| Classic Swine Fever(CSF)caused by the classical swine fever virus(CSFV) is a highly contagious viral diseas of pigs and is classified by the the Office International des Epizoofies(OIE)as a notifiable disease. CSF poses great risks to the swine industry worldwide. Hog Cholera Lapinized Virus(HCLV) has been widely applied to prevent of CSF in China since 1957(Zhou TC,1980)which is a worldly received golden standard attenuated vaccine. It also contributed greatly to the control of CSF in Europe. However, it can not be distinguished between the immuned pigs by vaccine and infected pigs on serology. Development of marker vaccine will be benefit for eliminating CSF.By inserting FLAG genes into the E1 protein of the HCLV, we got a new virus full-length plasmid. Genomic RNA was transcribed in vitro and further transfected into BHK-21 and SK6 cell lines by electroporation. v C-FLAG were rescued successfully and identified by Immunohistochemistry, RT-PCR real time and QPCR experiment. At the same time, we set up one method for identifying the v C-FLAG by using FLAG monoclonal antibody.The v C-FLAG viruses were challenged in severl cell-lines by using freezing-thawing method. We found the SK6 cell-line is the optimal cell-line. We also established one method, using infected-cells mixed with nomal cells, to culture for virus propagation.Chimeric virus was proved stable expression of FLAG genes.Through rabbites experiment we have found the relationship between marker virus v C-FLAG and rabbit fever. The results showed that the marker virus v C-FLAG could induce rabbit fever reaction as C strain did. To confirm the case of antibodies in rabbit serum, the blood was collected and the serum was separated. The antibodies in rabbit serum significantly increased from the seventh day on. To study whether v C-FLAG would lead animals to produce antibodies against the FLAG or not, two methods were established. By comparing these two methods, we found the FLAG-BAP protein-coated ELISA plate was more sensitive than that of synthesis peptides FLAG. However, when using this method to identify the FLAG antibody in rabbit serum, it was not very effective. Then using the commercial rabbit anti-FLAG polyclonal antibody has proved that the method for the detection of rabbit anti-FLAG antibody was not obviously specific. This showed that the level of antibodies which v C-FLAG virus induced against the FLAG in rabbits might be low, and it did not reach the range of using monoclonal antibody for detection. It was possible that the period was too much short after immunizing the rabbit. It was also possible that this method was not suitable for the detection of rabbit antibody.Further study was needed in pigs, because the case of rabbit FLAG antibody did not directly reflect the response of pigs.To sum up,the FLAG marker chimeric virus v C-FLAG was rescued successfully. It can display the FLAG antigenic epitopes well. v C-FLAG cultured in virus-carrying SK6 cells mixed with normal SK6 cells would lead to a higher and stable titer. v C-FLAG strain maintains the typical characteristics of the C strain in rabbit, and it also can induce the production of antibodies against CSFV in rabbit serum. But the FLAG antibody in rabbits can not be detected by the methods to detect FLAG antibodies in mouse. More researches are being done to solve these problems. |
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