本文選題：禽流感 + 延遲裂解型沙門菌�。� 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：禽流感(Avian influenza,AI)因其高致病性導(dǎo)致禽類大批量死亡,不但給我國(guó)養(yǎng)禽業(yè)造成了巨大的經(jīng)濟(jì)損失,而且嚴(yán)重威脅人類的公共衛(wèi)生安全。迄今為止,控制禽流感的最有效的方法仍是接種疫苗,但由于該病毒的高突變性,普通的傳統(tǒng)疫苗很難達(dá)到理想的免疫效果,所以,對(duì)禽流感病毒(avian influenza virus,AIV)疫苗的研究成為國(guó)內(nèi)外學(xué)者的研究熱點(diǎn)。本研究選擇AIV的血凝素(Hemagglutinin,HA)頸部的一段高度保守的序列HA2片段,并將其與粘膜免疫佐劑大腸桿菌不耐熱腸毒素B亞單位(Escherichia coli heat-labile enterotoxin,LTB)基因融合,分別將HA2基因與HA2-LTB融合基因?qū)氲窖舆t裂解型沙門菌載體中,構(gòu)建兩組重組沙門菌并對(duì)其免疫效果進(jìn)行初步評(píng)價(jià)。具體研究?jī)?nèi)容及結(jié)果如下:(1)重組沙門菌χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB)制備及反應(yīng)原性分析本試驗(yàn)運(yùn)用現(xiàn)代分子生物學(xué)技術(shù),經(jīng)PCR擴(kuò)增目的基因、目的基因與載體連接轉(zhuǎn)化、雙酶切鑒定等方法構(gòu)建了重組質(zhì)粒pYA4545-HA2和pYA4545-HA2-LTB;通過(guò)脂質(zhì)體轉(zhuǎn)染細(xì)胞和Western-Blot等方法驗(yàn)證了HA2及HA2-LTB蛋白能夠成功表達(dá),并能夠與抗體特異性結(jié)合,具有良好的免疫原性;再將重組質(zhì)粒最終導(dǎo)入到沙門菌χ11218中,獲得重組沙門菌χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB),并驗(yàn)證其對(duì)阿拉伯糖具有依賴性,為下一步實(shí)驗(yàn)奠定基礎(chǔ)。(2)重組沙門菌χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB)免疫保護(hù)效果比較與評(píng)價(jià)將實(shí)驗(yàn)小鼠分為5組,每組15只。分別將成功制備的χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB)兩組沙門菌和本實(shí)驗(yàn)室已經(jīng)構(gòu)建的χ11218(pYA4545)沙門菌免疫小鼠,共口服免疫4次,同時(shí)設(shè)置對(duì)照組與H9N2亞型禽流感滅活疫苗組。最后一次加強(qiáng)免疫后一周,通過(guò)流式細(xì)胞術(shù)(Flow cytometry,FCM)和間接ELISA方法檢測(cè)免疫效果;通過(guò)滴鼻攻毒實(shí)驗(yàn),觀察小鼠的生命體征、體重變化及生存情況;攻毒兩周后,取各組小鼠肺組織,制成病理切片,通過(guò)HE染色比較各組病理變化。進(jìn)一步對(duì)兩組重組沙門菌的免疫保護(hù)作用進(jìn)行評(píng)價(jià)。結(jié)果顯示,小鼠口服免疫新型沙門菌χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB),能夠增加CD3+CD4+和CD3+CD8+細(xì)胞數(shù)量,促進(jìn)T細(xì)胞分化,提高CD3+CD4+T細(xì)胞分泌的IL-4、IL-17和IFN-r的水平,對(duì)CD3+CD8+T細(xì)胞分泌IFN-γ也有一定的促進(jìn)作用,還能提高樹突細(xì)胞CD40、CD80和MHCⅡ的表達(dá)量,而且對(duì)B細(xì)胞分泌型IgA的表達(dá)也有一定的影響。與Control組及χ11218(pYA4545)組相比較,重組沙門菌組和滅活疫苗組,血清中細(xì)胞因子及特異性抗體IgG濃度較高,腸道灌洗液中的特異性抗體IgA含量也相對(duì)較高。針對(duì)沙門菌LPS特異性抗體的檢測(cè)中,重組沙門菌χ11218(pYA4545-HA2)、χ11218(pYA4545-HA2-LTB)及χ11218(pYA4545)組均有較高濃度的LPS抗體。感染流感病毒后,各組小鼠體重變化趨勢(shì)基本相同,攻毒的前7天,各組體重均不同程度下降,在第8天達(dá)到最低,從第9天起,各組小鼠體重均逐漸恢復(fù),最后趨于穩(wěn)定。通過(guò)對(duì)小鼠肺指數(shù)及肺指數(shù)抑制率的計(jì)算,說(shuō)明重組沙門菌χ11218(pYA4545-HA2)和χ11218(pYA4545-HA2-LTB)對(duì)H9N2型禽流感具有抵抗作用。肺臟病理切片結(jié)果顯示χ11218(pYA4545-HA2)組、χ11218(pYA4545-HA2-LTB)組和滅活疫苗組肺組織相對(duì)完整,病理癥狀較輕。以上結(jié)果中,雖然重組沙門菌組的免疫效果較疫苗組差,但同樣證實(shí)了遞送HA2及HA2-LTB基因的延遲裂解型沙門菌對(duì)H9N2亞型流感病毒具有較好的保護(hù)作用。說(shuō)明HA2能夠作為作為禽流感病毒疫苗的靶標(biāo)基因,同時(shí)發(fā)現(xiàn)HA2融合LTB能夠增強(qiáng)機(jī)體對(duì)禽流感病毒的抵抗作用,以上結(jié)果為延遲裂解性沙門菌作為疫苗活載體提供了理論依據(jù),為禽流感分子免疫機(jī)制的研究奠定了基礎(chǔ)。
[Abstract]:Avian influenza (AI), because of its high pathogenicity, causes large poultry deaths, not only causes huge economic losses to poultry industry in China, but also poses a serious threat to human health safety. So far, the most effective way to control avian influenza is still vaccinated, but because of the high mutagenicity of the virus, conventional traditional vaccines. It is difficult to achieve the ideal immune effect, so the research on the avian influenza virus (AIV) vaccine has become a hot spot of research at home and abroad. This study selected a highly conservative sequence of HA2 fragment in the neck of AIV (Hemagglutinin, HA), and used it with the mucosal immune adjuvant of Escherichia coli. Escherichia coli heat-labile enterotoxin (LTB) gene fusion was used to import the HA2 gene and HA2-LTB fusion gene into the carrier of the delayed lysate Salmonella. Two groups of recombinant Salmonella were constructed and their immune effects were evaluated. The specific contents and results were as follows: (1) the recombinant Salmonella Chi 11218 (pYA4545-HA2) and Chi 11218 (pYA4). 