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Lipovitellin in Rosy Barb (Puntius Conchonius) is a Pattern Recognition Receptor with an Opsonic Activity

Author: ZhangJun
Tutor: ZhangShiZuo
School: Ocean University of China
Course: Marine biology
Keywords: Yolk protein Lipovitellin Pattern recognition factor Opsonin Antibacterial activity
CLC: S917.4
Type: Master's thesis
Year: 2010
Downloads: 20
Quote: 0
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Abstract


Lipovitellin (Lipovitellin, Lv), is one of the major product of vitellogenin (Vitellogenin, Vg) absorbed by the oocytes after hydrolysis, is a macromolecular sugar lipoprotein. The paper roses without gills (Puntius conchonius) egg material by DEAE-23 ion exchange column chromatography and Superdex 200 gel filtration column chromatography Lv, native polyacrylamide gel electrophoresis (Native-PAGE) LV molecular weight of about 370 kDa, denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showed three bands, the molecular weight is 125 kDa, respectively, 110 - kDa and 76 kDa. Sugar, phosphorus, lipid-specific staining experiments showed that the barb fish Lv rose rich in sugar, fat, and phosphorus content of less. Lv the amino acid composition analysis results show that the taimen, rainbow trout, and medaka fish Lv amino acid composition is very similar to mass spectrometry analysis showed that from the 125 kDa and 110kDa with the resulting peptide sequences with carp yolk fibrinogen B1 LV heavy chain matches from LV light chain of 76 kDa bands resulting peptide sequences with carp vitellogenin B1 match. By amino acid composition analysis and mass spectrometry results can be identified through the double-step chromatography purified protein is indeed rose barb lipovitellin. Barb Lv immunization of New Zealand white rabbits with purified rose polyclonal antiserum found the Lv gender specificity, but anti-the Lv serum can detect female Rose barb plasma protein immunoblotting (Western blotting) antiserum Vg, using double immunodiffusion experiments also found that the tissue-specific Lv. Lv has been considered the major nutrients oogenesis and embryonic development process, but other aspects of the less functional studies. Fish mature the egg routed the water combine to form a fertilized egg and sperm belong to in vitro fertilization, embryonic and larval development in the water, but the water there a lot of fish pathogens and viruses, the immune system not to develop improved embryos and juveniles is a serious threat, the papers proved by experiments Lv also played in the embryonic and larval development stage resist pathogen invasion a opsonized immune recognition factor. Lv fluorescein isothiocyanate (FITC) labeled with Escherichia coli (E. coli) and Staphylococcus aureus (S.aureus) after incubation fluorescence microscopy of E. coli and S. aureus can green fluorescence; eggs the homogenate and Lv solution was incubated with Escherichia coli and Staphylococcus aureus, and then washed to remove unbound proteins, and finally eluted with 4M urea The Western blotting analysis of the binding protein of the bacterial surface, resulting in the eggs homogenate and Lv solution eluate after the two bacterial action can be detected to Lv, proof of the above two experiments LV with Escherichia coli and Staphylococcus aureus can be combined. For a better understanding of the lipovitellin with bacterial binding mechanism, do ELISA assay to investigate the bacterial surface which molecules lipovitellin identification. Experimental results show that, Lv can recognize non-self components, and pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (lipopolysaccharide, LPS), lipoteichoic acid (lipoteichoic acid LTA), peptidoglycan (peptidoglycan, PGN), specific binding, and the combination of a concentration-dependent manner. The above results show Lv is a multivalent pattern recognition receptors. The paper also macrophage phagocytosis of bacteria experiments Lv macrophages engulf bacteria. Define two parameters to determine macrophage phagocytosis phagocytic capacity (phagocytic the Ability, PA), i.e. phagocytic macrophage number of bacteria account for the percentage of the total number of all statistical cells; another phagocytic index (phagocytic index, PI), and that the average number of each statistic phagocytosis of bacteria. The results found that, compared with the control, PA and PI after the Lv processing experimental group has significantly improved (p lt; 0.05), the Lv able to promote macrophage phagocytic the bacterial ability, may have a similar function of opsonin.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquatic basic science > Aquatic Biology > Aquatic Zoology
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