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[Expression of NDV HN protein in rice and development of a semi-quantitative rapid method for detection of antibodies].
- Source :
-
Sheng wu gong cheng xue bao = Chinese journal of biotechnology [Sheng Wu Gong Cheng Xue Bao] 2022 May 25; Vol. 38 (5), pp. 1981-1993. - Publication Year :
- 2022
-
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 Mly Ⅰ and Xho Ⅰ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by Nae Ⅰ and Xho Ⅰ. 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 Eco RⅠ and Hin d 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≥4log <subscript>2</subscript> , 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.
Details
- Language :
- Chinese
- ISSN :
- 1872-2075
- Volume :
- 38
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Sheng wu gong cheng xue bao = Chinese journal of biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 35611743
- Full Text :
- https://doi.org/10.13345/j.cjb.210629