[Screening and Applications of qPCR Primers for apomucin Gene of Echinococcus multilocularis].

Objective: To screen for the optimal qPCR primers for Echinococcus multilocularis apomucin gene (Em-apo) and analyze Em-apo expression. Methods: Primers were designed based on 4 Em-apo sequences from GeneDB. Primer specificity and PCR efficiency were determined, based on which the optimal primer pairs were selected. Alterations of Em-apo expression in 1 000 E. multilocularis protoscoleces treated with albendazole（5 µg/ml） and insulin（100 ng/ml） were separately assessed using the selected primers. DMSO used in albendazole dilution and in PBS insulin dilution were used as the control. Results: Specific primers for Em-apo-1, Em-apo-2/3, Em-apo-4 and actin were selected. qPCR melting curves revealed a single peak for each primer pair and an amplification efficiency from 95% to 101%. The qPCR showed increased expression of Em-apo-1（1.51±0.27）, Em-apo-2/3 （1.39±0.30） and Em-apo-4（1.14±0.18） after albendazole treatment in comparison to the DMSO control（1.00）（P>0.05 among the three genes）; and an unaltered Em-apo-1 expression, slightly decreased Em-apo-4 expression, and significantly decreased Em-apo-2/3 expression（0.73±0.09） after insulin treatment in comparison to the PBS control (P>0.05 among the three genes）. Conclusion: The selected specific primers for Em-apo genes can be used to analyze the gene expression by qPCR. Treatment with albendazole and insulin show certain effects on the expression of Em-apo genes in E. multilocularis protoscoleces.

MRI quantitative study and pathologic analysis of crush injury in rabbit hind limb muscles.

BACKGROUND: To explore the value of quantitative magnetic resonance imaging (i.e., T2 map technique) in the diagnosis of crush injury in rabbit hind limb muscles. MATERIALS AND METHODS: We established a rabbit hind limb crush injury model and performed examinations on magnetic resonance imaging (MRI) (T1WI, T2WI, and T2 map), muscle pathology, serum level of muscular troponin (sTnI), and urine myoglobin (Myo) at 1, 3, 7, 14, and 30 d after injury to investigate the correlation of MRI, library examination, and histopathology. RESULTS: T2WI of the injured muscle showed high signal intensity at 1, 3, and 7 d after crush injury and the T2 value continued to rise. The pathologic findings of the muscle included swollen and ruptured cells, expanded extra-cellular space, inflammatory reactions, and fine muscle fiber regeneration. The serum sTnI and urine Myo were high. At 14 d, these indices returned to normal gradually. The T2WI changes and T2 value were positively associated with the pathological changes of the muscles, serum sTnI and urine Myo. However, the signal intensity of T1WI did not vary significantly at different time points. CONCLUSION: T2WI and T2 value from T2 mapping are very useful methods of choice to evaluate the distribution and extension of the affected muscles. The high signal intensity on T2WI of the affected muscles after crush injury may be due to an increased extracellular space, local inflammation, and incomplete muscle fiber regeneration.

Protective effects of ischemic postconditioning on intestinal mucosa barrier function in rabbits with crush injury of hind limb: an experimental study.

OBJECTIVE: To explore the protective effects of two types of ischemic postconditioning (IP) on intestinal mucosa barrier in rabbits with crush injury of the hind limb. METHODS: This study was conducted between August and December 2008 in the Department of Trauma Surgery, Daping Hospital, Third Military Medical University, Chongqing, China. The model of crush injury to the hind limb of rabbits was firstly developed by a 25 kg object with the right hind limbs fixed by wooden splints, and then two types of IP were established, including occluding/opening the common iliac artery and vein alternatively (traditional IP, IP A) and binding/loosening the proximum of the injured hind limb alternatively (modified IP, IP B). Thirty-six male New Zealand white rabbits were randomly divided into three groups: IP A group, IP B group and control group, with 12 rabbits in each group. The serum levels of diamine oxidase (DAO) and intestinal fatty acid-binding protein (I-FABP) were detected at 2, 6, 12 and 24 hours after injury. Pathological changes of ileum were examined at 24 hours after injury. RESULTS: The serum levels of I-FABP at 2, 6, 12 and 24 hours after injury in both IP A and IP B groups had a significant decrease, compared with control group. DAO levels also showed the same change trend at 2 and 6 hours after injury, but showed no significant difference between two IP groups. No difference in pathological changes of ileum was found among the three groups. CONCLUSIONS: IP can protect intestinal mucosa barrier function on the model of hind limb crush injury in rabbits. Meanwhile the modified IP B shows the same protection as the traditional IP A, and is worth applying in clinic.

[Construction, expression and immunogenicity of eukaryotic vectors based on goat pox virus P32 gene].

The full-length P32 gene and the truncated P32 gene (MP-32) were amplified from the recombinant plasmid pMD-P32 by polymerase chain reaction (PCR) and cloned into pcDNA3. 1(+) and pcDNA3.1-CpG respectively. The recombinant plasmids (pcDNA3.1-P32, pcDNA3.1-CpG-P32 and pcDNA3. 1-CpG-MP32) were transfected into BHK-21 cells by using lipofectin. The expressed P32 protein was confirmed by indirect immunofluorescence assay (IFA). The BALB/c mice were immunized with these recombinant plasmids by intramuscular injection. The specific antibodies aginst CPV were detected by ELISA kit weekly. The murine splenic T lymphocyte subgroups CD4+ and CD8+ were detected by flow cytometry. Results showed that the P32 protein was expressed successfully in vitro. After 2 weeks post im munization, the specific IgG antibodies against CPV were detected in the vaccinated mice. The percentage of CD4+ /CD8+ T cells was significantly higher than that of the control. In conclusion, these constructed eukaryotic vectors could induce humoral and celluar immune responses in mice.

[High-level expression of the potential vaccine antigen TSO18 of Taenia solium in Pichia pastoris].

TSO18 gene was subcloned into the Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-TSO18 was transformed into P. pastoris GS115 by electroporation so that the plasmid will be integrated with chromosome of P. pastoris. The P. pastoris strains containing multi-copy recombinant were screened by G418 and induced by methanol. The expression product was analyzed by SDS-PAGE, Western blot, deglycosylation, and purified by Sephadex column, and was used to immunize mice. The results indicated that the target protein was efficiently expressed in P. pastoris, and glycosylated moderately, and had immunological activity. In a 5 liter fermentor, the expression level of the target protein was up to 2.54 mg/mL. These results will benefit for the development of genetically engineering vaccine.