Prokaryotic expression and preparation of the polyclonal antibodies of E protein and domain Ⅲ in Zika virus / 中国人兽共患病学报

To clone,express and purify the E Protein and EDⅢ of Zika virus in E.coli and prepare two kinds of polyclonal antibodies,the virus was amplified by Vero E6 cell culture.Total RNA was extracted by RT-PCR and reverse transcribed into cDNA.The prokaryotic expression vectors pET32a/E and pET28a/EDⅢ were constructed by cDNA sequence of E and EDⅢ gene.Then,recombinant plasmids were transformed into E.coli BL21 and induced by IPTG,and purified by Ni+ column affinity chromatography.BALB/C mice were immunized with purified recombinant proteins.Antiserum was collected and titer was determined by indirect ELISA.Western blot was used to detect the specificity.Results showed that the recombinant proteins were successfully expressed and purified.The titer of the polyclonal antibodies both reached 1:409 600.Western Blot analysis showed that the polyclonal antibodies could specifically recognize the recombinant proteins.Thus,the specific polyclonal antibody were successfully prepared,laying a foundation for further study on the pathogenesis,detection methods and immune strategies of Zika virus.

Construction and characterization of the stk and stp1 knockout mutant in streptococcus suis type 2 / 医学研究生学报

Objective Serine /threonine kinases (STK) and phosphatases (STP) regulate various physiological activities of prokaryotes by reversible phosphorylation of proteins .This paper aimed to study the effects of simultaneous deletion of the stk and stp1 genes on the biological characteristics and pathogenicity of streptococcus suis type 2, the Chinese virulent strain 05ZYH33. Methods The double mutant of the stk and stp1 genes of 05ZYH33 was constructed by homologous recombination .The biological characteristics of the wild strain 05ZYH33 and the mutant strain Δstk/stp1 were compared.The effects of the stk and stp1 deletion on bacterial virulence was analyzed using cell adhesion assay , anti-phagocytosis assay and the mouse model of infection . Results RT-PCR showed that the stk and stp1 genes were replaced by the spectinomycin resistance gene Spc r and the mutant strain was successfully constructed .Experi-ments of biological characterization revealed gradually increased value of 05ZYH33 and Δstk/stp1 at 2 hours after inoculation and a plateau period at 7 hours.The logarithmic phase of the mutant strain (A600≈0.4) was 1 hour later than that of the wild one , and the bacterial den-sity of the former was lower than that of the latter after the plateau pe -riod (0.8 vs 1.0).On the blood plates of 05ZYH33 and Δstk/stp1 were observed greyish, round, semitransparent, wet and smooth-sur-faced tiny bacterial colonies , around which there were hemolysis rings with no significant differences in colony morphology and hemolytic ac -tivity.In the experiment on pathogenicity , the mice of the 05ZYH33 group all died within 12 hours while 9 of the 30 mice in the Δstk/stp1 group died within 12 hours and all died within 24 hours. Conclusion The simultaneous deletion of the stk and stp1 genes may mainly affect the regulation of the proteins associated with bacte -rial proliferation and division.

Study on molecular epidemiology of major pathogenic Streptococcus suis serotypes in middle part of Jiangsu province / 中华流行病学杂志

