Objective To analyze the evolution and genetic characteristics of Echovirus 11 (Echo11) strains isolated from patients with hand food and mouth disease ( HFMD) in Henan province. Methods Enterovirus strains were isolated from stool samples of patients with HFMD from year 2010 to 2012.The sequences of VP1 region of all positive strains were amplified , sequenced and then analyzed by BLSAT to determine their genotypes .The entire VP1 coding regions of 10 Echo11 strains were amplified by RT-PCR, and the homology analysis was conducted among them and other Echo 11 strains published in Gen-Bank.A phylogenetic tree was constructed to evaluate the evolution of Henan isolates and the prevalence of different genotypes .Results From year 2010 to 2012 , 184 non-Enterovirus 71 ( EV71 ) strains and non-Coxsakievirus A16 (CA16) strains causing HFMD were isolated, 10 (5.43%) of with were Echo11 strains. There were 876 nucleotides ( nt) in the complete VP1 gene sequence , encoding 292 amino acids ( aa) .The 10 Echo11 strains shared 93.1%-100% homologies in nt sequences and 97.3%-100% in aa sequences. The homology analysis indicated that Henan strains and prototype strains were highly similar but different with the homology of 77.8%-78.8% in nt and 90.8%-91.8% in aa, respectively.Conclusion Echo11 was one of the pathogens causing HFMD in He-nan province and genotype A was the dominant genotype .

Objective To construct single chain antibody specific to membrane protein of Schistosoma japonicum by gonetic engineering technique. Methods The V\-H (heavy-chain variable region) and V\-L (light-chain variable region) genes were amplified by PCR from the genomic DNA of NP11-4 cell line, and sequenced by Sanger's method. The ScFv was constructed in pTHA90 vector using V\-H and V\-L genes, then expressed by IPTG. Results The V\-H and V\-L genes were obtained through PCR. The DNA sequences showed that V\-H and V\-L were new variable region genes of antibody. They were registered by GenBank. A ScFv gene with (Gly4Ser) 3 intralinker in the pTHA90 vector was successfully constructed. The ScFv was expressed as thioredoxin-fused proteins about 36\^2 kDa. Conclusion A specific ScFv against the membrane protein of Schistosoma japonicum was constructed and expressed.

Objective The aim of this study was to evaluate the commercial SepsityperTM kit and serum separator tube coupled with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry for direct identification of microorganisms in a blood culture system.Methods A total of 138 clinical blood samples from clinical laboratory in Renji hospital were tested with two methods respectively from April to June of 2016.Performance of the assays were compared against that of conventional bacterial culture as a reference.Results A total of 138 nonduplicate positive blood culture samples were collected,including 70 (53.03％) gram negative samples,57 (43.18％) gram positive samples,3 fungus samples,2 mixed samples,and 6 false positive samples which were excluded from further analysis.The accuracy rate of SepsityperTM kit and serum separator tube was 91.67％ and 84.09％ in rapid identification of pathogen from blood samples,83.33％ and 61.36％ in correct identification to species level.The accuracy rate of SepsityperTM kit and serum separator tube was 98.57％ and 95.71％ in identifying gram-negative bacteria,87.72％ and 78.59％ in identifying gram-positive bacteria,respectively.The turnaround time for identification of each sample was 40 min by the commercial SepsityperTM kit and 25 min by serum separator tube.Conclusions MALDI SepsityperTM kit has shown slightly higher accuracy rate in identification of pathogen from blood sample than serum separator tube,but the difference is not significant (91.67％ vs.84.09％,P＞0.05).Compared with MALDI SepsityperTM kit,serum separator tube is a rapid,easy,and cost-effective pretreatment method for direct identification of microorganisms from blood cultures using MALDI-TOF mass spectrometry.

OBJECTIVETo clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen.

METHODSVP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected.

RESULTSVP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay.

CONCLUSIONSVP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; Enterovirus A, Human ; genetics ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Hand, Foot and Mouth Disease ; immunology ; Humans ; Immunoglobulin M ; blood ; Recombinant Proteins ; genetics ; immunology