Proliferative Comparison Of Chinese Attenuated Lapnized Strain On The Immortalized Swine Umbilical Vein Endothelial Cells And Bovine Testicular Cells

classical swine fever attenuated vaccine play an important role in the control of classical swine fever epidemic in large-scale in the 2060s. Europe, Asia, and a few Latin American countries are widely used attenuated C strain and agreed that a strong strain of immunity, particularly the safety of immunization can prevent piglets, suckling pig and swine breeding. Vaccination is an important means of preventing classical swine fever, the most commonly used in China is the classical swine fever attenuated vaccine However , swinery those had been immuned by Hog cholera Lapinised Vaccine failed in immunization during 2090s , caused difficulties about preventing classical swine fever virus effectively. This aim was that we found the best cell for proliferating hog cholera lapinised virus by propagation compartion immortalized endothelial cell with bovine testicular cells. obtained the following findings.1. Culturing primary Bovine testicular cells. Firstly selected the bovine testis, because of cells with activity. The isolation and culture of primary Bovine testicular cells by way of enzyme-digesting and direct-tissue-culturing detect cells in order to get normal bovine testicular cells, there are two cell morphologies which contains fibroblast-like and endothelial-like.2.Establishing mould comparisons between bovine testicular cells and immortalized swine endothelial cell infected hog cholera lapinised strain. Bovine testicular cells was serial subcultivated to the seventh generation and tested first generation of cells and immortalized the 70th generation of vascular endothelial cells. The classical swine fever was diluted in accordance with the instructions and then added its 1mL to these two cell and cultured 90min, collected the venom, adding 2-5mL containing 2% FBS in DMEM culture medium to maintain for 72 hours. By for RT-PCR and indirect immunofluorescence assay, results showed that HCLV proliferation existed in cell. And then bovine testicular cells after post-inoculation were serial sub cultivated to the sixth generation; immortalized endothelial cells after post-inoculation were serial sub cultivated to the 110 generation till happened to cellular atrophy. At the same time established mould immortalized endothelial cells mould after infected classical swine fever Chinese strain.3.Using 6～8 week old female Balb/c mice before animals immunation testing and divided into 16 groups randomly, which made of bovine testicular cells after post-inoculation group; bovine testicular cell supernatant after post-inoculation group; immortalized vascular endothelial cells after post-inoculation group; immortalized endothelial cell supernatant after post-inoculation group; negative control group; control positive group. Immunization dose of supernatant from these two cells was 1mL, cellular immunity dose was 1x106。After two weeks by the way of intraperitoneal injection after a tail blood were vaccinated 2 times, the last time booster immunization by empennage injection. the immune serum to be collected and detected antibody by indirect ELISA.Then by the way of fluorescence real-time PCR of relative quantitative method which showed that the virus in immortalized endothelial cells was more than bovine testicular cells.Sum up, immortalized swine endothelial cells were more easier than bovine testicular cells for proliferation HCLV , which had a foundation of manufacturing new classical swine fever attenuated vaccine.