<p><b>OBJECTIVE</b>To determine the prevalence of Streptococcus suis and major pathogenic serotypes in middle part of Jiangsu province.</p><p><b>METHODS</b>Tonsillar specimens from 303 slaughtered pigs aged 6 to 8 months were investigated for the presence of Streptococcus suis and major pathogenic serotypes by polymerase chain reaction (PCR) method. Bacteriological examination compared with molecular genetics identification for three Streptococcus suis isolates were also done.</p><p><b>RESULTS</b>The overall carrier rate of Streptococcus suis was up to 88.0%, with the percentages of serotype 1(14), 2(1/2), 7 and 9 were 9.6%, 8.5%, 11.3% and 29.5% respectively in 2005. While in 2006, the prevalence of Streptococcus suis was 82.5%, with capsular types 1 (14), 2 (1/2), 7 and 9 were accounted for 17.6%, 2.4%, 25.8% and 20.0% of all the specimens. All the three isolates belonged to Streptococcus suis serotype 2,named 2a, 2f and 14e, which exhibiting the virulent phenotype cps2+/gdh+/mrp-/lepf-/sly-/fbps+/orf2+/89k-, cps2+/lgdh+/mrp-/epf-/sly-/fbps-/orf2-/89k- and cps2+/gdh+/mrp-/epf-/sly-/fbps/orf2-/ respectively. These isolates were all susceptible to amoxicillin, ampicillin, penicillin and resistant to amikacin and tetraycline. Clinical signs were not noted in BALB/c mice and rabbit.</p><p><b>CONCLUSION</b>Prevalence of the Streptococcus suis among the healthy herds in the areas was very high, with various capsule types of Streptococcus suis involved in the same herds, and the virulent phenotype of these 3 isolates were very different from those prevalent Streptococcus suis serotype 2 virulent isolates frequently discovered from the epidemic areas.</p>

Construction of RevS gene knock-out mutant of Streptococcus suis serotype 2 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To construct a gene knock-out mutant of response regulator named RevS in Streptococcus suis serotype 2 virulent strain 05ZYH33, and to investigate the effects of its deletion on the biological characters of this pathogen and the pathogenesis to mice and piglets.</p><p><b>METHODS</b>Recombinant gene knock-out vector consisting of Spc(r) cassette was constructed and flanking was constructed consisting of Spc(r) cassette with flanking homology regions to the RevS genes while the isogenic RevS-deficient mutant was screened by allelic replacement. The effects of RevS deletion on the basic biological characters of 05ZYH33 including growth stability, colonial morphology, haemolysis, Gram staining, growth curve and protein expression were examined in vitro. The mice and piglets were infected with 10(8) CFU wild virulent and mutant isolates.</p><p><b>RESULTS</b>PCR analysis confirmed that the coding genes of RevS were replaced completely by Spc(r) cassette and the basic biological characters of 05ZYH33 did not undergo any apparent change. Balb/c mice infection assay indicated that RevS play a role in the pathogenesis of Streptococcus suis infections, while no remarkable difference was observed in the piglets' pathogenesis infection rates between mutant isolates deltaA05ZYH33 and wild-type isolates 05ZYH33.</p><p><b>CONCLUSION</b>The mutant of Streptococcus suis 05ZYH33 response regulator was successfully constructed, while the mutation did not obviously affect the bacterial biological characters, while the knock-out mutant of RevS was shown to be attenuated in pathogenesis to mice and piglets.</p>

Detection of virulence-associated factors of Streptococcus suis by multiplex PCR assay / 中华流行病学杂志

<p><b>OBJECTIVE</b>To rapidly and sensitively detect the four virulence-associated factors of Streptococcus suis, a multiplex PCR was developed.</p><p><b>METHODS</b>In the process of this reaction, four distinct DNA targets were amplified. One target was based on the serotype 2 (and 1/2) specific cps gene and the others were based on Streptococcus suis mrp, epf (epf*) and sly gene, encoding the MRP, EF(EF*) and Sly proteins of Streptococcus suis. 72 isolates, which including 48 strains of Streptococcus suis and 24 strains of negative control, and 49 clinical specimens were detected by the multiplex PCR assay.</p><p><b>RESULTS</b>All PCR products were detected by electrophoresis on 1.2% agarose gels. With the 48 Streptococcus suis strains, the positive detection rates of cps2+, mrp+, epf+, epf*+ and sly+ were 16/48, 14/48, 12/48, 3/48 and 26/48,respectively. The results were confirmed by bacteriological examination. There were no specific amplification products including 49 clinical specimens and 24 negative control strains.</p><p><b>CONCLUSION</b>The results demonstrated that multiplex PCR was a highly specific and sensitive diagnostic tool for the detection of virulence-associated factors of streptococcus suis.</p>