Construction And Immunogenicity Of Recombinant Eukaryotic Expression Plasmid Of VP60 Of Rabbit Hemorrhagic Disease Virus

Rabbit viral hemorrhagic disease (RHD), i.e. rabbit plague, is a kind of virulent virus disease caused by rabbit hemorrhagic disease virus (RHDV). The typical symptom of RHD mainly present the internal organs hemorrhage, die acutely within 2-3 days, with the morbidity reaching up to 90-100% and the mortality rate is 95% of the 3-month-old rabbit. The RHD occurred firstly in Wuxi and Jiangyin, Jiangsu, China in 1984,then rapidly spreaded in other areas of china and gradually extended to the other countries, which caused great damage to the farmed rabbit industries. As the antibiotics is invalid to control RHD and the immunomodulatory function of the interferon could not completely cure the RHD. At present, it mainly relys on the immune prevention to control the RHD and commonly used vaccine is tissue inactivated vaccine of RHD on the market. Altho μ gh the tissue inactivated vaccine plays an important role in controlling the rabbit blast, it has many disadvantages such as high cost, easy to poison, difficult to save. Compared to the traditional vaccines, Gene vaccine has more advantages, such as high security, lower cost,etc. In this experiment, VP60 gene was cloned from RHDV Sichuan virus strain (Y8504) to construct the recombinant eukaryotic plasmid, and study its immunogenicity, in the hope of laid a foundation for development of RHD gene vaccine.According to the VP60 sequence published on GenBank RHDV to design specific primers,the capsid protein VP60 gene of rabbit hemorrhagic disease virus (RHDV) was amplified by RT-PCR,The fragment size is 1740bp,And the product after purification was linked into the pMD19-T vector. Then the recombinant plasmid (pMD19-T-VP60) was sequenced and digested by Ecd R I and Xho I to obtain the VP60 gene fragment. The fragment of VP60 gene was inserted into the eukaryotic expression vector pVAX-1 to construct nucleotide vaccine plasmid pVAX1-VP60 and identified via PCR and double restriction enzyme digestion. To investigate the immunogenicity of the constructed plasmid pVAXl-VP60,25d old healthy rabbits were used to intramuscularly inject (dose:500μg/each). Meanwhile, three other experimental groups received an_jntramuscular injection of the same volume of pVAX-1,RHDV tissue inactivated vaccine and PBS.After immunized 7d,14d,21d,28d,35d and 42d, blood was collected from each rabbit in four groups to test the specific antibody level by indirect ELISA. The results showed that there was no specific antibody for RHDV in PBS group and pVAX-1 group, but there was a high specific antibody both in pVAXl-VP60 group and RHD tissue inactivated vaccine group, and antibody level of serum have no significant difference (p>0.05). For detecting the tissue distribution of the pVAXl-VP60 in rabbits, three rabbits were killed random after immunized 3d,7d,14d,21d,28d,35d and 42d, and collected heart, liver, spleen, lung, kidney and leg muscle to test the distribution of plasmid within tissues. The results showed that the gene of VP60 could distribute in all these tissues in each period after immunized. The challenge experiment was conducted by intramuscular injection with 0.5 mL/each rabbit RHDV at 108 plaques after immunized 28 days. The relative protection rates were 0%,0%,90%,85% in PVAX-1, PBS, RHDV tissue inactivated vaccine and the pVAXl-VP60 group, respectively. These results suggested that the recombinant eukaryotic expression plasmid pVAX1-VP60 possessed good optimun immunity.