本文選題：豬瘟疫苗 + 口服免疫　； 參考：《上海海洋大學》2015年碩士論文

【摘要】：豬瘟(classical swine fever,CSF)是由豬瘟病毒(Classical swine fever virus,CSFV)引起的一種豬的高度接觸性傳染病。該病呈急性或慢性感染,在許多國家和地區(qū)均有發(fā)生,給養(yǎng)豬業(yè)造成極大的危害。目前,疫苗接種仍是控制豬瘟疫情的主要手段�，F(xiàn)階段我國使用的豬瘟疫苗主要有豬瘟兔化弱毒株、家兔脾淋組織苗、乳豬腎細胞苗等注射型疫苗。國外報道豬瘟疫苗口服免疫獲得了很好的效果,經(jīng)口服免疫,可減少對豬只的應激且省時省力。因此研究擬通過篩選理想的保護劑,制備可經(jīng)口服途徑免疫的豬瘟疫苗。本研究采用兩種方法制備口服抗原:以豬瘟病毒疫苗株與重組LTRG蛋白及凍干保護劑配伍,制備凍干抗原;另外利用殼聚糖-海藻酸鈉包埋重組CSFV E2蛋白,制備微囊抗原;兩種抗原分別口服飼喂小鼠,以細胞免疫指標和體液免疫指標評價抗原的免疫效果,篩選理想的抗原保護劑。1蛋白抗原包埋及免疫活性檢測構(gòu)建CSFV E2基因的原核表達質(zhì)粒并表達蛋白,經(jīng)純化后將其作為抗原,與黏膜佐劑蛋白LTRG共同包埋制備殼聚糖-海藻酸鈉微球,然后口服免疫小鼠,檢測其免疫效果。結(jié)果,包埋E2和LTRG蛋白組小鼠的血清Ig G和黏膜Ig A水平均顯著高于對照組,高于E2蛋白包埋組,表明LTRG蛋白作為一種黏膜佐劑,增強了小鼠的免疫反應。此外,包埋E2和LTRG蛋白組小鼠的抗體水平顯著高于E2+LTRG蛋白組,說明殼聚糖-海藻酸鈉微球作為包埋載體,將抗原進行了有效的呈遞,從而誘導小鼠產(chǎn)生免疫反應。2凍干保護劑篩選及免疫效果評價研究配比了不同組分的凍干保護劑,包括明膠、乳糖和甘氨酸,與黏膜佐劑LTRG蛋白、CSFV按比例混合,經(jīng)優(yōu)化凍干工藝后,制備了4種CSFV的凍干苗�？诜庖咝∈�,測定血清Ig G和黏膜Ig A抗體效價。結(jié)果,含明膠和甘氨酸的試驗2組和含明膠的試驗4組經(jīng)凍干后外觀形態(tài)較好,水溶后呈現(xiàn)澄清狀態(tài);試驗2組和試驗4組的凍干苗經(jīng)口服免疫,誘導小鼠產(chǎn)生的Ig G抗體水平極顯著高于CSFV組、CSFV+LT組及其他試驗組;二免7天后試驗2組小鼠產(chǎn)生的Ig A抗體極顯著高于其他組。而且,試驗2組小鼠脾臟的IFN-γ和IL-4細胞因子的表達量也極顯著高于其他組。表明,在凍干過程中,明膠和甘氨酸對豬瘟病毒起到了良好的保護作用,提高了豬瘟病毒疫苗經(jīng)口服途徑的免疫效果。利用殼聚糖-海藻酸鈉包埋制備重組蛋白微球抗原,能誘導機體產(chǎn)生良好的體液免疫反應和細胞免疫應答,證實包埋蛋白抗原具有良好的免疫效果。配伍不同組分的豬瘟病毒疫苗保護劑,制備的凍干疫苗,能有效提高機體免疫抗體水平,證實制備的凍干保護劑配方對豬瘟病毒有較好保護作用。本研究為豬瘟疫苗凍干保護劑篩選和不同劑型疫苗的開發(fā)奠定了基礎。
[Abstract]:Classical swine virus (CSFV) is a highly contagious disease of swine caused by classical swine fever virus (CSFV). The disease is acute or chronic infection, which occurs in many countries and regions, causing great harm to pig industry. At present, vaccination is still the main means to control the outbreak of swine fever. At present, the main vaccines used in China include attenuated strain of hog fever rabbit, splenic tissue vaccine of rabbit, kidney cell vaccine of suckling pig and so on. Oral immunization against swine fever vaccine has been reported in foreign countries. Oral immunization can reduce stress and save time and effort in pigs. Therefore, the aim of this study is to prepare an oral vaccine against classical swine fever (CSFV) by screening ideal protective agents. In this study, oral antigens were prepared by two methods: the recombinant LTRG protein and freeze-dried protective agent were mixed with CSFV vaccine strain to prepare freeze-dried antigen, and the recombinant CSFV E2 protein was encapsulated with chitosan sodium alginate to prepare microencapsulated antigen. Two kinds of antigens were fed orally to mice respectively. The immune effects of the two antigens were evaluated by cellular and humoral immune indexes. The prokaryotic expression plasmid of CSFV E2 gene was constructed by screening the ideal antigen protectant, 1 protein encapsulation and immunoassay, and the protein was purified and used as antigen. Chitosan-sodium alginate microspheres were prepared by embedding with mucosal adjuvant protein LTRG. The results showed that the levels of serum IgG and mucosal IgA in E2 and LTRG protein group were significantly higher than those in control group and E2 protein group, indicating that LTRG protein, as a mucosal adjuvant, enhanced the immune response of mice. In addition, the antibody levels of mice in E2 and LTRG protein groups were significantly higher than those in E2 LTRG protein group, indicating that chitosan and sodium alginate microspheres were used as entrapment carriers to present antigens effectively. In order to induce the immune response of mice, the screening of lyophilized protectants and the evaluation of immune effect were carried out. The lyophilized protectants of different components, including gelatin, lactose and glycine, were mixed with mucosal adjuvant LTRG protein in proportion to CSFV. Four kinds of CSFV freeze-dried seedlings were prepared after optimized freeze-drying process. Mice were immunized orally and the titers of serum IgG and mucosal IgA antibody were determined. The results showed that group 2 with gelatin and glycine and group 4 with gelatin had better appearance after freeze-drying and showed clear state after water solubilization, and the freeze-dried seedlings of test group 2 and test 4 were immunized orally. The level of IgG antibody induced in mice was significantly higher than that in CSFV LT group and other test groups, and the level of IgA antibody in group 2 was significantly higher than that in other groups after 7 days. Moreover, the expression of IFN- 緯 and IL-4 cytokines in spleen of experimental group 2 was significantly higher than that of other groups. The results showed that gelatin and glycine had a good protective effect on CSFV during freeze-drying, and the immune effect of CSFV vaccine was improved by oral route. The recombinant protein microsphere antigen was prepared by encapsulation of chitosan and sodium alginate, which could induce good humoral immune response and cellular immune response. It was proved that the encapsulated protein antigen had a good immune effect. The freeze-dried vaccine prepared with different components of CSFV vaccine could effectively improve the immune antibody level of the body. It was proved that the lyophilized protective agent formula had a better protective effect on CSFV. This study laid a foundation for the screening of freeze-dried protective agents for swine fever vaccine and the development of different dosage forms of vaccine.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.4

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