【作者】 崔珂；

【导师】 于永利；

【作者基本信息】 吉林大学 ， 免疫学， 2010， 硕士

【摘要】 口蹄疫病毒可以引起偶蹄动物急性传染性接触性疾病,牛、羊、猪等家畜都是对此病毒敏感,其中牛最为易感。接种口蹄疫疫苗是预防和治疗口蹄疫的重要方法,通常在疫苗中加入佐剂来增强疫苗的免疫活性。CpG ODN是一种包含非甲基化的胞嘧啶和鸟嘌呤核苷酸(CpG基序)的寡聚脱氧核苷酸,能够激活机体免疫系统,可以分为A型、B型、C型、N型四种类型,其中B型和C型都能通过刺激B细胞增生,并促使B细胞分泌多种细胞因子。为了研究CpG ODN能否成为重组口蹄疫病毒疫苗的分子佐剂,我们采用重组牛Asia1型口蹄疫病毒A10蛋白疫苗,研究了自行设计的CpG 02的分子佐剂功能,结果表明:CpG 02能够增效重组A10蛋白疫苗免疫作用,CpG 02与重组A10蛋白在剂量上有配比相关性。CpG 02对于A10蛋白疫苗有分子佐剂的效果,但能否作为A10蛋白疫苗理想的分子佐剂还有待进一步探讨。更多还原

