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

White spot disease caused by white spot syndrome virus (WSSV) leads to devastating losses in shrimp farming. The WSSV envelope proteins, such as VP28 and VP19, can be used as subunit vaccines that can efficiently protect shrimp against WSSV disease. However, other reports found that VP19 has no protection against WSSV. To detect the functions of VP28 and VP19 and find a method to prevent this disease, plasmid vectors plevp28 and plevpl9, which efficiently expressed VP28 or VP19 in red swamp crayfish Procambarus clarkii, were constructed. The results of quantitative real-time PCR and western blot show that after injection into the crayfish, the vectors expressed VP28 or VP19 in the crayfish. The vp28 or vpl9 signal was detected on the fifth day post injection, and maintained its expression for 30 days. The mortality of the crayfish with plevp28 showed obvious decline compared with the controls (pIe and PBS injection). However, plevp19 did not affect the mortality of the crayfish compared with the controls. Furthermore, only VP28 was found tightly bound to the host haemocytes under immunocytochemistry. The results suggest that the VP28 protein might protect shrimp from the virus through competitive inhibition. Oral administration of Escherichia coli containing plevp28 could also protect crayfish from white spot disease. The vector plevpl9 was not effective against WSSV. This vector may be developed into a feasible way to prevent and cure white spot disease in aquaculture.Then we choose a C-type lectin from crayfish to detect its function by over expression this protein using the vector with a promoter of immediate-early gene (iel) from white spot syndrome virus. In innate immunity of invertebrates, C-type lectins show their important activity in defending the invasion of pathogenic microorganisms. In our study, a novel C-type lectin called PcLec4 is cloned from the red swamp crayfish Procambarus clarkii. PcLec4 contains a putative signal peptide of 17 amino acids followed by a single carbohydrate recognition domain (CRD). The mRNA level of PcLec4 increased after infection of WSSV or Vibrio anguillarum. Recombinant PcLec4 couldn't agglutinate neither Gram-positive nor Gram-negative bacteria, but it could bind to all of them. A vector with the promoter of WSSV iel gene was used to express the protein in vivo and a higher tolerance to the pathogenic bacteria was detected in the crayfish injected with the vector. To the best of our knowledge, this might be the first to use this kind of vector to over express a C-type lectin to detect its function in crayfish.