545-HA2-LTB) preparation and reactivity analysis, the recombinant plasmid pYA4545-HA2 and pYA4545-HA2-LTB were constructed by using modern molecular biology technology, PCR amplification of the target gene, the target gene and the vector, and the double enzyme digestion method. The HA2 and HA2-LTB proteins were verified by liposome transfected cells and Western-Blot. The recombinant plasmid was finally introduced into the Salmonella Chi Chi 11218, and the recombinant Salmonella Chi Chi 11218 (pYA4545-HA2) and Chi 11218 (pYA4545-HA2-LTB) were obtained. The recombinant plasmid was proved to be dependent on the Arabia sugar, which was the basis for the next experiment. (2) the recombinant Salmonella x 11218 (pYA4545-HA2) and the comparison and evaluation of the effect of Chi 11218 (pYA4545-HA2-LTB) immune protection, the experimental mice were divided into 5 groups, with 15 mice in each group. The mice were successfully prepared by Chi 11218 (pYA4545-HA2), Chi 11218 (pYA4545-HA2-LTB) two group and the already constructed Chi 11218 (pYA4545) Salmonella mice were immune to 4 times and set up at the same time. In the control group and the H9N2 subtype avian influenza inactivated vaccine group, the immune effect was detected by flow cytometry (Flow cytometry, FCM) and indirect ELISA after the last week of immunization. The vital signs, weight change and survival of the mice were observed by the intranasal attack test. After two weeks of attack, the lung tissue of each group was taken to make a pathological cut. The immunological protection of two groups of recombinant Salmonella was evaluated by HE staining. The results showed that the oral immunization of new Salmonella x 11218 (pYA4545-HA2) and Chi 11218 (pYA4545-HA2-LTB) could increase the number of CD3+CD4+ and CD3+CD8+ cells, promote the differentiation of T cells and improve the I of CD3+CD4+T cells. The level of L-4, IL-17 and IFN-r also promoted the secretion of IFN- gamma by CD3+CD8+T cells, and also increased the expression of CD40, CD80 and MHC II in dendritic cells, and also had a certain influence on the expression of B cell secretory IgA. Compared with the Control group and the Chi 11218 (pYA4545) group, the recombinant Salmonella and inactivated vaccine group, and the serum cell factors in the serum. The concentration of sub and specific antibody IgG was high, and the specific antibody IgA content in the intestinal lavage fluid was relatively high. In the detection of Salmonella LPS specific antibody, the recombinant Salmonella Chi 11218 (pYA4545-HA2), Chi 11218 (pYA4545-HA2-LTB) and Chi 11218 (pYA4545) group had a higher concentration of LPS antibody. After infection of influenza virus, the weight of mice in each group The change trend was basically the same. The weight of each group declined in the first 7 days and reached the lowest in the eighth day. From the ninth day, the weight of the mice in each group gradually recovered and finally tended to be stable. Through the calculation of the lung index and the lung index inhibition rate of the mice, the recombinant Salmonella x 11218 (pYA4545-HA2) and the X 11218 (pYA4545-HA2-LTB) to the H9N2 type were calculated. The results of lung pathology showed that the lung pathological sections showed the Chi 11218 (pYA4545-HA2) group, the lung tissue of the Chi 11218 (pYA4545-HA2-LTB) group and inactivated vaccine group was relatively complete and the pathological symptoms were lighter. The results of the above results showed that the immune effect of the recombinant Salmonella group was worse than the vaccine group, but it also confirmed the delayed lysis of the delivery of HA2 and HA2-LTB genes. The Salmonella type has a good protective effect on H9N2 subtype influenza virus. It shows that HA2 can be used as a target gene for avian influenza virus vaccine, and that HA2 fusion LTB can enhance the body's resistance to avian influenza virus. The above results provide a theoretical basis for the delayed lysis of Salmonella as a live carrier of the vaccine, for avian influenza. The research of molecular immune mechanism has laid the foundation.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王臣;郭香玲;李小康;吳庭才;李德元;陳溥言;;胸腺五肽和法氏囊活性五肽的融合表達(dá)及其對(duì)禽流感滅活疫苗的佐劑效應(yīng)[J];生物工程學(xué)報(bào);2015年05期

，

本文編號(hào)：1968481