Interaction Of DTMUV E Protein With Cell Protein Detected By Yeast Two-Hybrid System

In 2010, a newly infectious disease caused by Tembusu virus(TMUV) was obeserved in many egg-laying and breeder duck farms in China, resulting in sharply egg production drop in affected egg-laying and breeder ducks and neurological symptoms(ataxia and paralysis) in ducklings. Epizootic investigation revealed that natural infections with TMUV have been reported in a variety of domestic ducks including Peking duck, Cherry Valley duck and Shaoxing duck, Jinyun duck, Longyan duck, Jinding duck and Khaki-Campbell duck. With the different infected ages changed, the mortality rates was between 5%～30%. The morbidity rates was 90%. TMUV infection occurred in almost all duck breeder areas and lead to serious economic loss.DTMUV is a single-strand positive RNA virus, its genome consists of three structural proteins, named capsid(C), pre-membrane(Pr M), envelope glycoprotein(E), as well as seven nonstructural proteins(NS1, NS2 A, NS2 B, NS3, NS4 A, NS4 B and NS5). As the major surface protein of Tembusu Virus, the envelope(E) protein is involved in many events, such as viral attachment, fusion, penetration, host range, cell tropism,virulence and host membrane fusion. Meanwhile the envelope E protein is a major antigen in eliciting neutralizing antibodies during the immune response. To discover new E interaction with host factors may not only expond the existing network between Tembusu virus and host,but also open a new way for preventing and eontrolling disease. The research content was divided into following two parts:1. Constructing the c DNA library of duck embryo fibroblasts(DEF)DEF was cultured using SPF duck embryo at 10 day. The total RNA of DEF was extracted using Trizol method, and the total RNA was used for synthesis of first strand c DNA. Double strands c DNA was synthesized by LD-PCR technique. After purified by using CHROMA SPIN TE-400 column, the double strands c DNA were cloned into plasmid p GADT7. Then homologous recombination mediated approach was used to construct c DNA library of DEF in Y187 yeast cells. The results showed that the titer of c DNA library was 3× 107 cfu/ m L, The inserted fragments were between 500～2250 bp in size with the average of inserted fragments about 1.0 kb. The constructed c DNA library may be useful for yeast two-hybrid experimentand provide a data of studying the interaction between E protein of DTMUV and host proteins.2. Screening host proteins that interact with E proteinRNA was extracted using Trizol method. According to RT-PCR method, double-stranded c DNA was synthesised by reverse transcription. The amplified fragment that was digested by Eco R I and Bam H I enzyme was cloned into the digesting p GBKT7. The product was transformed into DH5α competent cells, coated plates, select monoclonal colony by PCR.The positive plasmid was confimed to be constructed as expectation by enzyme digestion and sequence reaction. The recombinant plasmid p GBKT7-E and empty vector p GBKT7 were transformed into strain Y2 H independently. The transformants growing on SD/-Trp 、SD/-Trp/X-α-Gal/Ab A showed that plasmid p GBKT7-E was qualified with self-activation and toxicity or not. The c DNA library was screened using plasmid p GBKT7-E as a bait. Positive prey plasmids were obtained and analysised. The result showed that plasmid p GBKT7-E had no toxicity to the yeast cells and could not induce self-activation in yeast two-hybrid system.Moreover interacting proteins of ANTX1、PRDX1、PRS7、MORC3 were screened from the library.