Polyclonal antibody preparation and expression in liver tissues of transactivated protein 5 of hepatitis C virus nonstructural 5A / 西安交通大学学报·英文版

Objective: To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods: In Escherichia coli BL21, the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG), and it was analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyclonal antibody, with which we studied the function of NS5ATP5 by determining the different liver tissues with the streptavidin-perosidase (SP) immunohistochemistry method. Results: Recombinant NS5ATP5 (molecular weight, 65 kD) and polyclonal antibody were successfully prepared. NS5ATP5 expression in the liver of patients with chronic HCV infection was much higher than that of a normal person, and it was detected mainly in the cytoplasm. Conclusion: The findings of the expression difference between HCV patients and normal people led to a novel diagnostic marker to detect HCV infection.

The immunostimulatory study on peripheral blood mononuclear cell and CD4+ T lymphocyte of HBV infections that were activated by CpG oligonucleotide / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the effects of CpG oligodeoxyribonucleotide (ODN) as adjuvant on the immune responses in PBMC and CD4+ T cell with chronic hepatitis B virus.</p><p><b>METHODS</b>The selected 20 infections were averagely divided two groups. The frequency of IFN-gamma secreting PBMC and CD4+ T cell in immune tolerant phase and in the immune clearance phase that had stimulated by CpG ODN, HBsAg and Mixture [CpG ODN + HBsAg] were analyzed by enzyme linked immune spot (ELISOT).</p><p><b>RESULTS</b>The PBMC and CD4+ T cell were differently incubated by CpG ODN, HBsAg and M [CpG ODN + HBsAg]. The number of IFN-gamma spot differently are 3 +/- 8, 339 +/- 429, 375 +/- 496, 1 +/- 4, 5 +/- 16 and 5 +/- 12; the results of immume tolerance are 3 +/- 8, 361 +/- 153, 375 +/- 276, 0 +/- 2, 2 +/- 2 and 4 +/- 4; but the results of immune clearance are 3 +/- 21, 289 +/- 345, 405 +/- 656, 2 +/- 14, 8 +/- 40 and 7 +/- 30. The IFN-gamma spots statistical analysis of PBMC were differently incubated by HBsAg and M, the total is P = 0.720, The IFN-gamma spots statistical analysis of CD4+ T cell were differently incubated by HBsAg and M, the total is P = 0.890, The IFN-gamma spots statistical analysis of PBMC and CD4+ T cell were differently incubated by M, the total is P = 0.000.</p><p><b>CONCLUSIONS</b>The ability that CpG ODN can not significantly increase the IFN-gamma secreting of PBMC and CD4+ T cell that were incubated by HBsAg to the infection in immune tolerant phase and in the immune clearance phase, but the PBMC outweighed The CD4 T cell.</p>

Application of pulse-field gel electrophoresis analysis in source-tracking of food-borne disease caused by Vibrio parahaemolyticus / 中华预防医学杂志

<p><b>OBJECTIVE</b>To apply pulse-field gel electrophoresis analysis(PFGE) in analysing a case of food poisoning caused by Vibrio parahaemolyticus.</p><p><b>METHODS</b>PFGE using restriction enzyme Not I was employed in molecular subtyping of thirty strains of V. parahaemolyticus isolated from a case of food poisoning in Guangzhou city and PFGE patterns were analyzed by using BioNumerics Version 4.0 software to perform cluster analysis. Pattern profiles were compared by using the Dice coefficient and unweighted pair group method with arithmetic averages (UPGMA).</p><p><b>RESULTS</b>Thirty strains were of the same type of pulsotype.</p><p><b>CONCLUSIONS</b>Molecular subtyping by PFGE might disclose the epidemiological relationships of the strains from humans, food and the environment, giving a strong molecular epidemiological evidence and a support for the source-tracking of outbreak events.</p>

Application of pulse-field gel electrophoresis analysis in the source-tracking of cholera epidemics / 中华流行病学杂志

<p><b>OBJECTIVE</b>To apply pulse-field gel electrophoresis analysis(PFGE) in the analysis of cholera outbreak events and to determine the molecular epidemiological characteristics of Vibrio cholerae ( V. cholerae) isolates.</p><p><b>METHODS</b>PFGE using restriction enzyme Not I was employed in the molecular subtyping of forty-one strains of V. cholerae isolated in cholera outbreak events from 2003 to 2005 in Guangzhou area and PFGE patterns were analyzed by BioNumerics Version 4.0 software to perform cluster analysis. Pattern profiles were compared by utilizing of Dice coefficient and UPGMA(unweighted pair group method with arithmetic averages). Comparison of PFGE typing results was performed with phage-biological typing and pathogenicity-associated genes typing.</p><p><b>RESULTS</b>In cholera outbreak events, PFGE could discriminate epidemiologically related and unrelated strains, having more discriminatory power than phage-biological typing and pathogenicity-associated genes-typing.</p><p><b>CONCLUSIONS</b>Molecular sub-typing by PFGE could disclose the epidemiological relationships of strains from humans and the environment, providing molecular epidemiological evidence and support for the source-tracking of cholera outbreak events.</p>

Analysis of characteristics of major pathogenicity-related genes of Vibrio cholerae isolated in Guangzhou area from 2001 to 2005 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To apply multiplex polymerase chain reaction (MPCR) assay and sequencing in study of the carrying status of four pathogenicity-related genes of Vibrio cholerae (V.cholerae) and the variation of ctxA.</p><p><b>METHODS</b>Primers targeting cholera toxin sub-unit A gene (ctxA), toxin-coregulated pilus gene (tcpA), accessory cholera enterotoxin gene (ace), zonula occludens toxin gene (zot) were designed and the MPCR method was applied to detect the pathogenicity-related genes of 276 strains of V.cholerae isolates. The amplified fragments of ctxA gene were sequenced and the genetic homology of the amplified fragments of ctxA was analyzed.</p><p><b>RESULTS</b>Of the 276 strains of V.cholerae, 93.9% strains from human sources belong to the pathogenicity-related genes type A (ctxA(+)tcpA(+)ace(+)zot(+) type) and 6.1% belong to pathogenicity-related genes type C (ctxA(-)tcpA(-)ace(-)zot(-) type). Type A strains from clinical sources were isolated from patients with mild to severe symptom and carriers, among which 68.5% were isolated from patients with mild symptom and 21.9% from carriers. All 63.6% of type C strains from clinical sources were isolated from patients with mild symptom and 36.4% from carriers. The proportion of type C strains that caused mild symptom was higher than that of type A strains. Of the 78 strains isolated from the environment, 9.0% strains belong to pathogenicity-related type A and 35.9% belong to the pathogenicity-related genes type B (ctxA(-)tcpA(-)ace(+)zot(+) type), while 55.1% belong to pathogenicity-related genes type C. The sequencing results showed little genetic variation among the amplified fragments for ctxA.</p><p><b>CONCLUSION</b>MPCR disclosed the polymorphic status of pathogenicity-related gene patterns in V.cholerae isolates of Guangzhou, providing effective means for further study on evolution of pathogenicity-related genes among V.cholerae isolates from human and environmental sources. This study also offers significant guidance for effective prevention, control and warning against cholera epidemic in local area.</p>