10.3969/j.issn.2095-4344.2013.25.010

ObjectiveTo detect the levels of bone morphogenetic protein-2(BMP-2) mRNA in gunshot fracture healing of the rabbits.MethodsA model of primary treating gunshot fracture of the rabbit with external fixator and the real time fluorescence quantitative reverse transcription polymerase chain reaction(FQ-RT-PCR) technology with SYBR Green I were established.The levels of BMP-2 mRNA 1,2,3 and 4 weeks after operation were detected with FQ-RT-PCR respectively.ResultsIn the control group,BMP-2 mRNA of the bone tissue began to rise at the first week after fracture and the peak of mRNA expression appeared at the second week.The expression returned to normal at the forth week.In the experimental group,the BMP-2 mRNA rose more slowly that it arrived peak stage at the third week and was significantly lower in experimental group than that in control group at the same phases.ConclusionReal time FQ-RT-PCR is a good method for measuring the levels of BMP-2 mRNA during gunshot fracture healing.The mRNA expression rose more slowly and was significantly lower in gunshot fracture than in common fracture at the same phases.

Modified ORF5 (MORF5) and ORF6 gene of PRRSV were cloned into two multiple cloning sites of MVA transfer vector pLR-gpt to construct the recombinant plasmid pLR-MORF5/ORF6. Homologous recombination between pLR-MORF5/ORF6 and the wtMVA on BHK-21 cell line was mediated with liposome by infecting the cell with 0.01 MOI wtMVA two hours before transfecting the recombinant plasmid into the cell. When the cytopathic effect (CPE) was obvious, virus was collected from the cell plate and the recombinant virus was selected with drug selecting medium (2% MXHAT). After 12 cycles of selection, rMVA with a selection marker Eco gpt was obtained and named as rMVAgpt-MGP5/M. By infecting BHK-Cre expressing Cre recombinant enzyme, the Eco gpt marker in rMVAgpt-MGP5/M was deleted and this rMVA was named as rMVA-MGP5/M. The insertion of MORF5 and ORF6 into the MVA genome was confirmed with PCR analysis and the expression of MGP5 and M protein was identified with Western blot and IFA. Through biological study on the recombinant MVA, no obvious difference was observed between rMVA-MGP5/M and the wtMVA regarding to the CPE and growth curve. The recombinant MVA constructed in this study could coexpress the modified GP5 and M protein and the expressed product had good immunocompetence. Furthermore, the insertion of the MORF5 and ORF6 into MVA genome had no obvious effect on the replication and biological characteristics of this virus.
Animals ; Cell Line ; Genetic Vectors ; genetics ; Porcine Reproductive and Respiratory Syndrome ; prevention & control ; Porcine respiratory and reproductive syndrome virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Recombination, Genetic ; Swine ; Transfection ; Vaccines, DNA ; genetics ; immunology ; Vaccinia virus ; classification ; genetics ; metabolism ; Viral Envelope Proteins ; biosynthesis ; genetics ; Viral Matrix Proteins ; biosynthesis ; genetics

UNLABELLEDA novel double expression shuttle vector named pLR-gpt was constructed for marker-free recombinant modified vaccinia virus Ankara generation. A delectable Eco gpt marker was adopted with Cre/LoxP DNA recombination system and a BHK-21 cell line that can express Cre enzyme. Eco gpt gene controlled by P7.5 promoter from Vaccinia virus was cloned between two LoxP sites in the same direction. Additionally, two multiple cloning site under control of other two Vaccinia virus promoters were constructed outside LoxP sites. With this new transfer vector, Eco gpt marker in rMVA can be deleted on BHK-Cre with interaction between Cre enzyme and LoxP sequence. In order to verify the efficacy of this system, ORF5 and ORF6 gene of Porcine reproductive and respiratory syndrome virus (PRRSV) NJ-a strain were cloned into two multiple cloning sites of pLR-gpt to construct recombinant plasmid pLR-ORFS/ORF6. Homologous recombination between pLR-ORF5/ORF6 and wtMVA on BHK-21 cell was mediated by liposome by infecting cells with 0.01 MOI wtMVA two hours before transfection. After twelve cycles of selection, recombinant MVA with selecting marker Eco gpt was obtained and named as rMVAgpt-GP5/M. By infecting BHK-Cre, the Eco gpt marker in rMVAgpt-GP5/M was deleted and this rMVA was named as rMVA-GP5/M. Expression of GP5 and M protein was identified with Western blotting and IFA. Results from PCR and biological study for rMVA indicated that Eco gpt marker was completely deleted.

CONCLUSIONSdouble expression transfer vector for marker-free recombinant Modified vaccinia virus Ankara generation was successfully constructed, and works well in MVA expression system.

