Cloning And Sequence Analysis Of Envelope Gene (Small Membrane Gene) Of Three Porcine Epidemic Diarrhea Virus

In this paper total RNA of three different original porcine epidemic diarrhea virus (PEDV) were isolated. Then one pair of primers was designed based on the enunciated genome of PEDV in GenBank. Using total RNA as a template the envelope gene was amplified by a reverse transcribe polymerase chain reaction (RT-PCR). The amplified products were ligated into T vector respectively and sequence was determined after identification by PCR. The result of sequence comparison is: among three strains, the homologies of the nucleotide sequence and amino acid sequence are 99. 6%-99.8% and 98. 7%-100%; in comparison of the three strains with NC-003436 and AF353511 in GenBank data, the homologies of the nucleotide sequence and amino acid sequence are 99.8%-100% and 98.7%-100%. And porcine epidemic diarrhea virus .(PEDV) strain was passaged and purified. This is the first time in the study of PEDV to add dextran sulfate (DS) into MEM, which maintain growth of cell after inoculating PEDV (strain QD). The aim to add DS is to quicken the pathological changes. The different concentrations of DS are 100μg/ml , 50μg/ml and 25μg/ml. After adding DS, PEDV has been passaged for 17-20 generations. Then we do plaque to pick out single clone to purify the virus. The consistency of 50μg/ml is fittest to do plaque, the 25μg/ml is second and the 100μg/ml is last. It' s obvious that DS adding can quicken the pathological changes and shorten the time of harvest virus. And it can be a referenced way to study longtime harvest virus. The diagnosis of PED in China mainly depends on ELISA and other serological methods. In this paper the envelope gene can be amplified steadily and specifically by the designed primers. And the PCR based on the primers may be useful for the diagnosis of PED.