Research On The Preparation Of Liposomes Containing Recombinant E Protein Vaccine And Immune Effect Against Tembusu Virus In Ducks

Since April 2010, a newly emerged infectious disease in ducks which characterized by severe egg production drop has spread rapidly to all of the south-east provinces of China.Then, the infectious disease spreaded to Shandong, Anhui, Fujian, Henan, Hebei and other provinces, causing serious economic losses in the Chinese duck industry. To October 2010, the causative agent has been identified as duck Tembusu virus(DTMUV) through the joint efforts of lots of experts in our waterfowl system.The main clinical symptoms of ducks catched the TMUV disease were acute anorexia, retarded growth, neurological dysfunction and the mortality rate up to 10-30%. The pathological features were that ovary, meninges, pancreas, trachea, heart, liver, spleen, lungs, bleeding, and other organs and tissues hemorrhaged, especially the follicle bleeding, degenerated and ruptured.The immune prevention is one of the important measures to control the DTMUV disease. Therefore, to develop an effective and safe vaccine for the prevention of the disease is imperative.To obtain an effective vaccine candidate against duck Tembusu viral(DTMUV) disease in the Chinese duck industry, liposomal vaccines containing recombinant E protein were prepared and assessed in this study.Firstly, the recombinant plasmid PET28a-E was transformed into BL21(DE3). Isopropyl ?-D-thiogalactopyranoside(IPTG) was added to a concentration of 1m M in the culture broth, after induced at 37 °C for 5 hours to get the E protein. A high-affinity Ni-NTA column was used to purify the E protein. The further purified protein was evaluated by SDS-PAGE and Western blot. The protein E was concentrated by PEG6000 to 2.2 mg·m L-1.Then, reverse-phase evaporation method was used to prepare the liposomal vaccine and protamine aggregation was applied to evaluate encapsulation efficiency, and other safety evaluation. The ratio of phospholipid-to-cholesterol(M) was 1:1. In order to avoid lipid peroxidation, a certain amount of vitamin E should be added. The encapsulation efficiency was confirmed to be approximately 52%. LPS concentration in the liposomal vaccine formulation was lower than 2.3 EU/mg. The peroxide value of liposomal vaccine formulation was 0.12. The above indexes conformed to the stipulation of Chinese Pharmacopoeia.Finally, the effect of liposomal vaccine was evaluated by animal experiment compared with Freund's adjuvant vaccine. Eighty-four 1 age of cherry valley ducks were randomly divided into seven groups: group 0(control), group 1(L+E 1st day once), group 2(L+E 1st, 7th day twice), group 3(L+E 7th day once), group 4(F+E 1st day once), group 5(F+E 1st, 7th day twice), and group 6(F+E 7th day once).Serum samples were collected from each ducks at weekly intervals before and after vaccination. Indirect ELISA assay was used to assess the titers of anti-E protein antibody. The results indicates that the antibody that could be due to either the original vaccination or viral challenge levels of group 2(L+E, immunized at the 1st and 7th day) were higher than the other groups, and its peak appeared at the 4th week and maintained at a high level for longer time. To assess the effect of liposome-E protein vaccine, the eighty-four cherry valley ducks were challenged i.m. with 1m L 102.4 50% tissue culture infective dose(TCID50) of virus at the age of 3 weeks. Meanwhile, six ducks were randomly chosen from each group to collect plasma from day 1 to day 5 post-challenge(p.c.), Oropharyngeal and cloacal swabs were collected from ducks on day 5 and day 7 post-challenge for virus titration. The titer was detected by indirect ELISA. According to the result of antibody level, group 2(L+E, immunized at the 1st and 7th day) and group 5(F+E, immunized at the 1st and 7th day) were selected for the detection of viremia and virus shedding. As to the viremia and DTMUV shedding, positive for the virus were detected in other groups from day 1 to 5 p.c., whereas none was detected in group 2(L+E twice). These results indicated that liposomal vaccine immunized twice could provide better protection against DTMUV challenge in ducks than other groups.In a word, this study revealed the potential of liposome vaccines containing recombinant E protein in the clinical prevention of DTMUV, which will play an important role in thecontrol and eradication of DTMUV in China.