Expression Of Three Tetraspanins And Their Roles In WSSV Infection

White Spot Syndrome Virus(WSSV) is currently the most serious shrimp pathogens, which has brought huge losses to shrimp industry worldwide. So far there is no effective treatment to this virus. So exploring the mechanism and pathogenesis of WSSV infection is essential for finding way to control WSSV. Tetraspanin are conservative four transmembrane glycoproteins that are widely expressed in many species. By forming a protein web, tetraspanin involved in many activities, such as cell growth, signal transduction, as well as they play important roles in the immune processes like pathogen infection, tumor cell migration. This paper analyzes the role of tetraspanin in WSSV infection, which concludes the following aspects:Chapter 1. A pair of primers was designed according to the sequence of Tetraspanin-3 gene of Fenneropenaeus chinensis from Gen Bank. The Tetraspanin-3 fragment was amplified by PCR and cloned into E. coli expression vector p BAD/g IIIA successfully. Then the p BAD/g IIIA–T3L was transformed into E.coli-Top 10 cells, and sequenced to confirm the correctness. After L-Arabinose induction at 37 ℃, the protein was purified using Co2+-column affinity chromatography. The expressed protein was confirmed by MALDI-MS assay. The interaction between purified Fc T3 L and WSSV structure protein was analyzed by Far-western-blot, and the result indicated that Fc T3 L can interact with VP26. Furthermore, ELISA assay showed that the interaction activity increased with the amount of protein.Chapter 2. A pair of primers was designed according to the sequence of CD63 gene of Fenneropenaeus chinensis from Gen Bank. The fragment of LEL of Fc CD63 was amplified by PCR and cloned into E. coli expression vector p BAD/g IIIA successfully. Then p BAD/g IIIA–Fc CD63 L was transformed into E.coli-Top 10 cells and induced to express protein. The expressed protein was confirmed by MALDI-MS assay. Western-blot assay indicated that Fc CD63 L can interact with VP28, and both VP28 C fragment and VP28 N fragment can interact with Fc CD63 L. ELISA assay showed that the interaction was correlated with the protein concentration.Chapter 3. CD63 c DNA sequence of Litopenaeus vannamei was cloned using RACE method. The amplified sequence is 1472 bp, with its ORF 744 bp, encoding 247 amino acids. Bioinformatics analysis showed that the sequence of Lv CD63 has 93% similarity with Penaeus monodon and 92% similarity with Fenneropenaeus chinensis. Real- time PCR analysis showed that the m RNA levels of Lv CD63 expressed in the tissues of haemocytes, gill, epithelial, heart, lymphoid, hepatopancreas, stomach, intestines, muscle and nerve. Among these tissues the highest expression level was showed in the tissue of haemolymph, followed by epithelial tissue, hepatopancreas, and nerve, by contrast the muscle tissue showed the lowest expression level. After WSSV challenged, the expression levels in the tissues of gill and epithelial were both up-regulated, however the expression level in hepatopancreas were down-regulated. Western-blot analysis showed that Lv CD63 can interact with VP28, and both VP28 N and VP28 C fragments can interact with Lv CD63. Flow cytometry analysis showed that Lv CD63 has a little distributed in the surface of hemocytes. Neutral Experiments showed that Lv CD63 can protect shrimp from WSSV infection.