Cell Line ; Cloning, Molecular ; DNA, Recombinant ; genetics ; Escherichia coli Proteins ; genetics ; Genetic Vectors ; genetics ; Pentosyltransferases ; genetics ; Porcine respiratory and reproductive syndrome virus ; genetics ; Vaccinia virus ; genetics ; Viral Envelope Proteins ; genetics ; Viral Matrix Proteins ; genetics

In order to obtain a virus-like particle vaccine both for porcine parvovirus (PPV) prevention and growth-promotion, VP2 gene of PPV NJ-a strain was amplified with PCR, and four copies of synthetic somatostatin gene were fused to the N-terminal of VP2 gene. The fused gene was cloned into pFast-HT A to construct the recombinant plasmid pFast-SS4-VP2, then the pFast-SS4-VP2 was transformed into DH10Bac competent cells and recombined with shuttle vector Bacmid, followed by identification with blue-white screening and PCR analysis for three cycles, and the positive recombinant was named as rBacmid-SS4-VP2. The positive Sf-9 cells were transfected with rBacmid-SS4-VP2 by Lipofectamine to produce recombinant baculovirus. When the cytopathic effect (CPE) was obvious, the transfected Sf-9 cell was harvested, and the positive recombinant virus was named as rBac-SS4-VP2. The insertion for the target gene into baculovirus genome was confirmed with PCR. SDS-PAGE and Western blotting revealed that the calculated protein of approximately 68 kDa was in the expressed in the insect cells. The Sf-9 cells infected with rBac-SS4-VP2 were stained positive against PPV antibody using the indirect immunofluorescence assay (IFA). Moreover, the virus particle self-assembly was observed under electron microscopy. 90 four-week-old mice were immunized by the recombinant protein coupled with different adjuvants alhydrogel, IMS and oil. VP2-specific ELISA antibodies, PPV-specific neutralizing antibody, somatostatin antibody and growth hormone levels were examined to evaluate the immunogenicity of this virus like particle. Results indicated that mice groups immunized rSS4-VP2 protein with alhydrogel and IMS developed similar humoral immune response comparing with inactived PPV vaccine. Mice group immunized with rSS4-VP2 generated higher level of SS antibody and growth hormone comparing with negative control, mice receiving rSS4-VP2 with alhydrogel developed the highest antibody titre than all other groups, while the oil group developed the lowest antibody level. This study provides not only a new rout for production of safe and effective virus like particle subunit vaccine, but also the foundations for peptide presentation and multivalent subunit vaccine design.
Animals ; Antigens, Viral ; biosynthesis ; genetics ; Artificial Gene Fusion ; Baculoviridae ; genetics ; Capsid Proteins ; biosynthesis ; genetics ; Mice ; Parvoviridae Infections ; prevention & control ; Parvovirus, Porcine ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Somatostatin ; genetics ; Swine ; Vaccines, Virus-Like Particle ; biosynthesis ; immunology ; Viral Vaccines ; biosynthesis ; immunology ; Virion ; genetics ; immunology

Objective:To analyze the epidemiological characteristics of acute occupational poisoning in Yunnan province, so as to provide basis for formulating prevention and control measures of acute occupational poisoning in Yunnan province.Methods:In December 2019, the information of acute occupational poisoning events reported in Yunnan province from 2004 to 2019 was collected, and the epidemiological distribution, event classification, industry characteristics, poison types and poisoning causes were analyzed.Results:A total of 47 acute occupational poisoning incidents were reported in Yunnan province from 2004 to 2019, with 562 poisoning cases and 51 deaths (case fatality rate of 9.07%) . The regions with the largest number of reported incidents were Kunming and Qujing, with 12 incidents (25.53%) and 10 incidents (21.28%) respectively; The majority of incidents was relatively large (31 incidents, 65.96%) , and the industry was mainly distributed in the chemical industry (19 incidents, 40.43%) and metallurgy (15 incidents, 31.91%) . The most poisonous poisons were carbon monoxide (10 incidents, 21.28%) and arsine (9 incidents, 19.15%) . The main causes of poisoning included not using personal protective equipment or poor equipment (25 incidents, 53.19%) , failure to formulate or violate safety operating procedures (15 incidents, 31.91%) .Conclusion:Acute occupational poisoning incidents occur from time to time in Yunnan province, and the fatality rate is high. Therefore, it is necessary to strengthen the supervision of key areas and industries.

Objective:To analyze the epidemiological characteristics of acute occupational poisoning in Yunnan province, so as to provide basis for formulating prevention and control measures of acute occupational poisoning in Yunnan province.Methods:In December 2019, the information of acute occupational poisoning events reported in Yunnan province from 2004 to 2019 was collected, and the epidemiological distribution, event classification, industry characteristics, poison types and poisoning causes were analyzed.Results:A total of 47 acute occupational poisoning incidents were reported in Yunnan province from 2004 to 2019, with 562 poisoning cases and 51 deaths (case fatality rate of 9.07%) . The regions with the largest number of reported incidents were Kunming and Qujing, with 12 incidents (25.53%) and 10 incidents (21.28%) respectively; The majority of incidents was relatively large (31 incidents, 65.96%) , and the industry was mainly distributed in the chemical industry (19 incidents, 40.43%) and metallurgy (15 incidents, 31.91%) . The most poisonous poisons were carbon monoxide (10 incidents, 21.28%) and arsine (9 incidents, 19.15%) . The main causes of poisoning included not using personal protective equipment or poor equipment (25 incidents, 53.19%) , failure to formulate or violate safety operating procedures (15 incidents, 31.91%) .Conclusion:Acute occupational poisoning incidents occur from time to time in Yunnan province, and the fatality rate is high. Therefore, it is necessary to strengthen the supervision of key areas and industries.

