表达Tsol18抗原的口服减毒鼠伤寒沙门氏菌重组活载体疫苗的构建及其免疫学研究

发布时间：2018-06-15 06:33

本文选题：猪带绦虫六钩蚴 + Tsol18　；参考：《中国农业科学院》2008年博士论文

【摘要】： 囊尾蚴病(Cysticercosis)是由猪带绦虫(Taenia solium)的幼虫囊尾蚴(Cysticercus cellulosae)寄生于人或猪等而引起的人畜共患寄生虫病,是公认的世界经济病之一。囊尾蚴病在中国大部分地区尤其在少数民族地区是一个重要的公共卫生问题,严重威胁着人体健康,不仅如此,猪囊尾蚴病的广泛存在极大影响了我国畜产品在国际市场上的竞争力,给畜牧业造成重大经济损失,猪囊尾蚴病的免疫防治势在必行,然而在猪囊尾蚴病疫苗研究中,疫苗抗原的选择和来源一直困扰着兽医工作者。Tsol18是猪带绦虫六钩蚴(T.solium oncosphere)阶段特异性表达的抗原,主要在六钩蚴感染中间宿主的早期阶段分泌,其免疫血清在体外试验中能杀死六钩蚴,是猪囊尾蚴病疫苗研制的主要候选抗原之一。在已有的研究中,Tsol18重组蛋白在原核系统中多以包涵体形式表达,给蛋白质的复性和纯化带来许多困难,并且包涵体活性较低、保存期短、降解快,因而限制了Tsol18在猪囊尾蚴病疫苗研制上的应用。鼠伤寒沙门氏菌(Salmonella typhimurium)是一种常见的侵袭性胞内菌,通过基因工程方法减毒后对宿主致病性显著降低,但仍保留良好的侵袭力,可将免疫抗原直接递呈给免疫细胞而诱导特异性的免疫应答反应,减毒鼠伤寒沙门氏菌作为口服疫苗载体是目前疫苗免疫研究的热点之一。为了优化猪囊尾蚴病疫苗候选抗原Tsol18重组蛋白,探索猪囊尾蚴病口服重组活载体疫苗的免疫应答,研究其在猪囊尾蚴病免疫预防上的作用,进行了本项研究。收集成熟的猪带绦虫虫卵,经孵化和激活后,提取六钩蚴总RNA,设计特异性引物RT-PCR扩增Tsol18基因片段,并同义突变Tsol18基因片段中四个大肠杆菌低利用率密码子,截去其N端的16个氨基酸的信号肽编码序列,构建原核表达载体pGEX-4T-Tsol18并表达,进行GST-Tsol18重组蛋白的SDS-PAGE、Western blot及稳定性分析,并以GST-Tsol18重组蛋白为抗原制备兔高免血清;将改造的Tsol18基因片段插入Asd+的组成型表达载体pYA3341,构建重组载体pYA3341-Tsol18,将重组载体电转化入缺失腺苷酸环化酶(Δcya)、环腺苷酸受体蛋白基因(Δcrp)以及天冬氨酸-β-半醛脱氢酶(Δasd)的减毒鼠伤寒沙门氏菌X4550,研究重组活载体疫苗X4550(pYA3341-Tsol18)表达Tsol18重组蛋白的免疫原性、生长稳定性、口服安全性及在小鼠体内的分布情况,ELISA检测免疫小鼠血清和肠液中抗Tsol18抗体的产生情况,流式细胞术分析其脾细胞亚型的分化,ELISA检测免疫猪血清中抗Tsol18抗体的动态变化,探讨重组疫苗可能的免疫应答。结果显示改造后的GST-Tsol18重组蛋白多以可溶性形式表达,蛋白表达量占菌体总蛋白的24 %,Western blot证明GST-Tsol18重组蛋白具有良好的抗原性,4℃保存120 d只有20.1 %降解,显示重组蛋白稳定性有很大改善。重组疫苗4550(pYA3341-Tsol18)在没有选择压力的情况下连续培养100代后,随机挑选的重组疫苗株全部都能生长和表达Tsol18重组蛋白,表明重组疫苗生长稳定;BALB/c鼠口服重组疫苗株2.0×1012 cfu 30天后,存活率100 %,证实重组疫苗口服安全可靠,小鼠口服免疫后第21天在脾脏仍能测到大量的重组疫苗株,表明重组疫苗在体内能够长久存活,有利于刺激机体产生持久的免疫应答;ELISA检测显示在二免后四周小鼠血清中抗Tsol18 IgG抗体的OD值达到0.645,二免后六周小鼠肠液中有抗Tsol18分泌性IgA抗体产生,表明重组疫苗能诱导小鼠产生较强的抗体水平和激发多种免疫应答方式;免疫小鼠的CD4+、CD8+T淋巴细胞数目明显增多,CD4+/CD8+ T细胞比值也显著增高(P0.01),提示口服疫苗4550(pYA3341-Tsol18)可能同时诱导Th1和Th2免疫应答反应。猪免疫后20 d抗Tsol18 IgG抗体水平开始上升,在二免后30天抗体OD值达到1.025,表明重组疫苗能诱导猪产生抗猪囊尾蚴病的免疫应答效应。本研究获得了具有高效表达、良好免疫原性和稳定性的猪囊尾蚴病疫苗候选抗原GST-Tsol18重组蛋白,构建了携带Tsol18重组蛋白的减毒鼠伤寒沙门氏菌疫苗4550(pYA3341-Tsol18),初步进行了重组疫苗的免疫学研究,为猪囊尾蚴病基因工程疫苗的研制和应用开拓了新的思路。
[Abstract]:Cysticercosis (Cysticercosis) is a human zoonosis parasitic disease caused by the parasitic cysticercus of Taenia solium (Cysticercus cellulosae) parasitic on human or pig. It is one of the most recognized world economic diseases. Cysticercosis is an important public health problem in most areas of China, especially in ethnic minority areas. Heavy threat to human health, not only that, the widespread existence of cysticercosis has greatly affected the competitiveness of livestock products in the international market, causing major economic losses to animal husbandry, and the immune prevention and control of cysticercosis of swine cysticercosis is imperative. However, in the study of the cysticercosis vaccine, the selection and source of vaccine antigen has been plaguing beasts. .Tsol18, a medical worker, is a specific antigen expressed at the stage of the six T.solium oncosphere of the Taenia solium. It is secreted at the early stage of the intermediate host of the six cercaria. The immune sera can kill six of the cercaria in the experiment in vitro. It is one of the major candidate antigens for the development of the cysticercosis vaccine. In the previous study, the recombinant Tsol18 is reorganized. Protein expression in the prokaryotic system is mostly expressed in the form of inclusion body, which brings many difficulties to the renaturation and purification of protein, and the inclusion body activity is low, the preservation period is short, and the degradation is fast. Therefore, the application of Tsol18 in the development of the cysticercosis vaccine is limited.
Salmonella typhimurium (Salmonella typhimurium) is a common invasive intracellular bacteria, which reduces the pathogenicity of the host by genetic engineering methods. But it still holds good invasiveness. It can direct the immune antigen directly to immune cells and induce specific immune response. The attenuated Salmonella typhimurium is the mouth. In order to optimize the recombinant protein of the vaccine candidate antigen Tsol18 for the vaccine of cysticercosis of porcine cysticercosis, the immune response of the oral recombinant live vector vaccine for cysticercosis of porcine cysticercosis was explored and its role in the immunization of cysticercosis of porcine cysticercosis was studied.
