The Study On Manufacture DNA Adjuvants And Their Immune Enhancing Effect To Canines

Objective: The experiment was conducted to enhance the immunity of canine vaccine by utilizing the immunostimulatory of CpG, and to select some kind of CpG-ISS with good immune enhancing effect on the clinic veterinary application, and what's more, to research the immunostimulatory effect of the DNA adjuvants to canine parvovirus inactivated vaccine and canine distemper attenuated vaccine.Methods: 1. Constructed four kinds of CpG-ODN adjuvants, and extracted natural bacterial CpG-DNA adjuvant from the coliform groups of Tibetan Yak, then detected the purification and concentraction of the five adjuvants. CpG-ODN] was sent to the company for sequence analysis when the result of PCR was positive. Animal toxicity experiment was done with mouse .2. Observed the immune enhancing effect of different CpG-ODNs and bacterial DNA to canine parvovirus inactivated vaccine. Seventy laboratory canines were averagely assigned to seven groups in the experiment.Take bacterial DNA CpG-ODN, CpG-ODN2 CpG-ODN3 and CpG-ODN4 respectively as adjuvant group, only canine parvovirus inactivated vaccine group and un-immuned group. On the fifteenth day after the second immunization, we detected the HI antibody, and compared the immune enhancing effect of every adjuvant, then selected the best CpG-ODN sequence.3. Researched the immune enhancing effect of bacterial DNA and CpG-ODN i to canine distemper attenuated vaccine. Fourteen laboratory canines were assigned into four teams, and inoculated with bacterial DNA CpG-ODN1 and aluminium hydroxide as adjuvant, there are four canines in every team. The control team with two laboratory canines was innoculated only with canine distemper attenuated vaccine. On the sixth and eleventh day We detected canine distemper virus of sera lymphocyte with RT-PCR, and on the third eighth fifteenth thirtieth and forty-fifth day sampled blood, detected the viralneutralizing antibody with microserum neutralization test.Results: 1. We constructed successfully four kinds of CpG-ODN adjuvants, and extracted natural bacterial DNA adjuvant, their purification and concentraction are qualified. The results showed that: CpG-ODN1 which was detected by PCR was positive; The result of sequence analysis showed the plasmid had contained aim sequence; No toxic action was found in the animal toxicity test, which suggested that all of them could be used into animal clinical experiments.2. The sequence of HI antibody titer from high to low was: ODN1 team bacterial DNA team ODN4 tearm ODN3 team only vaccine teanu ODN2 team and un-immuned team. The staticis results demonstrated: there were significant difference between ODN1 and other six team(P<0.01), so was the bacterial DNA team; There was no difference between ODN3 and ODN4 team(P>0.05); there was no difference between ODN2 and only vaccine immuned team((P>0.05).3. The results of RT-PCR showed that: on the sixth day they were all positive in alhydrogel hydroxide team and vaccine innoculated team. Only two canines of the bacterial DNA team and one of the CpG-ODN 1 team were positive. On the eleventh day only two canines of the alhydrogel hydroxide team and one of the vaccine immuned team were positive. It suggested indirectly that: bacterial DNA and CpG-ODN] adjuvants made CDV antibody improve more rapidly than aluminium hydroxide.4. The microserum neutralization test showed that: every immune method could induce specific antibody. The neutralizing antibody titer from high to low was: CpG-ODN 1 team bacterial DNA team aluminium hydroxide team and only vaccine immune team. The staticis results showed that: there were difference between CpG-ODN 1 and bacterial DNA team(P<0.05), but there were significant difference between CpG-ODN 1 and the other two teams (P<0.01), so was the bacterial DNA team. There wasn't difference between the aluminium hydroxide and vaccine immuned team(P>0.05). At the same time, it was found that bacterial DNA and CpG-ODN 1 could induce neutralizing antibody earlierthan alumi...