乳酸菌pSIP409-pgsA-3M2e-HA2的制备及其抗H9N2亚型禽流感免疫效果的研究

发布时间：2018-06-20 07:55

本文选题：禽流感 + 乳酸菌　；参考：《吉林农业大学》2016年硕士论文

【摘要】：禽流感(Avian influenza,AI)是全球范围内广为流行的禽类急性高度传染性疾病之一,近几十年来不仅给我国的养禽业造成了重大的经济损失、而且引起威胁人类健康的严重公共卫生学问题。目前,接种疫苗仍然是控制禽流感的最有效的方法,由于传统的疫苗多为灭活疫苗,虽安全性较好、但其免疫效果不理想,所以研制新型的口服黏膜免疫的通用型微生物制剂将具有更广阔的应用前景。本研究选用乳酸菌作为外源蛋白的递送载体,将禽流感病毒(Avian influenza virus,AIV)的血凝素基因HA2与主要保护性抗原M2e蛋白基因融合在一起,研制了HA2融合M2e基因的重组新功能型乳酸菌并对其免疫效果进行初步评价。具体研究内容及结果如下:(1)乳酸菌p SIP409-pgs A-3M2e-HA2、p SIP409-pgs A-HA2的制备及反应原性分析本文通过酶切、连接、转化等方法制备了两个重组质粒p SIP409-pgs A-3M2e-HA2和质粒p SIP409-pgs A-HA2。将该质粒电转入受体菌植物乳杆菌(Lactobacillus plantarum)NC8中,并用诱导肽Spp IP诱导表达,利用Western blotting分析乳酸菌表达蛋白的反应原性,结果表明新功能型乳酸菌p SIP409-pgs A-3M2e-HA2和p SIP409-pgs A-HA2所表达的外源蛋白均具有良好的反应原性,为下一步实验奠定基础。(2)乳酸菌pSIP409-pgsA-3M2e-HA2和pSIP409-pgsA-HA2免疫保护效果比较与评价将实验雏鸡分6组,每组20只。分别将已成功制备的p SIP409-pgs A-3M2e-HA2和p SIP409-pgs A-HA2两组乳酸菌和本实验室已经构建的p SIP409-pgs A-3M2e和的p SIP409-pgs A共四种乳酸菌对雏鸡进行初次免疫和加强免疫,同时设计空白对照组与H9N2亚型禽流感灭活疫苗组。初免从21天开始,连续3天,间隔14天之后加强免疫,连续3天。间隔3天后杀鸡取样,通过流式细胞术和淋巴细胞增殖检测各组雏鸡脾脏中的T淋巴细胞的活化与增殖情况,从而来检测新功能型乳酸菌对雏鸡的免疫保护效果。此外,分别收取各组雏鸡的血清和气管灌洗液,并用间接ELISA方法检测雏鸡特异性的Ig G抗体和分泌型抗体SIg A(Secretory immunoglobulin A,SIg A)的表达水平。雏鸡加强免疫后一周后通过滴鼻方式感染鹤源和鸭源两种不同源的H9N2亚型禽流感病毒,攻毒后观察雏鸡的精神状态并记录雏鸡的体重变化。攻毒后10天宰杀雏鸡并取肺脏和喉头,制成病理切片,通过HE染色观察并比较肺脏和喉头病理变化。更进一步检测并评价新功能型乳酸菌对雏鸡的免疫保护效果。结果显示,雏鸡口服免疫新功能型乳酸菌p SIP409-pgs A-3M2e-HA2、p SIP409-pgs A-HA2和p SIP409-pgs A-3M2e后雏鸡的脾脏中的T淋巴细胞数量相比空白对照组显著增高,其中p SIP409-pgs A-3M2e-HA2组和p SIP409-pgs A-HA2组差异极显著(P0.001)。血清及气管灌洗液中的特异性抗体的表达水平p SIP409-pgs A-3M2e-HA2组和p SIP409-pgs A-HA2组差异极显著(P0.05)。观察和比较各组雏鸡感染病毒后精神状态结果显示p SIP409-pgs A-3M2e-HA2组和p SIP409-pgs A-HA2组雏鸡先呈现精神不振、采食量下降等状态,但随后逐渐恢复正常,而空白对照组则持续呈现发病状态。感染两种病毒后雏鸡体重变化规律基本相同,结果显示p SIP409-pgs A-3M2e-HA2和p SIP409-pgs A-HA2组雏鸡体重开始逐渐下降,第6天达到最低,随后体重开始增加,而空白对照组雏鸡体重持续下降。肺脏及喉头病理切片结果显示p SIP409-pgs A-3M2e-HA2和p SIP409-pgs A-HA2组雏鸡的病理症状较轻。以上结果表明新功能型乳酸菌对H9N2亚型流感病毒的不同毒株均有保护作用,并且p SIP409-pgs A-3M2e-HA2和p SIP409-pgs A-HA2乳酸菌的免疫保护效果更好。说明HA2作为禽流感病毒的血凝素的保守抗原针对不同的禽H9N2亚型流感病毒都有较强的保护效果,同时发现了共表达M2e基因的融合新功能型乳酸菌更能增强机体抵抗AIV攻击的能力,以上结果为研制抗禽流感的新功能型微生态制剂提供了理论依据。
[Abstract]:Avian influenza (AI) is one of the most popular avian acute infectious diseases in the world. In recent decades, it has not only caused serious economic losses to the poultry industry in China, but also caused serious public health problems threatening human health. At present, vaccination is still the most effective way to control avian influenza. Because the traditional vaccine is mostly inactivated vaccine, although the safety is good, but its immune effect is not ideal, so the development of a new type of oral mucosal immune general microbial preparation will have a wider application prospect. This study selects the lactic acid bacteria as the delivery carrier of foreign protein and Avian influenza virus (AIV). The fusion of the hemagglutinin gene HA2 and the main protective antigen M2e protein gene has been developed and the recombinant new functional lactic acid bacteria with HA2 fusion M2e gene are developed and its immune effect is preliminarily evaluated. The specific contents and results are as follows: (1) the preparation of P SIP409-pgs A-3M2e-HA2, the preparation of P SIP409-pgs A-HA2 and the analysis of the reactivity Two recombinant plasmids P SIP409-pgs A-3M2e-HA2 and plasmid P SIP409-pgs A-HA2. were prepared by over enzyme digestion, connection and transformation. The plasmid was transferred into the NC8 of Lactobacillus plantarum (Lactobacillus plantarum), and was induced by inducible peptide Spp IP. The exogenous proteins expressed in the new functional lactic acid bacteria P SIP409-pgs A-3M2e-HA2 and P SIP409-pgs A-HA2 all have good reactivity and lay the foundation for the next experiment. (2) the comparison and evaluation of the immune