Screening Of The Receptor Gene Of WSSV Using Yeast Two-Hybrid Systems

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, so, Understanding the infection mechanism and finding the gene of receptor is very important for the treatment of viral disease.The study of screening gene of receptor was peforming in this experiment.In this research the author constructed a full-length cDNA library using total RNA from gill of Fenneropenaeus chinensis by homologous recombination. Total RNA was isolated by the NucleoSpin?RNA II Kit.The first-strand cDNA was synthesized by using MMLV reverse transcriptase with CDS III Oligo(dT) primers and sequencely switching template. The double-strand cDNA were synthesized and amplified by long-distance PCR (LD PCR).The ds cDNA was purify with a BD CHROMA SPIN?TE-400 Columns. The ds cDNA and pGADT7-Rec vector were ligation by homologous recombination method, and transfer competent cells of AH 109 strain. According to calculating, the cDNA library contained 2.1xl06 clones and the titer of the amplified library was 7.3 107 cfu/ml.By using PCR identification, the cDNA library contained approximatedly 90% recombinant clones.The two DNA-BD Fusion Vectors of VP28 and VP281 were constructed The PCR primers which encompass the whole gene of VP28 and VP281 in WSSV genome, meanwhile included the endonuclease site Nde I, Pst I; After digestion with endonuclease, the PCR products were ligated into pGBKT7, and then transformed E. coli DH5a. Identify insert-containing plasmids by restriction and PCR. Extracting bait plasmid and transformed competent yeast cells of AH109 or Y187. The result of testing the DNA-BD Fusion for Transcriptional Activation indicated that VP281 is active which will not used in a two-hybrid library screening and VP28 is inactive which is suitable for screening.Mating the library host strain with the bait strain containing VP28 and Select for yeast diploids expressing interacting proteins in TDO and QDO medium.At last6 positive clones were obtained. By sequencing, 2 cDNA fragments were screening.The size of cDNA inserts are 105bp and 360bp. The two cDNA fragments have no significant homologous sequence by comparing with GeneBank Database using the BLAST program in www.ncbi.nlm.nih gov. The analysis of open reading frame (ORF) indicated that the two cDNA sequences are not complete.