| White spot syndrome virus is a major pathogen of shrimp farming, and hamper the shrimp aquaculture industry severely. The binding of Virus and receptor is the first step for entry of virus, then it will reproduce in the host cells. Understanding the infection mechanism and finding the gene of receptor is very important for the treatment of viral disease. The gene of virus attachment protein 37 is cutted and used in the yeast two-hybrid system III in this study, aiming at screening and identification of the receptor gene.VAP1 is one of the attachment proteins which has binding activity in the envelope. It was tested that VAP1 as bait protein in the yeast two-hybrid system had self-activity. There are transmembrane and hydrophobicity region in the C-end of VAP1, then we plan to cut it which corresponds with 34 amino acids of the C-end. In the genome of WSSV, 102 bases was cutted in the gene of VAP1, a pair of primer was designed by using primer premire software, meanwhile the endonuclease site Nde I and Pst I was included; After digestion with endonuclease, the PCR products were ligated into pGBKT7,and then transformed the competent E. coli DH5α. After the extraction of the transformed plasmid, PCR and double-endonuclease was used to identify it. After ensuring the recombinant, we measured the sequence and the rusult is right. The recombind plasmids were extracted and transformed the competent cells: AH109 and Y187. Dropout substrate had been used to identify it and the result is that the bait fusion protein expressed did not act the reportor genes of HIS3 and ADE2, but acted MEL1. HIS3 and ADE2 can be used in the yeast two-hybrid system.We used AH109 [cDNA library] and Y187 [pGBKT7-VAP1] to do yeast fusion. After screening in the dropout medium of TDO and QDO, and culturing repeatly in QDO, and identifing by PCR, one positive clone was obtained. The cDNA fragment was sequenced and it has 649 bases. Analysing the sequence at +2 ORF, the sequence has one ORF before TAA, which may be the protein coded by the DNA. Analysing it by BLASTP in NCBI, the protein it expressed has the highest homology with an unnamed protein product (59%). Thehomology with protease is 38%, and the cell wall mannoprotein acceptor of B1-6 glucan is 26%. In all probability, the protein is one kind of protease from the binding of virus and branchial membrane protein. Their binding is very special like the enzyase and substrate.The branchial of shrimp is the target organ of WSSV, the receptor protein which can combine with virus should exist among the branchial membrane protein of shrimp, so it's very important of branchial membrane protein to study the interaction mechanism of WSSV and receptor. It has been approved that P53 branchial membrane protein has combining activity with WSSV by ELISA. P53 branchial membrane protein had been measured by MALDI-TOF-MS-MS mass spectrum, and the function of the conserved domain of it had been analysed by BLASTP.This study altered the gene of VAP1 for the first time and screened the gene interacting with it by using yeast two-hybrid system which is initiated to study the receptor protein of WSSV. It's a new found that the protein expressed by the gene has high homology with protease by using bioinformatics. P53 branchial membrane protein of shrimp has high homology with ATP synthase P-subunit by analyzing spectrum. The function and binging activity of the protein need to be further analyzed through clone and expression. |