protection effect of lactic acid bacteria pSIP409-pgsA-3M2e-HA2 and pSIP409-pgsA-HA2, the experimental chickens were divided into 6 groups, each group was 20. SIP409-pgs A-3M2e-HA2 and P SIP409-pgs A-HA2 two groups of lactic acid bacteria, P SIP409-pgs A-3M2e and P SIP409-pgs A four kinds of lactic acid bacteria were first immunized and strengthened in chicks. At the same time, the blank control group and the H9N2 subtype avian influenza inactivated vaccine group were designed. The initial immunity began from 21 days, 3 days in a continuous period and 14 days interval. After 3 days of immunization, the chickens were sampled for 3 days. The activation and proliferation of T lymphocytes in the spleen of the chickens were detected by flow cytometry and lymphocyte proliferation, so as to detect the protective effect of the new functional Lactobacillus on the chickens. ELISA method was used to detect the expression level of Ig G antibody and secretory antibody SIg A (Secretory immunoglobulin A, SIg A). After a week of immunization, chickens infected with two different sources of the crane source and duck source were infected with the H9N2 subtype of avian influenza virus. After attack, the spirit state of the chicks was observed and the body weight changes of the chicks were recorded. 10 days after the attack, the chicks were slaughtered and the lungs and larynx were taken to make pathological sections. HE staining was used to observe and compare the pathological changes of the lungs and larynx. The immune protection effect of the new functional lactic acid bacteria to the chicks was further detected and evaluated. The results showed that the chickens were immunized with new functional lactic acid bacteria P SIP409-pgs A-3M2e-HA2, P SIP409-pgs A-H The number of T lymphocytes in the spleen of A2 and P SIP409-pgs A-3M2e was significantly higher than that in the blank control group, and the difference between the P SIP409-pgs A-3M2e-HA2 group and P SIP409-pgs A-HA2 group was very significant (P0.001). Very significant (P0.05). Observation and comparison of the mental state of chickens infected with virus in each group showed that the chicks of the P SIP409-pgs A-3M2e-HA2 group and the P SIP409-pgs A-HA2 group first presented the state of discomfort and feed intake, but then gradually recovered to normal, while the blank control group continued to appear in the present state. The change of the body weight of the chickens after the two viruses was infected. The rules were basically the same, the results showed that the body weight of P SIP409-pgs A-3M2e-HA2 and P SIP409-pgs A-HA2 group began to decline gradually, the sixth days reached the lowest, then the body weight began to increase, while the blank control group of chick body weight continued to decrease. The pathological section of lung and throat pathology showed the pathology of P SIP409-pgs A-3M2e-HA2 and P SIP409-pgs A-HA2 group. The above results showed that the new functional lactobacillus has protective effect on different strains of H9N2 subtype influenza virus, and the immune protection effect of P SIP409-pgs A-3M2e-HA2 and P SIP409-pgs A-HA2 lactic acid bacteria is better. It shows that HA2 as the conserved antigen of the hemagglutinin of avian influenza virus is directed against the different avian influenza virus of different avian influenza viruses It has a strong protective effect. At the same time, the new functional lactic acid bacteria, which co express M2e gene, can enhance the ability of resisting AIV attack. The above results provide a theoretical basis for the development of new functional microecosystem for anti avian influenza.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S855.3

【相似文献】