【Abstract】 Foot and mouth disease (FMD) is an acute, febrile, highly contagious diseasescaused by foot and mouth disease virus (FMDV). The disease mainly affects domesticcloven-hoofed animals, including cattle, swine, sheep as well as more than 70species of wild animals, characterized by fever, lameness, and vesicular lesions on thetongue, feet, snout and teats.The transmission of the disease is mainly through respiratory route by aerosolized viruses. FMD dose not kill animals usually but sickensinfected animals and severely reduces the production of meat and mike, resulting ineconomic disaster. FMDV is the type species of the Aphthovirus genus of thePicornaviridae family. By electron microscopy, the FMD virion appears to be a roundparticle with a smooth surface and the diameter of the virion is about 25 nm. Sevenserotypes (A, O, C, Asia1, and South African Territories 1, 2, and 3) have beenidentified. In China, the main popular serotypes are O, A, C and ZB (Yunnan Baoshantype).Vaccination is the mian method of prevention of FMD. The killed FMD vaccinehas been successful in reducing the number of disease outbreaks in many parts of theworld. However, there are several disadvantages associated with the use of the killedvaccines, such as the requirement of a cold-chain to preserve vaccine stability and therisk of viral release during vaccine production. With the development ofbiotechnology, new vaccines were developed, such as genetic engineering subunitvaccines, synthetic peptide vaccines, DNA vaccines and live vector vaccine.Due tothe weak immunogenicity, the newly development vaccines require the appropriateadjuvants to boost their immunogenicity. For the present,the most widely adjuvantused in human and animal is the aluminum adjuvant. However, alum salts arerelatively weak adjuvants and rarely induce cellular immune responses. Highaluminium levels in the body predominately affect the brain, causing fatalneurological syndrome and dialysis-associated dementia .For the limitations of thealuminum adjuvant, the new adjuvants such as carbohydrate, bacterial derivatives, CpG ODN and other adjuvants are highlyexpected.It has been reported that mycobacterial DNA had adjuvant activity, and thisadjuvant activity was correlated with a higher content of CpG motifs present inbacterial nucleic acids. Synthetic oligodeoxynucleotides (ODN) containingunmethylated CpG motifs act as immune adjuvants, accelerating and boostingantigen-specific immune responses. CpG motifs promote the induction of Th1 andpro-inflammatory cytokines and support the maturation/activation of professionalantigen presenting cells (particularly plasmacytoid dendritic cells).Co-administeringCpG ODN with a variety of vaccines has improved the resultant humoral and/orcellular immune responses. CpG ODN has been obtained good results in theanti-bacterial, viral, anti-tumor as vaccine adjuvants.Recombinant A10 protein designed in our lab.is one of the bovine vaccinesagainst foot and mouth disease designed in our lab. In order to enhance theimmunological effect of the A10 protein, we immunized guinea pigs withrecombinant A10 protein vaccine with CpG ODN and Montanide ISA 206. Thisarticle was divided in to the following parts:1. Screening the optimum CpG ODNIn order to screen the CpG OND which has the optimum adjuvant effect for therecombinant A10 protein, we chose the different CpG ODN designed in our lab tostimulate splenocytes of three guinea-pigs respectively and detect the effects of CpGODN on proliferation of guinea-pig splenocytes by MTTassay. We found CpG 02 hadthe optimum effet to stimulate the proliferation of guinea-pig splenocytes, so wechose the CpG 02 as adjuvant of the recombinant A10 protein and immuneguinea-pigs to observe the adjuvant effect of CpG 02.2. Purityand concentration analysis of CpG 02In order to prepare vaccine, we analysed the concentration and purity of CpG 02to test whether this batch of CpG 02 degraded or not. From the figure of 20%denaturing PAGE, we can see that there was no degradation band and the CpG 02 wasclear. So the purity of CpG 02 met the experiment requirement. We measuredconcentration of CpG 02 by using ultraviolet spectrophotometer, and theconcentration was 0.5mg/ml.3. Concentration, purity and molecular weight analysis of recombinant A10protein. Firstly, we measured concentration of the A10 protein by lowry assay and themeasured result was 2.73 mg/ml. Subsequently non-denaturing SDS-PAGE anddenaturing SDS-PAGE electrophoresis were carried out for A10 protein, we analysedthe purity of A10 protein was 91.64% and the molecular weight was 38.25 KD byGIS gel image processing system. From the foregoing, this batch of A10 protein canbe used as protein vaccine for animal immunization.4.The enhancement of CpG ODN onA10 protein.Firstly, we measured the antibody of guinea-pigs in different time points byELISA. According to the ELISA results, when the bovine Asia1 Inactived FMDV ascoating antigen, the antibodies of guinea-pigs in A10-100 ?g + CpG 02 group wasobviouslyhigher than the other adjuvant groups and the A10-200 ?g group (except forinactivated FMDV group), the results showed that when CpG 02 with certain dose ofA10 protein immuned guinea-pigs, CpG 02 had the optimum adjuvant effet. WhenA10 protein as coating antigen,the antibodies of guinea-pigs in A10-100 ?g + CpG 02group were obviously higher than the other groups (especially in 91 day aferimmunization), the results showed that CpG 02 could enhance and prolong A10protein-Specific Immune Response.Next,we measured the antibody of guinea-pigs from -2 day to 91 day afterimmunization by ELISA. According to the ELISA results,when the bovine Asia1Inactived FMDV as coating antigen,the antibody of guinea-pigs in A10-100 g +CpG 02 group was obviously higher than A10-200 ?g group(P<0.05) (except forinactivated FMDV group) and the antibody still rased on day 91 afterimmunization.The antibody of other groups reached to the highest level on andrespectively decreased to the foot on day 42 and 72.When A10 protein as coatingantigen,the antibody of guinea-pigs in adjuvant groups were higher than theA10-200 g group and the antibody in A10-100 ?g + CpG 02 group washighest.Observation on Immunological efficacy of CpG 02 adjuvant A10 proteinAgainstFMDV,CpG 02 have adjuvant effect toA10 protein.The conclusions are got from the results analysis in this paper as following: whenCpG 02 is used with A10 protein,CpG 02 can enhance immunogenicity of A10protein and A10 protein-Specific Immune Response. To exert the optimum adjuvanteffect, CpG 02 shoud be used with the proper dosage ofA10 protein(100 ?g).When changed the proper dosage of A10 protein,CpG 02 does not effectively exertadjuvant effect,and even inhibits immunogenicity of vaccine protein.更多还原