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Objective To analyze the biological characteristics of clinical isolates of coxsackievir-us A6 (CVA6), a pathogen of hand,foot and mouth disease (HFMD), and to provide reference for vaccine development. Methods CVA6 strains were isolated from 21 stool and throat swab specimens of patients with HFMD in Yunnan Province and then identified. Their growth characteristics, plaque morphology and virulence to suckling mice were analyzed. Results Five CVA6 strains, named CVA6-129, CVA6-113, CVA6-57, CVA6-94 and CVA6-162, were isolated and all belonged to D3 subtype. Only the CVA6-129 strain could proliferate rapidly in Vero and KMB17 cells and the proliferation peaked 30 h after inoculation. The infectious titer of the CVA6-129 strain was 7. 54 lgCCID50 (50％ cell culture infective dose) / ml in KMB17 cells. Different morphologies of plaques were formed by the CVA6-129 strain in Vero and KMB17 cells at the same time points, which were small and round with clear edges in Vero cells, and large and irregular with blurry edges in KMB17 cells. Suckling mice were susceptible to CVA6 via intramuscular and intraperito-neal injection. The most common symptoms in infected suckling mice were reduced mobility, hind limb pa-ralysis and quadriplegia. CVA6 infection could result in death in severe cases. Conclusions This study isolated five CVA6 strains from a number of clinical samples of suspected HFMD cases, of which the CVA6-129 strain showed potential as a vaccine candidate.
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Objective To optimize the PCR primer sets for Simian virus 40 (SV40) detection and establish an assay method for SV40 which is of high sensitivity, strong specificity, broad applicability. Methods Two pairs of PCR primers were designed of based on 21 different SV40 strains genome by Primer Premier 5.00 software, and the features of two pairs of PCR primers were analyzed by Oligo software (version 6.71), conservative nucleotide of two pairs of PCR primers and the PCR amplification product were analyzed by DNAMAN software (version 6.0.40). Two pairs of new-built PCR primers were compared with those derived from China pharmacopoeia (Clip) in these aspects. The detection sensitivity of four pairs of PCR primers were analyzed using different SV40 DNA diluent as PCR template. The detection specificity of four pairs of PCR primers were analyzed using sterile water, Vero cell DNA, SV40 DNA as PCR template, respectively. Results The sequences of the new PCR primer sets VP1 and T are conservative for 21 Strains. The sequences of PCR primer sets GCVP1 and GCT are conservative for SV40 strains whose accession No. is J02400, NC_001669, AF316139 and AF316141. As far as the same diluent SV40 DNA template is concerned, the PCR amplification efficiency of PCR primer set VP1 and T is higher than that of PCR primer set GCVP1 and GCT. There are non-specific band in nucleic acid electrophoresis for amplification products of PCR primer sets GCVP1 and GCT, whereas there are no non-specific band in nucleic acid electrophoresis for amplification products of PCR primer sets VP1 and T. Conclusion The new assay method for SV40 nucleic acid sequence has many better qualities than those in Chp such as high sensitivity, strong specificity, broad applicability, conservation of primers and their amplification products and so on.
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Objective To analyze the factors of Protein A/G affinity column,influencing purification effect,and get the best condition to improve application effect of Protein A/G affinity column. Methods We Purified anti-HCV-IgG serum with different disposal modality,different sample input modality and different application number of affinity column before detecting and analyzing the purified samples. Results The Protein A/G affinity column had the best purified effect after using saturated ammonium sulfate to first purification,which increased the affinity column adsorption effect within 30 minutes adsorption. Conclusion Using antibody with first purification and adding the adsorption time could improve utilization rate of affinity column and prolong the service life.
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To explore the possibility and condition of differentiation of bone marrow mesenchymal cells (BMSCs) to neural cells in vitro, BMSCs from whole bone marrow of rats were cultured. The BMSCs of passage 3 were identified with immunocytochemical staining of CD44 ( + ), CD71 ( + )and CD45(-). There were type Ⅰ and type Ⅱ cells in BMSCs. Type Ⅰ BMSCs were spindleshaped and strong positive in immunocytochemical staining of CD44 and CD71, whereas flat and big type Ⅱ BMSCs were lightly stained. The BMSCs of same passage were induced to differentiate into neural cells by β-mercaptoethanol (BME). After induction by BME, the type Ⅰ BMSCs withdrew to form neuron-like round soma and axon-like and dendrite-like processes, and were stained positively for neurofilament (NF). The type Ⅱ BMSCs did not change in the BME medium and were negatively or slightly stained of NF.