Cloning And Prokaryotic Expression Of Phosphoprotein Gene Of Avian Paramyxovirus Type 1 Isolated From Muscovy Duck

Avian paramyxovirus type 1 (APMV-1) is a member of the genus Rubulavirus, subfamily Paramyxovridae, family Paramyxovridae, in the order Mononegavirales, which is one of the most serious infectious diseases of poultry in the world. APMV-1 commonly referred to as Newcastle disease virus (NDV) . Now the newcastle disease (ND) is considered as a potent infectious disease by the world organization for animal health. Therefore the research of which becomes more and more significance.Since 2002, a new infectious disease has prevailed in Muscovy duck in Fujian and Zhejiang provinces in China. The 8～40 ages were attacked by the disease, which were characterized with obvious nervous signs, decompensation and diarrhea. The morbidity and the mortality was 30%,52%, respectively, and which was identified to be caused by APMV-1．Tow pairs of primers were designed according to the sequence of the phosphoprotein (P) gene of the ZJ1 strain avian paramyxovirus type 1 from Genebank. The P gene was succeedly amplified by RT-PCR from the avian paramyxovirus type 1 (FPl/02) isolated from Muscovy duck in Fujian. The result showed that the whole P gene is 1441 nt, and contains an open read frame (ORF) which encodes a polypeptide of 395 amino acids. The cloned P gene shared a high homology of 97．8% in nucleotide acid sequence and 96．5 % in deduced amino acid sequence with the counterpart gene of the ZJ1 strain, respectively. Meanwhile, In virtue of biology software and correlative analysis tools online, a series of bioinformatics analysis were performed on the structure and function of P protein including prediction of the physical and chemical properties, domain, motif and dimensional structure etc. The results indicated that P protein did not had any signal peptides and known coiled-coils regions. In this experiment, the correct sequence of the phosphoprotein (P) gene was used as templates for polymerase chain reaction (PCR) to amplify the P gene with the primers containing the EcoRI and XhoI, respectively. Then it was cloned into the plasmid pGEX-5X-1．The recombinant plasmid was identified by restriction enzymes analysis and nucleotide sequence analysis, the results showed that the P gene has been correctly cloned into plasmid pGEX-5X-1, the prokaryotic expression vector of pGEX-5X-1/P was successfully constructed.The corrcet recombinant pGEX-5X-1/P was transformed into Escherichia coli BL21 (DE3) with inducement of IPTG The results measured by SDS-PAGE manifested that it could be highly expressed, the expressed existed in the form of solubility. Target protein was purified by GST affinity chromatography. This purified protein was proven to be specifically recognized by the positive serum of p gene of APMV-1 by using indirect ELISA and Western blotting tests.This trail has successfully cloned the full segment of P gene, and made it get high expression in the prokaryotic expression system. All these work will lay foundation for the research about the immunogenecity of phosphoprotein protein of APMV-1 to different host, the function in cross-species pathogenesis of APMV-1, along with development of genetic engineering vaccine and new diagnosis reagent.