Based on gram positive enhancer matrix displaying technology, we designed and evaluated a bacteria-like particle vaccine against swine type O Foot-and-mouth disease virus. Three optimized genes of type O Foot-and-mouth disease virus strain Mya98 were cloned into recombinant prokaryotic expression vector pQZ-PA and renamed as pQZ-BT1B-PA, pQZ-BT2B-PA and pQZ-B (T1BT2) 4B-PA, fused with an anchor protein (PA) binding to Gram-positive enhancer matrix (GEM) particles specifically. The protein expression was identified with SDS-PAGE and Western blotting, and then purified with GEM particles. Five-week old female mice were randomly divided into six groups and all the immunization was developed according to subcutaneous injection. Mice in the first three groups were injected with 50 μg/dose GEM-BT1B, GEM-BT2B and GEM-B (T1BT2) 4B, respectively. Mice in the fourth group were immunized with commercial peptide vaccine as positive control. The fifth group vaccinated with host E. coli transformed with pQZ-PA fulfilled as negative control. Mice in the last group injected with sterile PBS served as blank control. The humoral immunity of recombinant protein vaccine was evaluated with peptide-specific antibody and LPB antibody. The cellular immunity was evaluated with lymphocyte proliferation test and cytokine expression detection. SDS-PAGE and Western blotting showed that the most part of soluble target fusion protein have been purified and displayed on GEM particles. Vaccine GEM-B (T1BT2) 4B stimulated mice produce not only higher level of specific antibody against peptide and Foot-and-mouth disease virus specific liquid phase blocking antibody, but also more vigorous spleen lymph proliferation and higher levels of Th1 type cytokines. To summarize, vaccine of GEM-B (T1BT2) 4B possessed good immunogenicity and opened a new way for further Foot-and-mouth disease virus subunit vaccine design.

Objective: To understand the epidemiological characteristics of imported acute infectious diseases between 2008 and 2017 in the border areas of Yunnan province. Methods: All the cases occurred between January 2008 and December 2017 and related information was from the Chinese CDC infectious disease report information management system, according to definition of imported cases diagnosed by clinicians. Epidemiological characteristics of the imported cases of related information were gathered. Results: A total of 13 157 imported acute infectious diseases were reported from the border areas of Yunnan province, which accounted for 6.03% (13 157/218 284) of the total number of acute infectious diseases in the same areas from 2008 to 2017. Malaria, dengue fever and hand-foot-mouth disease were accounted for 56.05% (7 374/13 157), 21.82% (2 871/13 157) and 4.62% (608/13 157), of all the case, respectively. The number of imported malaria cases decreased annually. However, dengue fever showed a sharp increase. Peaks of the epidemics appeared as: May for malaria and October for dengue fever. Male patients were accounting for the majority (73.22%, 9 634/13 157), so as the patients with Chinese nationality (54.91%, 7 225/13 157). The age distribution appeared as: 67.12% (8 829/13 157) for the 15-44 year olds and 19.26% (2 535/13 157) were children below 14 years of age. Proportions of occupation appeared as: farmers (45.23%, 5 596/13 157), migrant workers (21.30%, 2 802/13 157) and children living at home (11.12%, 1 463/13 157). Most of the imported cases were coming from Myanmar and appearing in the following three counties: Ruili city, Tengchong city, and Yingjiang of Yunnan province. Cities/counties that with number of imported cases more than 10% of the local reported cases, would include Ruili city, Tengchong city, Zhenkang county and Mangshi of Yunnan province. Conclusions Imported acute infectious disease was a serious public health problem in Yunnan province, 2008-2017. The main imported acute infectious diseases were malaria, dengue fever and hand-foot-mouth disease. The majority imported cases were accounting for Chinese, male, young adults and farmers. It is also important for immigration workers to carry out surveillance, prevention and control programs on infectious diseases when working in neighboring countries.

Objective To understand the epidemic characteristics of different virus types of hand, foot and mouth disease (HFMD) in Zhaotong City, and provide guidance and recommendations for the prevention and control of HFMD, and to analyze seasonal characteristics of different virus types of HFMD in Zhaotong City. Methods The epidemiological characteristics of different virus types of HFMD in Zhaotong City from 2014 to 2017 were analyzed using the concentration and circular distribution methods. Results The main pathogens detected were EV71, Cox A16 and other enteroviruses, which were 216, 182, and 294, respectively, accounting for 57.45%, 73.44%, and 67.11%. M was 0.86, indicating that EV71 had strong seasonality. The Rayleigh test showed statistically significant differences (Z = 99.53, P <0.001). ā = 157 °, the peak day of onset was May 10, similar to untyped (May 16), the peak period was April 21-June 1, and the epidemic period was April 1-June 21. Conclusion According to the incubation period of hand-foot-mouth disease and the period of time during which the vaccine develops protective effects, vaccination of hand-foot-mouth disease vaccine at the peak period has a good guiding significance for the timeliness and pertinence of vaccination.