/B and mecA was 9.1% (12/82) and 46.6% (82/176), respectively. But the incidence of qacA/B genes in methicillin-resistant S. aureus (MRSA) was significantly higher than that in methicillin-sensitive S. aureus (MSSA) (14.6% versus 4.3%). These qacA/B positive strains were classified as 8 spa types and the main classes were t037 and t042. The PFGE typing divided these strains as A to G types,and the A₁,B,G types were actually the same clone. Furthermore, the SCCmec gene typing confirmed these mecA-carried strains as SCCmecâ…¢.Conclusion: These strains were sampled mainly from patients in surgical wards. And neurosurgical ICU or ward could be considered as a source of MRSA carrying qacA/B genes. spa type t037 may beis likely to be an epidemiological clone. Despite of low distribution rate of qacA/B in S. aureus in this area, monitoring is still warranted due to the significant higher frequency in MASA strains. PRAT TWO DIRECT MOLECULAR CLONING TO CONSTRUCT RECOMBINANT ADHU5-040-FSP27AND ITS AMPLIFICATION AND PURIFICATIONMouse fsp27 gene belongs to cell death-inducing DNA fragmentation factor-α-like effector (CIDE)family member. Previous studies have found that it possibly is a key factor on the pathway of triglyceride metabolism. Thus it may be close associated with the initiation and development of nonalcoholic fatty liver disease (NAFLD). How to introduce this gene into cells in vitro or in vivo is a crucial step for detailed investigation.Objective: the aim of this study was to construct recombinant AdHu5-040-fsp27 (cds) using direct molecular cloning method and obtain high titer and purity of viruses for laboratory and clinical trials.Methods: the targeted gene- mfsp27 was first cloned into pShuttle and located between two rare REs (I-ceuI and PI-sceI). After digestion, gel ligation and transformation, recombinants were selected and confirmed by specific REs cutting and sequencing. The candidate recombinant plasmid was then linearized and rescued in HEK293 cells. After infection and expansion, the virus crude stocking was collected and purified by modified cesium chloride gradient centrifugation method. The virus MOI was measured by series dilution of the stocking. Results: recombinant AdHu5-040-fsp27 (cds) was successfully constructed. The titer (O.D. measurement) was as high as to 1×1013VP/mL and MOI was 1.44×1011,the ratio of the tow values was 69.44. The recombinant virus had potent ability to infect HEK293 cells. Conclusions: Direct molecular cloning to construct recombinant AdHu5-040-fsp27 (cds) is better than homologous recombination method on less time-spending and high positive clones as well as less mutation of target genes. The modified cesium chloride gradient centrifugation method is simple and efficient to obtain high-titer purified and high-activity viruses.