Adenoviral-Mediated Transfer Of The CSFV E0 Gene Into The Goat Mammary Gland

Classical swine fever(CSF) is a highly infectious disease caused by Classical swine fever virus(CSFV), and the prevalence of CSF has brought great losses to the swine industry in the whole world. At present, except for some countries and areas that have been eliminated CSF, the main strategy to control CSF is still vaccination with live attenuated vaccines. However, the prevalence pattern of CSF has changed greatly, and those live attenuated vaccines are not satisfying enough to protect pigs from infection. Moreover, with the development of genetic engineering vaccine and the progress in exogenous expression of recombinant proteins, it has become an unavoidable trend to explore safer and more effective new type vaccines. Therefore, in the present study a recombinant adenovirus Ad-hisE0, which contains a CSFV E0 gene, was constructed. And the expression of E0 gene mediated by Ad-hisE0 in Bovine mammary epithelial cells and goat mammary gland has been examined, thus offered some technical supports for further development of CSFV genetic engineering subunit vaccines and mammary gland bioreactor.1. Construction of Ad-his E0 recombinant virus contains the CSFV E0 geneUsing the pMD-18 T as a template, the CSFV E0 gene with a signal peptide and a His tag sequence in its upstream was amplified by PCR, and was subsequently inserted into an adenovirus shuttle vector pAdTrack-CMV-hisE0. Then the recombinant plasmid pAd-hisE0 was constructed by recombining the pAdTrack-CMV-hisE0 with pAdEasy-1. After transfecting the pAd-hisE0 into 293 A cell line, the recombinant adenovirus Ad-hisE0 with high titer was obtained.2. The examination of the availability of Ad-hisE0The 293 A cells were infected by the resulting recombinant adenovirus Ad-hisE0, and the expression of Green fluorescent protein proved that the Ad-hisE0 could infect cells effectively. In addition, the result of quantitative Real-time PCR showed that the CSFV E0 gene could express in Ad-hisE0 infected 293 A cells.3. The expression of Ad-hisE0 in Bovine mammary epithelial cellsThe confirmed Ad-hisE0 was used to infect Bovine mammary epithelial cells in vitro, and then the expression products were analyzed by Western blotting. The result revealed that there are two different molecular mass of the recombinant fusion protein—26 kD and 48 kD, demonstrated that Ad-hisE0 can introduce the CSFV E0 gene into the mammary epithelial cells.4. The expression of Ad-hisE0 in goat mammary glandIn order to transduce the goat mammary gland, lactating goats were infused with recombinant virus Ad-hisE0 through the teat canal. After 48 h, the milk sample was collected and analyzed by Western blotting. The result also showed two specific protein band demonstrating that the recombinant adenovirus Ad-hisE0 can introduce the CSFV E0 gene into the goat mammary gland, and thus enabling the secretion of the recombinant protein.