Construction Of Full-length CDNA Clone Of Porcine Epidemic Diarrhea Virus And Replicon

Porcine epidemic diarrhea is an acute and highly contagious intestinal infectious disease of swine, which is caused by porcine epidemic diarrhea virus(PEDV). Acute enteritis, vomiting, watery diarrhea and loss of appetite are the basic characteristics of this disease. Pigs of all ages are susceptible, but PED is most serious in piglet. The incidence rate and mortality rate of piglets can be 90%-100%. Although it was first reported in Europe, this disease has become widely pandemic in many Asian countries which include China, Korea, Philippines, and so on. It has already been one of infectious diseases of the swine industry. Coronaviruses reverse genetics system is a very useful tool in researching on viral replication, transcription mechanism and the function of each gene. Therefore, on the basis of PEDV AJ1102 strain, the construction of the reverse genetic operating system of PEDV was carried out. The main contents are as follows:1. Construction of full length c DNA clone of PEDVAccording to the sequence of PEDV AJ1102, PCR primers were designed and six fragments spanning the full genome of PEDV AJ1102 strain were amplified by RT-PCR and SOE-PCR. The six fragments were named A, B, C, D, EF and G. At the same time, the right CMV promoter sequence was introduced in the A fragment 5’end. Through modifying the p Belo BAC11 vector, the intermediate plasmid p Belo-AJ1102-5’-3’ was constructed by introducing of CMV promoter sequence, PEDV 5’ end, multiple clone sites, PEDV 3’ end, HDV sequence and the BGH termination sequence. Then the construction of full-length c DNA clone of PEDV was accomplished when the acquired six fragments were cloned into the intermediate plasmid p Belo-AJ1102-5’-3’, which could lay the foundation for acquiring the infectious clone of PEDV AJ1102 strain.2. Construction of PEDV replicon plasmidBased on the establishment of the full-length c DNA clone of PEDV AJ1102, the c DNA clone of PEDV replicon was constructed. By using PCR, the S, ORF3, E and M gene in the EF fragment were deleted and the N protein gene and its upstream TRS sequence related to the porcine epidemic diarrhea virus replication were retained. While the reporter gene Luc and EGFP were introduced respectively. The two modified EF fragments were named H fragment(H-EGFP) and I fragment(I-Luc) respectively. Then the H fragment and I fragment were respectively cloned into the intermediate plasmid p Belo-ABDG. After inserting C fragment, the replicon plasmid p Belo-AJ1102-EGFP-rep and p Belo-AJ1102-Luc-rep containing Luc gene and EGFP gene respectively were constructed.