To construct secretory expression vector of PPV NS1 gene, the fragment of PPV NS1 gene coding for major antigen region of the NS1 protein was amplified by PCR and inserted into multiple clone site of eukaryotic expression vector pPICZalpha-A. The recombinant pPICZalpha-A-NS1 plasmid was transferred into P. pastoris strain GS115 mediated by electro transform. Recombinant P. pastoris strain GS115 was induced to express the fusion protein by methanol. The expressed and purified protein was analyzed by SDS-PAGE and Western Blot. The recombinant protein was highly-expressed and showed a good immunoreactivity. The indirect ELISA method was developed for detecting antibodies against PPV by checkerboard titration assay. The result showed that the optimal concentration of coated antigen was 3.2 microg/mL and the best dilution of serum was 1 : 80. The positive cut-off value of the ELISA assay was OD450 > 0.4 and OD450 positive serum/OD450 negative serum > 2.0. Compared with HI and commercial ELISA kits, the assay revealed 94.2% and 92.1% agreement respectively. The assay demonstrates good specificity and sensitivity, and can be applied in the detection of porcine parvovirus.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Parvoviridae Infections ; diagnosis ; immunology ; veterinary ; virology ; Parvovirus, Porcine ; genetics ; immunology ; isolation & purification ; Recombinant Proteins ; genetics ; immunology ; Swine ; Swine Diseases ; diagnosis ; immunology ; virology ; Viral Nonstructural Proteins ; genetics ; immunology

Objective:This study aimed to construct a lentiviral expression vector for microRNA-194 and investigate its effect on the metastasis of human osteosarcoma cell line U2-OS. Methods:Pri-and mature miR-194 amplified by PCR were inserted into the plenty-GFP vector and identified by restriction endonuclease digestion and nucleotide sequencing. The osteosarcoma cell line U2-OS was transfected with the lentivirus. Then, the stable transfected cells were used in Transwell and wound healing assay. Results:Restric-tion analysis and sequencing showed that the recombinant lentiviral expression vector was constructed correctly. The titers of obtained overexpression and suppression expression recombinant lentivirus were 1.5*108 and 4*108 TU/ml. Cell metastasis ability was signifi-cantly different in different experimental groups (P<0.01). Conclusion:The lentiviral expression vector for microRNA-194 was suc-cessfully constructed. MicroRNA-194 could influence the metastasis of the osteosarcoma cell line U2-OS;thus, it could be further ex-plored as a potential target in osteosarcoma therapy.

The aim of this study is to establish the method of loop-mediated indirect PCR assay for detection of Reproductive and Respiratory Syndrome Virus (PRRSV) infection and differentiation of highly pathogenic PRRSV (HP-PRRSV) and lowly pathogenic PRRSV (LP-PRRSV). Based on the alignments of ORF2 gene sequences and ORFla gene sequences of PRRSV Chinese isolates deposited in GenBank, two pairs of specific probes were designed and labeled to both ends of the soybean Lectin gene fragment by PCR, respectively. The probe-labeled soybean Lectin genes were used to be reporter genes for detection and differentiation of PRRSV. After one round strand displacement reaction, the reporter genes were amplified by reverse PCR. The specific PCR products were 193bp, 355bp for HP-PRRSV and 193bp, 442bp for LP-PRRSV, respectively. The method could detect 5. 6 TCID50/mL LP-PRRSV RNA and 18 TCIDs0/ mL HP-PRRSV RNA, and co-infection did not affect detection sensitivity. No amplification was observed with other porcine originated pathogens including CSFV, PPV, PRV, PCV2, ETEC and Haemophilus parasui. Twenty clinical samples were used for comparative testing with conventional PCR. Fourteen samples were found positive for PRRSV by the loop-mediated indirect PCR, of which 4 were LP-PRRSV, 9 HP-PRRSV and 1 LP/HP-PRRSV co-infection, consistent with the conventional PCR test results. In conclusion, the loop-mediated indirect PCR is a simple, rapid, sensitive and specific etiologic diagnosis tool, and suitable for the differential diagnosis of HP/LP-PRRSV, especially for identification of mixed infection of HP/LP-PRRSV.
Animals ; Coinfection ; veterinary ; DNA Primers ; genetics ; DNA, Complementary ; genetics ; Diagnosis, Differential ; Genes, Reporter ; Genetic Markers ; genetics ; Humans ; Porcine Reproductive and Respiratory Syndrome ; diagnosis ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; isolation & purification ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; veterinary ; Sensitivity and Specificity ; Swine ; Time Factors ; Viral Proteins ; genetics

