Cloning And Expression Of Prawn Coat-ε, Clathrin Coat AP17 And Clathrin Heavy Chain And Its Role In WSSV Infection

The main objects of this paper are Fenneropenaeus chinensis Coat-ε, Litopenaeus vannamei clathrin coat AP17 and prawns clathrin heavy chain. The full-length sequences of coat-ε, clathrin coat AP17 and clathrin heavy chain were cloned and the role of gene in WSSV infection was studied. This paper contains the following three parts:Part one: To obtain the full-length sequence of coat-ε, the rapid amplication of cDNA ends(RACE) was performed to get the sequence of 3’ and 5’ end. The full-length sequence of Coat-ε is 1402 bp, and the open reading frame is 1008 bp which is predicted to code 335 amino acids. The amino acid sequence has no signal peptide and transmembrane structure. Phylogenetic trees were constructed. RT-qPCR analysis indicated that Coat-ε was expression in all tested tissues, and the highest expression was showed in muscle. The recombinant expression vector pBAD/gIIIA-Coat-ε was constructed. The expressed protein was agreed with the expected size by SDS-PAGE detection. Far-Western blotting and ELISA analysis showed that the coat-ε interacted with WSSV structural protein VP26, VP28 N, VP28 C. Flow cytometry and cell ELISA assay indicated that Coat-ε protein antibody had an inhibitory effect for WSSV binding with hemocytes. Neutralization experiment in vivo showed that coat-ε was related with the expression of immediate early gene 1(ie1) and late gene VP28, and Coat-ε antibody delayed the WSSV infection.Part two: To obtain the full-length sequence of clathrin coat AP17 of Litopenaeus vannamei(LvCCAP17), RACE technique was performed to get the sequence of 3 ’and 5’end and splicing by DNAMAN. The full-length sequence of LvCCAP17 is 842 bp which is encoding 142 amino acids. The amino acid sequence of LvCCAP17 has no signal peptide and transmembrane structure. Furthermore, the phylogenetic tree was constructed. RT-qPCR analysis indicated that LvCCAP17 was detected in all tested tissues of Litopenaeus vannamei. The transcriptional expression level of LvCCAP17 in epithelium and hepatopancreas was significantly up-regulated after WSSV infection. The recombinant expression vector pBAD/gIIIA-AP17 was constructed. The expressed protein was agreed with expected size through SDS-PAGE detection and mass spectrometry analysis confirmed that the recombinant protein was AP17. Far-Western blotting and ELISA assay showed that LvCCAP17 interacted with rVP26 and rVP37. Silencing of LvCCAP17 gene by double-strand RNA(dsRNA) interference significantly delay of cumulative mortality rate in WSSV infected shrimp and reduced the expression level of ie1 and vp28. These results indicated that clathrin-meated endocytosis is responsible for WSSV infection.Part three: The full-length of Fenneropenaeus chinensis clathrin heavy chain(FcCHC) and Litopenaeus vannamei clathrin heavy chain(LvCHC) were amplified according to the primers based on CHC of Penaeus monodon. The full length of LvCHC and FcCHC is 5052 bp, encoding 1684 amino acids. Bioinformatics analysis indicated that these two proteins are hydrophobic protein and have no signal peptide and transmembrane structure. Domain analysis found that the domain of LvCHC and FcCHC have a great different with CHC of other species. RNAi experiments showed that silencing of LvCHC reduced the cumulative mortality rate in WSSV infected shrimp. Results suggested that LvCHC is involved in the infection of WSSV.