Search

The swine influenza virus (SIV) strain A/Swine/TianJin/01/2004(H1N1) (A/S/TJ/04) was rescued successfully by an eight-plasmid rescue system. The cDNAs of SIV 8 gene segments were synthesized by RT-PCR and cloned into the RNA polymerase I/II bidirection expression vector PHW2000 independently, resulting in 8 recombinant plasmids. The 8 recombinant plasmids were cotransfected into COS-1 cell, 30 h later TPCK-trypsin was added to 0.5 microg/mL. The COS-1 cell and supernatant were harvested 48 h after cotransfection and were inoculated into the allantoic cavity of 9-day-old specific-pathogen free (SPF) chicken eggs. The allantoic fluid of dead eggs was harvested and passaged 3 generations in SPF chicken eggs to get infective virus. The successful rescue of A/S/TJ/04 SIV was identified by hemagglutination assay, hemagglutination inhibition assay, sequence analysis and electron microscope observation. The successful rescue of SIV built a platform for the research of the relationship between genome structure and function of SIV, the mechanisms of trans-species transmission of influenza virus and for the generation of new SIV as vaccine.

Eight-plasmid system was used for the generation of Avian influenza virus (AIV) strain A/Chicken/Shanghai/F/98 (H9N2) which was isolated in China in 1998. In this plasmid-based expression system, viral cDNA was inserted beteen the RNA polymerase I (pol I) promoter and terminator sequences. The entire pol I transcription unit was flanked by an RNA polymerase II (pol II) promoter and a poly (A) site. Twenty-four hours after the transfection of eight expression plasmid into cos1 cells, the supernatant and cos1 cells transfected were inoculated into the allantoic cavity of 10-day-old specific-pothgen-free (SPF) chicken eggs. The HA titer was determined after passage of the rescued virus in chicken eggs, and as high as that of the parental wild-type virus.

To map the interacting site of subunit Rpb2 to subunit Rpb3 of RNA polymerase II in fission yeast Schizosaccharomyces pombe, the yeast two-hybrid system was employed in this paper to screen the interacting clones between Rpb2 and Rpb3.4 fragments of Rpb2 cDNA were cloned into the Ga14 BD vector pAS2. The 4 clones were named as pAS2 Rpb2-1, 2-2, 2-3 and 2-4, respectively. The complete cDNA of Rpb3 was cloned into the Gal 4 AD vector pGADGH. The clone was named as pGADGH Rpb3. The two-hybrid plasmids pGADGH Rpb3 and pAS2Rpb2-1, 2-2, 2-3 or 2-4 respectively were cotransformed into host cell yeast Y190. The interaction positive cotransformants were identified by beta-gal activity assay. The beta-gal positive cotransformants were selected from pGADGH Rpb3 and pAS2Rpb2-4 two-hybrid system. DNA sequencing and alignment results showed that the interacting site of Rpb2 to Rpb3 located within the fragment from base 2701 to 2966 of Rpb2 cDNA, or within the C-termini polypeptide from amino acid 902 to 989 of Rpb2 protein.

Objective: To evaluate the applicability of detection of rpoB gene mutation in M. tuberculosis susceptibility testing., Methods: 87 M. tuberculosis isolates and 22 sputum specimens from patients with active pulmonary tuberculosis were detected by PCR-SSCP., Results: The sensitivity of PCR for rpoB gene amplification was 100 pg DNA and 5000 organisms. The rpoB gene could be detected in the all isolates tested. In comparison with conventional susceptibility testing methods, the sensitivity and specificity of PCR-"cold" SSCP analysis for detecting rifampin resistance in 87 M. tuberculosis isolates was 89.6% and 100%, respectively. Among 22 smear- and culture-positive sputum specimens, only 1 (4.5%) was positive by PCR, however, 6 (27.3%) of them were positive by nested-PCR. The "cold" SSCP results of these 6 specimens were corresponding to that of the susceptibility testing., Conclusions: The PCR-"cold" SSCP described here can easily and rapidly detect rifampin resistance of M. tuberculosis. After increasing the primer specificity and amplification sensitivity, the technique might be used for detection of M. tuberculosis rifampin resistance in clinical specimen directly.

Objective: To evaluate rpoB gene mutation in rifampin-resistant Mycobacterium tuberculosis and its relationship with rifampin resistance., Methods: Forty clinical isolates of Mycobacterium tuberculosis were analyzed by PCR-SSCP technique, with H37Rv reference strains as control group., Results: The sensitivity of amplication products of 411bp and 258bp were found to be 5 pg/microliters, 500 organisms per milliliter and 1 pg/microliter, 500 organisms per milliliter respectively. rpoB gene belongs to genus specificity. Characteristics of SSCP graph of 258bp fragment: Ten sensitive strains were the same as H37 Rv. Thirty strains of rifampin-resistant or multidrug resistance, including rifampin, were different from H37Rv except for three strains. Positive rate was 90%, while specificity 100%., Conclusions: Results showed that PCR-SSCP technique could detect rPOB gene mutation, which might associate with rifampin resistance and be helpful to rapid detection and research of rifampin-resistant Mycobacterium tuberculosis.