ABSTRACT
The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log2, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.
Subject(s)
Animals , Antibodies, Viral , Chickens , HN Protein/metabolism , Newcastle Disease/prevention & control , Newcastle disease virus/metabolism , Oryza/geneticsABSTRACT
To study the effect of chitosan on delayed outward potassium current(IK) in single ventricular myocytes of guinea pig and investigate its antiarrhythmic mechanism from ion channel view. Patch clamp technique with whole-cell configuration. Holding potential was -40mV,commanding potential was -60- + 70mV ,step pulse +10mV,stimulating frequency 1 Hz,duration 300 ms and stimulating interval 6s. The result showed that Chitosan inhibited IK in a dose -dependent manner. Conclusion :Chitosan can inhibite IK in single ventricular myocytes of guinea pig.
ABSTRACT
AIM To investigate the effect of N methyl berbamine(NMB)on ATP sensitive potassium currents( I KATP )in single ventricular myocytes of guinea pig. METHODS Patch clamp technique with whole cell configuration. Holding potential was -40 mV, commanding potential was -100～+50 mV and duration was 600 ms. Pipette solution contained 0 3 mmol?L -1 ATP. RESULTS NMB inhibited I KATP in a concentration dependent manner. When holding potential was -40 mV and command potential was 0 mV, I KATP were reduced from (0 46?0 09) nA, (0 43?0 15) nA, and(0 47?0 10) nA to (0 37?0 07) nA( n=4,P