Objective To investigate the effects of RNA interference (RNAi) targeting angiotensin Ⅱ Type 1 receptor (AT1R) and angiotensin-converting enzyme (ACE) on blood pressure and myocardial remodeling in spontaneous hypertensive rats (SHRs). Methods Saline (control), adenovims (Ad5) and recombinant adenoviral vectors (Ad5-ACE-shRNA, Ad5-AT1R-shRNA and Ad5-ACE-AT1R-shRNA expressing ACE, AT1R, ACE and AT1R gene-specific shRNA, respectively) were randomly administered by caudal intravenation to SHBs (n = 12 each group) at day 1 and 17. Normotensive Wistar-Kyoto rats (WKY) served as normal controls. Systolic blood pressure (SBP) of the caudal artery was measured daily. Expression of ACE and AT1R at mBNA levels in ventricle and aorta were evaluated by fluorescence quantitative PCR. Angiotension Ⅱ serum concentration was measured by ELISA at day 3 (n = 6 each group). The ratio of left ventricular to body weight (LVW/BW) and myocardial collagen content were measured, myocardial ultrastructure observed under transmission electron microscope at the study end. Results The caudal artery pressure of saline and Ad5 group was equally increased by about 26 nun Hg(1 mm Hg=0.133 kPa) compared to baseline (both P <0.05). Ad5-ACE-shRNA,Ad5-AT1R-shRNA and Ad5-ACE-AT1R-shRNA injection significantly reduced SBP (-24 mm Hg, -22 mm Hg and -26 mm Hg respectively, all P<0.05 vs. baseline) and the antihypertensive effect could last at least 15 days post each injection. SBP was not affected by saline and Ads injections. ACE and AT1 mRNA expressions at ventricle and aorta were significantly decreased in Ad5-ACE-shRNA, Ad5-ACE-AT1R-shRNA and Ad5-AT1R-shRNA, Ad5-ACE-AT1R-shRNA treated SHRs compared to those in saline and Ad5 groups (all P < 0.05) and was comparable to that in WKY group (P> 0.05). The LVW/BW ratio [(2.22±0.18)μg/mg, (2.23 ±0.19)μg/mg, (2.17±0.16) μg/mg] and myocardial collagen content [(1.291±0.019)μg/mg, (1.298±0.019) μg/mg, (1.276±0.019)μg/mg] in Ad5-ACE-shRNA, Ad5-AT1R-shRNA and Ad5-ACE-AT1R-shRNA treated SHBs were also significantly lower than those in saline treated [(3.23±0.13) μg/mg and(1.683±0.013) μg/mg, both P<0.05] and Ad5 treated SHRs [(3.25±0.12) μg/mg and (1.693±0.013) μg/mg, both P<0.05], but still higher than those of WKY group [(2.06±0.12) μg/mg and (1.258±0.019) μg/mg, both P < 0.05]. Myocardial ultrastructure was also significantly improved in all SHRs underwent RNAi treatments compared to saline and Ad5 treated SHRs. Conclusion RNAi targeting ACE and AT1R gene significantly inhibited myocardial and aortic ACE and AT1R mRNA expressions and resulted in prolonged antihypertensive effects and myocardial ultrastructure improvements in SHRsl. The RNAi technology may be a potential new strategy of gene therapy for hypertension.

ObjectiveTo establish blood stasis models in zebrafish using three inducers and select the optimal model for evaluating the activity of Notoginseng Radix et Rhizoma in promoting blood circulation. MethodArachidonic acid （AA）， ponatinib， and isoprenaline （ISO） were used to induce blood stasis models in zebrafish. A normal group， a model group， a positive drug group， and Notoginseng Radix et Rhizoma water extract freeze-dried powder groups at different concentrations were set up. The staining intensity of cardiac erythrocytes and the fluorescence intensity of cardiac apoptotic cells were calculated， the anti-thrombotic effect and anti-myocardial hypoxia activity of Notoginseng Radix et Rhizoma were evaluated. The activities of water extract and 70% methanol extract of Notoginseng Radix et Rhizoma were compared based on the preferred AA- and ISO-induced blood stasis models in zebrafish and the difference in the chemical composition was analyzed by UHPLC LTQ-Orbitrap MS/MS. ResultAfter induction by AA and ponatinib， the staining intensity of cardiac erythrocytes was reduced （P<0.01）， and the fluorescence intensity of cardiac apoptotic cells increased after the induction by ISO （P<0.01）. The freeze-dried powder of the water extract of Notoginseng Radix et Rhizoma could antagonize the thrombosis in the AA-induced model （P<0.01） and the myocardial apoptosis in the ISO-induced model （P<0.05）， while no significant improvement in the thrombosis was observed in the ponatinib-induced model. The freeze-dried powder of 70% methanol extract of Notoginseng Radix et Rhizoma could inhibit myocardial apoptosis in the ISO-induced blood stasis model （P<0.01）， and the effect was stronger than that of the freeze-dried powder of Notoginseng Radix et Rhizoma water extract. The difference in chemical composition lay in some saponins （such as ginsenoside Re）， amino acids， and acetylenic alcohols. ConclusionAA， ponatinib， and ISO all can serve as inducers for the blood stasis model in zebrafish. AA- and ISO-induced models can be used to evaluate the activity of freeze-dried powder of Notoginseng Radix et Rhizoma water extract in promoting blood circulation. The chemical compositions of the freeze-dried powders of Notoginseng Radix et Rhizoma extracted with water and 70% methanol are quite different. For the ISO-induced blood stasis model， the freeze-dried powder of Notoginseng Radix et Rhizoma extracted with 70% methanol has a stronger ability against myocardial hypoxia. Saponins and acetylenic alcohols may be closely related to the effects of promoting blood circulation and resolving blood stasis.