Dissertation
Dissertation > Agricultural Sciences > Aquaculture, fisheries > Aquatic basic science > Aquatic Biology > Aquatic Zoology

Cloning and Expression Analysis of NCCRP-1 AND IL-10 Genes in Grass Carp (Ctenopharyngodon Idellus)

Author DaiLiPing
Tutor JianJiChang
School Guangdong Ocean University
Course Aquaculture
Keywords Ctenopharyngodon idellus NCCRP-1 gene IL-10 gene prokaryotic expression immunohistochemistry
CLC S917.4
Type Master's thesis
Year 2011
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NCCRP-1 and IL-10 genes of grass carp (Ctenopharyngodon idellus) were cloned and expressed. Total RNA was extracted from the kidney in grass carp. The cDNA sequences of NCCRP-1 and IL-10 genes were obtained by homology cloning and RACE-PCR techniques. Then, NCCRP-1 and IL-10 genes were expressed in E.coli and analysed.NCCRP-1 gene was 900 bp long with 3 bp 5’UTR,183 bp 3’UTR and a 714 bp ORF which coded for 237 amino acids. NCCRP-1 in grass crap had the highest identity to NCCRP-1 in Cyprinus carpio, which is 88%. But the identity of NCCRP-1 in grass carp and others in mammals was very low. Prokaryotic expression systems of thepET-32a/Epin-NCCRP-1 was constructed and expressed in E.coli. The fusion protein was expressed mostly as inclusion body and purified by HisTrap HP affinity column. The western-blot analysis showed that the expressed proteins were definitely confirmed to be the aim proteins. NCCRP-1 had a molecular weight of about 47.8 kDa. Rabbit antiserum against fusion NCCRP-1 was acquired. The titer was 1:4000 assayed by the ELISA method. The analysis of immunohistochemical mathed showed that NCCRP-1 proteins in head kidey and spleen tissues cells were mainly distributed in the cytoplasm.IL-10 gene was 1365 bp long with 274 bp 5’UTR,501 bp 3’UTR and a 540 bp ORF which coded for 179 amino acids. IL-10 in grass crap had the highest identity to IL-10 in Hypophthalmichthys molitrix, which is 99%. But the identity of IL-10 in grass carp and others in mammals was very low. Prokaryotic expression systems of the pET-32a/Epin-IL-10 was constructed and expressed in E.coli. The fusion protein was expressed mostly as inclusion body and purified by HisTrap HP affinity column. The western-blot analysis showed that the expressed proteins were definitely confirmed to be the aim proteins. IL-10 had a molecular weight of about 41.5 kDa. Rabbit antiserum against fusion IL-10 was acquired. The titer was 1:4000 assayed by the ELISA method. The analysis of immunohistochemical mathed showed that IL-10 proteins in head kidey and spleen tissues cells were mainly distributed in the cytoplasm. The study provided theoretic base for the prevention of the diseases in grass carp and enriched the research of the molecular immunity in fish. Furthermore, the study established the foundation for the study on the biologic characterestic and the function of NCCRP-1 and IL-10 in fish innate immunity.

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