CDNA Library Construction And Expression Of Main Functional Genes Of Enzootic Nasal Tumor Virus For Goats

Enzootic nasal tumour (ENT; enzootic nasaladenocarcinoma, ENA) of sheep and goats is a contagious diseases characterized by neoplastic transformation of secretory epithelial cells in the respiratory tract of the mucosal nasal glands. Clinical signs include continuous nasal discharge, respiratory distress, exophthalmos, skull deformations, facial swelling, sneezing and snoring respiration and weight loss. The disease occurs naturally in all continents except Australia and New Zealand, the prevalence in sheep was found to be about 0.1%-15%. Epidemiological date indicates that ENT prevalence in the sheep areas such as Inner Mongolia, Hunan and Sichuan, etc, which seriously affected the development of germplasm resources of local goats. There has been little studied in the aetiology and pathophysiology of the ENT in China. The main research contents include three aspects, in order to give further insight regarding the properties of the ENTV.â… . cDNA library construction of enzootic nasal tumor virus of SC strain for goats and Characterization of the genome sequenceIn order to obtain the cDNA library of enzootic nasal tumor virus of SC strain for goats, the RT-PCR was carried out on nasal fluid from goats with enzootic nasal tumor (ENT), using primers designed referencing to enzootic nasal tumor retrovirus of goats(ENTV-2). The 6 PCR fragments were purified, cloned into the pMD18-T vector and sequenced. The results showed that the genome is 7,168 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. ENTV is highly related to the ENTV-2 sequenced in 2003, Alignment with ENTV-2 gag, pro, pol and env showed 88%,90%,93%,88% and 92% nucleotide homology respectively. ENTV closely related to the retrovirus (ENTV-1) associated with enzootic adenocarcinoma of sheep, and to jaagsiekte retrovirus and endogenous retrovirus. The main sequence differences between these viruses reside in a small regions in gag, the transmembrane (TM) region of env and the U3 LTR.II. Molecular Cloning and Expression of Functional Gene of enzootic nasal tumor virus of goats strain SCThe PCRs were carried out on the ENTV cDNA libratory, using primers designed from the ORF of gag, env, SU, TM, SD respectively. The PCR fragments 1854bp,1848bp, 1146bp,727bp,150bp were subcloned into pMD18-T simple vector and sequenced. The correct gag ORF was subcoloned into prokaryotic expressing vector pET-32a. The recombinant plasmid pET-gag(1854) was constructed successfully and transformed into E. coli rostta strains. The pET-gag(1854) was induced by IPTG and analysis by SDS-PAGE, the relative molecular weight of expressed product was 87ku, Consistent with the theoretical reasoning. These results will help in the design of a protective vaccine against ENTV infection and the development of ENT. Plasmid DNA (pEGFP-env,pEGFP-SU,pEGFP-TM,pEGFP-SD) were Transfected into COS-7, Fluorescence can be observed after 12h, foreign gene transcripts were detected by RT-PCR, all results showed that foreign gene were fusion expressed effectively in COS-7.