Crustacean expression vector

Abstract

Methods and constructs for genetic manipulation of one or more of shrimp, shellfish, mollusks, and fish are disclosed. The nucleic acid construct includes a promoter and an internal ribosome entry site of an insect picomavirus, such as a cricket paralysis-like picomavirus. One or more open reading frames can be operably associated with one or both of the promoter and the internal ribosome entry site, and one or more proteins or protein subunits can be expressed upon introduction of the construct into a host cell, such as into a shrimp. Method for producing immortalized crustacean cell lines using enhancer elements derived from shrimp and/or shrimp viruses are also described.

1 . A non-naturally-occurring nucleic acid construct for expressing a cistron in a crustacean cell, the construct comprising a virus promoter and an insect picomavirus (IPV) internal ribosome entry site (IRES), wherein the promoter and the IRES are selected such that when the cistron is operably linked with the promoter and the IRES, the cistron is capable of expression in the cell. 
     
     
         2 . The construct of  claim 1 , wherein the promoter and the TRES are derived from the same IPV. 
     
     
         3 . The construct of  claim 1 , wherein the promoter is a promoter of a cricket paralysis-like picomavirus. 
     
     
         4 . The construct of  claim 1 , wherein the IRES is a IRES of a cricket paralysis-like picomavirus. 
     
     
         5 . The construct of  claim 1 , wherein the IRES is the IRES of the 5′-untranslated region of a cricket paralysis-like picomavirus. 
     
     
         6 . The construct of  claim 1 , wherein the IRES is the IRES of the intergenic region of a cricket paralysis-like picomavirus having at least two ORFs. 
     
     
         7 . The construct of  claim 1 , wherein the promoter is an infectious hypodermal and hematopoietic necrosis virus (IHHNV) promoter. 
     
     
         8 . The construct of  claim 1 , wherein the IRES is a Taura syndrome virus (TSV) IRES. 
     
     
         9 . The construct of  claim 8 , wherein the IRES is the IRES of the 5′-untranslated region of TSV. 
     
     
         10 . The construct of  claim 8 , wherein the promoter is an IHHNV promoter. 
     
     
         11 . The construct of  claim 10 , wherein the promoter is at least one of the P2 and P61 promoters of IHHNV. 
     
     
         12 . The construct of  claim 8 , wherein the promoter is a white spot syndrome virus (WSSV) promoter. 
     
     
         13 . The construct of  claim 1 , wherein the promoter is a promoter of an IPV that infects shrimp. 
     
     
         14 . The construct of  claim 1 , wherein the IRES is an IRES of an IPV that infects shrimp. 
     
     
         15 . The construct of  claim 14 , wherein the promoter is a promoter of an IPV that infects shrimp. 
     
     
         16 . The construct of  claim 1 , wherein the construct further comprises an animal cell origin of replication site (ORS). 
     
     
         17 . The construct of  claim 16 , wherein the animal is a crustacean. 
     
     
         18 . The construct of  claim 17 , wherein the crustacean is a shrimp. 
     
     
         19 . The construct of  claim 18 , wherein the shrimp is a  Penaeus  shrimp. 
     
     
         20 . The construct of  claim 1 , further comprising a first open reading frame (ORF) operably associated with the promoter.