After incubating and activating the eggs of Taenia solium, the total RNA of six cercariae was extracted and activated, and the specific primer RT-PCR was designed to amplify the Tsol18 gene fragment and four low utilization codon of Escherichia coli in the synonymous mutation Tsol18 gene fragment. The encoding sequence of the signal peptide of 16 amino acids at the N end was truncated, and the prokaryotic expression vector pGEX-4T-Tsol18 was constructed. SDS-PAGE, Western blot and stability analysis of GST-Tsol18 recombinant protein were carried out and rabbit high serum was prepared with GST-Tsol18 recombinant protein as antigen, and the modified Tsol18 gene fragment was inserted into the constituent expression vector pYA3341 of Asd+, and the recombinant vector pYA3341-Tsol18 was constructed, and the recombinant vector was converted into the deletion adenylate cyclase (delta). CyA), the adenylate receptor protein gene (delta CRP) and the attenuated Salmonella typhimurium X4550 of aspartic acid beta semi aldehyde dehydrogenase (delta ASD). The immunogenicity, growth stability, oral safety and distribution of the recombinant protein of Tsol18 recombinant protein expressed by recombinant live vector vaccine X4550 (pYA3341-Tsol18) were studied. ELISA was used to detect immune mice. The production of anti Tsol18 antibody in serum and intestinal fluid, flow cytometry analysis of its splenic cell subtype differentiation, ELISA detection of immune pig serum anti Tsol18 antibody dynamic changes, to explore the possible immune response of the recombinant vaccine.
The results showed that the recombinant protein of the GST-Tsol18 was expressed in soluble form, the protein expression was 24% of the total protein, and Western blot showed that the recombinant protein of GST-Tsol18 had good antigenicity, the preservation of the recombinant protein at 4 C was only 20.1%, which showed that the stability of the recombinant protein was greatly improved. The recombinant vaccine 4550 (pYA3341-Tsol18) was not. All the randomly selected recombinant vaccine strains could grow and express Tsol18 recombinant protein after 100 generations of continuous culture under pressure, indicating that the recombinant vaccine was stable, and the survival rate was 100% after 30 days of 30 days after oral recombinant vaccine of BALB/c mice, which confirmed that the recombinant vaccine was safe and reliable, and the spleen was still in the spleen for twenty-first days after oral immunization. A large number of recombinant vaccine strains can be detected, indicating that the recombinant vaccine can survive in the body for a long time and stimulate the body to produce a lasting immune response. ELISA detection shows that the anti Tsol18 IgG antibody in the serum of mice in the four weeks after two exemptions reaches 0.645, and there is an anti Tsol18 secretory IgA antibody produced in the intestinal liquid of two weeks after exemptions, indicating the reorganization of the antibody. The vaccine could induce a strong antibody level and stimulate a variety of immune responses in mice; the number of CD4+, CD8+T lymphocytes in the immune mice increased significantly and the ratio of CD4+/CD8+ T cells increased significantly (P0.01), suggesting that oral vaccine 4550 (pYA3341-Tsol18) may induce Th1 and Th2 immune response simultaneously. The 20 d anti Tsol18 IgG after swine immunization The antibody level began to rise, and the antibody od reached 1.025 on the 30 day after two immunization, indicating that the recombinant vaccine could induce the immune response of pigs to cysticercosis cysticercosis.
The recombinant protein of the vaccine candidate antigen GST-Tsol18 with high expression, good immunogenicity and stability was obtained. The attenuated Salmonella typhimurium vaccine 4550 (pYA3341-Tsol18) carrying Tsol18 recombinant protein was constructed, and the immunological study of the recombinant vaccine was preliminarily carried out for the genetic engineering vaccine of cysticercosis porcine cysticercosis. The development and application of it have opened up a new way of thinking.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

【引证文献】