Genetic characteristics analysis of the hemagglutinin gene of measles virus in Shanghai， 2001‒2018 / 上海预防医学

ObjectiveTo analyze the genetic characteristics of the hemagglutinin （H） gene of measles virus （MeV） in Shanghai， 2001‒2018. MethodsNasopharyngeal swab specimens were collected from suspected measles cases reported in Shanghai from 2001 to 2018， and the isolation of measles virus was conducted with Vero/hSLAM cell line. RT-PCR amplification and sequencing were conducted after RNA extraction to analyze the genetic characteristics of the complete H gene. ResultsIn total， 5 665 nasopharyngeal swab samples were collected by suspected measles case surveillance from 2001 to 2018， and 1 394 measles virus strains were isolated. The homology of nucleotide acid and amino acid among 349 representative measles virus isolates was 87.4%‒100.0% and 85.1%‒100.0%， respectively. The homology of nucleotide acid and amino acid between representative measles virus isolates and China vaccine strain （S191） was 85.7%‒100.0% and 84.1%‒100.0%， respectively. All the sub-genotype H1a MeV isolates had an amino acid substitution （Ser240Asn）， which removed a predicted N-linked glycosylation site. ConclusionMost of the MeV isolates are sub-genotype H1a analyzed based on H gene， which are identical to those of the N gene. The predicted amino acid sequences of the H protein are relatively conserved at most of the functionally significant amino acid positions.

Role and mechanism of Leptospira interrogans vWF-A gene products binding to human collagen proteins / 中华微生物学和免疫学杂志

Objective:To investigate the role and mechanism of Leptospira interrogans ( L. interrogans) vWF-A gene products binding to human collagen proteins. Methods:Bioinformatic software was used to analyze the structure and function of the vWF-A genes (LA_0012, LA_0697 and LA_4207) of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai. Prokaryotic expression systems for the vWF-A domain segments in the vWF-A genes were generated. The target recombinant proteins, rLep0012, rLep0697 and rLep4207, were purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE. ELISA and surface plasmon resonance (SPR) were performed to detect the binding ability of the target recombinant proteins to humanⅠ, Ⅲ, Ⅳ and Ⅵ types of collagen proteins (hCOL1/3/4/6). Expression of the vWF-A genes at mRNA and protein levels were detected by real-time fluorescent quantitative RT-PCT and Western blot during infection of human umbilical vein endothelial cells (HUVEC) and mouse hemangioendothelioma endothelial cells (EOMA). Results:The products of vWF-A genes were vWF-A superfamily domain-containing surface or transmembrane proteins, but LA_0697 and LA_4207 genes also contained metal ion-dependent adhesion sites (MIDAS). The established prokaryotic expression systems efficiently expressed the target recombinant proteins and each of the proteins extracted by Ni-NTA affinity chromatography showed a single band in SDS-PAGE. ELISA results showed the strong binding of rLep0697 to hCOL3/6 and rLep4207 to hCOL1/4. SPR results showed the rapid binding and dissociation of rLep0697 with hCOL3/6 ( KD values=5.71×10 -8 and 5.89×10 -8 mol/L) and the rapid and stable biding of rLep4207 with hCOL1/4 ( KD values=6.4×10 -9 and 3.2×10 -9 mol/L). Expression of the vWF-A genes at both mRNA and protein levels were significantly elevated ( P<0.05) during infection of HUVEC and EOMA cells. Conclusions:The products of LA_0697 and LA_4207 genes could act as the adherence factors of L. interrogans during infection.

Identification of Sigma N signaling system target genes in Leptospira interrogans / 中华微生物学和免疫学杂志

Objective:To confirm the Sigma N transcription factor activity of a gene product encoded by LA2404 gene of Leptospira interrogans ( L. interrogans) and to identify the target genes of Sigma N signaling system. Methods:L. interrogans LA2404 gene and its regulated target genes were predicted using bioinformatic analysis according to the promoter sequence signature in Sigma N-regulated genes. A LA2404 gene-knockout (ΔLA2404) strain of L. interrogans was constructed based on homologous sequence recombinant of suicide plasmid. Real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to detect the changes in the expression of target genes at mRNA level in the ΔLA2404 mutant. A prokaryotic expression system for LA2404 gene was established and the target recombinant protein rSigma N was extracted by Ni-NTA affinity chromatography. Gel electrophoresis mobility shift assay (EMSA) was used to screen out the target genes regulated by rSigma N. Results:Pathogenic L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai carried one Sigma N gene and 22 Sigma N promoter sequence-containing target genes. Qualitative examination of the ΔLA2404 mutant by microscopy revealed no defect in motility and appearance. Expression of LA1188, LA2306, LA3426, LA1968, LA1313, LA3806 and LA0773 genes at mRNA level in the ΔLA2404 mutant was significantly down-regulated ( P<0.05), but no significant changes in the expression of other target genes at mRNA level were detected. EMSA results confirmed that rSigma N could bind to the promotor sequences of the target genes mentioned above. Conclusions:Sigma N transcription factor was encoded by LA2404 gene. LA1188, LA2306, LA3426, LA1968, LA1313, LA3806 and LA0773 genes contained Sigma N promoter sequence and the expression of them was regulated by Sigma N signaling system.