ABSTRACT
Cases of 2019-nCoV nucleic acid and antibody (IgM and IgG total antibody) after discharge from a hospital in Chongqing were continuously monitored. It was found that 5 cases of "re-positive" phenomenon, 5 cases of antibody were positive, and there was a trend of increasing with time. "Re-Positive" may be related to the following three factors. Children with asymptomatic infection had a long time of fecal detoxification.There were two consecutive nucleic acid tests "false negative" caused by various reasons.The virus clearance in patients was not complete, and the discharge standard was not conservative enough. The analysis of the causes of "Re-Positive" patients and the discussion of its infection will help us reveal more characteristics of this virus, and to provide a new basis for the discharge standard in the constantly updated diagnosis and treatment programme.
ABSTRACT
Objective To investigate the binding ability of the terminase large subunit of Pseudomonas aeruginosa bacteriophage PaP3 to the cos site. Methods The gene tls was amplified from the genome of bacteriophage PaP3 by PCR and subcloned into pMD18-T vector. Then the gene tls cut down from the vector was inserted into the plasmid pQE31 which could give a 6-His tag at the N-terminal of the expressed protein. The recombinant vector pQE-tls was transformed to E.coli. JM109, after induction with IPTG, the expressed bacteria were resuspended and sonicated, then after centrifugation, the inclusion body was obtained. The inclusion body was dissolved with lysis buffer, then the tagged protein was purified by Ni-NTA affinity chromatography and renatured by dialysis. Finally the DNA-binding ability of the fusion protein rTLS was determined by EMSA. Results The expression plasmid pQE31-tls was successfully constructed, and the target protein yield was up to 30% of the total bacterial proteins. After purification and renaturation, the fusion protein rTLS can partially bind the cos fragment. Conclusion The fusion protein rTLS was successfully expressed, purified and renatured. The rTLS has the specific DNA-binding activity. The present work lays the foundation for the further research of the gene tls.
ABSTRACT
Objective To identify some basic biological properties,optimal multiplicity of infection(MOI),one-step growth kinetics,serum neutralization rate constant of Pseudomonas aeruginosa phage PaP3 were determined, respectively. Methods The host bacteria were infected with PaP3 at six different MOI(0.0001,0.001,0.01,0.1,1 and 10,then lysated adequately, the phage titer of supernatant was determined.For one-step growth experiments,the host bacteria were infected with PaP3 at MOI=10, following centrifugation after 15 min adsorption,the pellet containing (partially) infected cells was resuspended in 3 ml of pre-warmed LB broth and incubated with 160rpm at 37℃.Samples(50?l) were taken at 10 min-intervals and immediately tittered by the double-layer agar plate method. A rabbit was immunized with purified PaP3 to prepare anti-PaP3 serum, reaction constants between antiserum and PaP1, PaP2, PaP3 were determined using cross neutralization test. Results 0.001 MOI-infected host bacteria gave the highest phage offsprings. One-step growth curve was determined according to the results of one-step growth experiments.Reaction constant was determined using neutralization test. Conclusion The optimal MOI of PaP3 was determined to be 0.001. One-step growth curve for PaP3 shows that the latent period is about 20 min, the rise period is 60 min, and the average burst size is about 31pfu/cell. Rate constant of reaction between antiserum and PaP3 is 262.