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Construction of the Expression Vector with a Promoter of Immediate-early Gene (ie1) from White Spot Syndrome Virus and Application of the Vector in Gene Function Analysis

Author: MouYi
Tutor: WangJinXing
School: Shandong University
Course: Zoology
Keywords: White spot virus IE1 promoter Eukaryotic expression vector C- type lectin Bacterial clearance Procambarus shrimp
CLC: S945.1
Type: Master's thesis
Year: 2011
Downloads: 20
Quote: 0
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Abstract


White spot disease in shrimp aquaculture, causing huge losses, the pathogen WSSV (white spot syndrome virus, WSSV). Host the infection process, the envelope protein VP28 plays a key role. It has been reported recombinant VP28 or VP28 expression plasmid has significant antiviral activity; while the envelope protein VP19 in some studies demonstrated its antiviral activity, and some studies show it has no antiviral activity. In order to further the study of anti-virus VP28 function to find an effective way to control methods, and to determine the function of VP19, we constructed containing respectively of WSSV iel promoter and VP19 or VP28 expression vector (pIevp28, pIevp28). The vectors were injected Procambarus shrimp, through real-time quantitative PCR and Western blot and other methods can be found in different persistent expression plasmid vector VP19 or VP28, since the fifth day stronger signal can be detected and continued until the thirtieth day while in the event of the virus after infection, compared with the control group injected plasmid VP28 crawfish mortality was significantly decreased after infection, and virus replication in the body is suppressed. Although the VP19 plasmid can Crawfishes transcription, but can not inhibit viral replication, no significant mortality differences in the control group. Immunohistochemistry results showed VP28 proteins and blood cells, membrane binding, and VP19 not. Based on these results, we believe that VP28 may be through competitive inhibition of the effects on the protection crawfish. Feeding E. coli containing the plasmid (E. coli) can also reduce the crawfish mortality. This may serve as a new approach to prevention and treatment of white spot virus applications. Then we take into account whether the plasmid can be used to study protein expression crawfish its function, so he chose Procambarus shrimp from a C-type agglutination always experiment. C-type lectin as a pattern recognition receptors, such as the recognition of foreign pathogens infection plays a very important role. In our study, Procambarus shrimp from cloned into a new body of C-type lectin called PcLec4. PcLec4 comprises a 17 amino acid residues of the signal peptide followed by a carbohydrate recognition domain (carbohydrate recognition domain, CRD). By white spot virus in crayfish or anguillarum (Vibrio. anguillarum) after stimulation of the C-type lectin mRNA levels showed significant upward trend on. After recombinant expression of the C-type lectin proteins do not agglutinate bacteria, but the bacteria in vitro binding assay, for both Gram-positive bacteria or Gram-negative bacteria have demonstrated a strong binding activity. After recombinant protein can promote the body to invade the body anguillarum clearance, in order to study the in vivo activity of lectins, we use contain white spot syndrome virus immediate-early genes (immediate-early gene) in the core promoter ie1 Regional expression vector, the PcLec4 open reading frame is inserted into the plasmid vector after iel promoter. The carrier has been tested in vivo overexpression crawfish PcLec4, and injected the expression vector crawfish compared with the control group, after the invasion when anguillarum showed a faster clearance rate and thus help the body to resist pathogen invasion. To our knowledge this is the first use of the so far this plasmid to express a crawfish C-type agglutination always study its function.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Fisheries Protection > Pest and Disease Control of the crustaceans > Shrimp
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