本文選題：豬帶絳蟲六鉤蚴 + Tsol18�。� 參考：《中國農(nóng)業(yè)科學(xué)院》2008年博士論文

【摘要】： 囊尾蚴病(Cysticercosis)是由豬帶絳蟲(Taenia solium)的幼蟲囊尾蚴(Cysticercus cellulosae)寄生于人或豬等而引起的人畜共患寄生蟲病,是公認(rèn)的世界經(jīng)濟病之一。囊尾蚴病在中國大部分地區(qū)尤其在少數(shù)民族地區(qū)是一個重要的公共衛(wèi)生問題,嚴(yán)重威脅著人體健康,不僅如此,豬囊尾蚴病的廣泛存在極大影響了我國畜產(chǎn)品在國際市場上的競爭力,給畜牧業(yè)造成重大經(jīng)濟損失,豬囊尾蚴病的免疫防治勢在必行,然而在豬囊尾蚴病疫苗研究中,疫苗抗原的選擇和來源一直困擾著獸醫(yī)工作者。Tsol18是豬帶絳蟲六鉤蚴(T.solium oncosphere)階段特異性表達(dá)的抗原,主要在六鉤蚴感染中間宿主的早期階段分泌,其免疫血清在體外試驗中能殺死六鉤蚴,是豬囊尾蚴病疫苗研制的主要候選抗原之一。在已有的研究中,Tsol18重組蛋白在原核系統(tǒng)中多以包涵體形式表達(dá),給蛋白質(zhì)的復(fù)性和純化帶來許多困難,并且包涵體活性較低、保存期短、降解快,因而限制了Tsol18在豬囊尾蚴病疫苗研制上的應(yīng)用。 鼠傷寒沙門氏菌(Salmonella typhimurium)是一種常見的侵襲性胞內(nèi)菌,通過基因工程方法減毒后對宿主致病性顯著降低,但仍保留良好的侵襲力,可將免疫抗原直接遞呈給免疫細(xì)胞而誘導(dǎo)特異性的免疫應(yīng)答反應(yīng),減毒鼠傷寒沙門氏菌作為口服疫苗載體是目前疫苗免疫研究的熱點之一。為了優(yōu)化豬囊尾蚴病疫苗候選抗原Tsol18重組蛋白,探索豬囊尾蚴病口服重組活載體疫苗的免疫應(yīng)答,研究其在豬囊尾蚴病免疫預(yù)防上的作用,進(jìn)行了本項研究。 收集成熟的豬帶絳蟲蟲卵,經(jīng)孵化和激活后,提取六鉤蚴總RNA,設(shè)計特異性引物RT-PCR擴增Tsol18基因片段,并同義突變Tsol18基因片段中四個大腸桿菌低利用率密碼子,截去其N端的16個氨基酸的信號肽編碼序列,構(gòu)建原核表達(dá)載體pGEX-4T-Tsol18并表達(dá),進(jìn)行GST-Tsol18重組蛋白的SDS-PAGE、Western blot及穩(wěn)定性分析,并以GST-Tsol18重組蛋白為抗原制備兔高免血清;將改造的Tsol18基因片段插入Asd+的組成型表達(dá)載體pYA3341,構(gòu)建重組載體pYA3341-Tsol18,將重組載體電轉(zhuǎn)化入缺失腺苷酸環(huán)化酶(Δcya)、環(huán)腺苷酸受體蛋白基因(Δcrp)以及天冬氨酸-β-半醛脫氫酶(Δasd)的減毒鼠傷寒沙門氏菌X4550,研究重組活載體疫苗X4550(pYA3341-Tsol18)表達(dá)Tsol18重組蛋白的免疫原性、生長穩(wěn)定性、口服安全性及在小鼠體內(nèi)的分布情況,ELISA檢測免疫小鼠血清和腸液中抗Tsol18抗體的產(chǎn)生情況,流式細(xì)胞術(shù)分析其脾細(xì)胞亞型的分化,ELISA檢測免疫豬血清中抗Tsol18抗體的動態(tài)變化,探討重組疫苗可能的免疫應(yīng)答。 結(jié)果顯示改造后的GST-Tsol18重組蛋白多以可溶性形式表達(dá),蛋白表達(dá)量占菌體總蛋白的24 %,Western blot證明GST-Tsol18重組蛋白具有良好的抗原性,4℃保存120 d只有20.1 %降解,顯示重組蛋白穩(wěn)定性有很大改善。重組疫苗4550(pYA3341-Tsol18)在沒有選擇壓力的情況下連續(xù)培養(yǎng)100代后,隨機挑選的重組疫苗株全部都能生長和表達(dá)Tsol18重組蛋白,表明重組疫苗生長穩(wěn)定;BALB/c鼠口服重組疫苗株2.0×1012 cfu 30天后,存活率100 %,證實重組疫苗口服安全可靠,小鼠口服免疫后第21天在脾臟仍能測到大量的重組疫苗株,表明重組疫苗在體內(nèi)能夠長久存活,有利于刺激機體產(chǎn)生持久的免疫應(yīng)答;ELISA檢測顯示在二免后四周小鼠血清中抗Tsol18 IgG抗體的OD值達(dá)到0.645,二免后六周小鼠腸液中有抗Tsol18分泌性IgA抗體產(chǎn)生,表明重組疫苗能誘導(dǎo)小鼠產(chǎn)生較強的抗體水平和激發(fā)多種免疫應(yīng)答方式;免疫小鼠的CD4+、CD8+T淋巴細(xì)胞數(shù)目明顯增多,CD4+/CD8+ T細(xì)胞比值也顯著增高(P0.01),提示口服疫苗4550(pYA3341-Tsol18)可能同時誘導(dǎo)Th1和Th2免疫應(yīng)答反應(yīng)。豬免疫后20 d抗Tsol18 IgG抗體水平開始上升,在二免后30天抗體OD值達(dá)到1.025,表明重組疫苗能誘導(dǎo)豬產(chǎn)生抗豬囊尾蚴病的免疫應(yīng)答效應(yīng)。 本研究獲得了具有高效表達(dá)、良好免疫原性和穩(wěn)定性的豬囊尾蚴病疫苗候選抗原GST-Tsol18重組蛋白,構(gòu)建了攜帶Tsol18重組蛋白的減毒鼠傷寒沙門氏菌疫苗4550(pYA3341-Tsol18),初步進(jìn)行了重組疫苗的免疫學(xué)研究,為豬囊尾蚴病基因工程疫苗的研制和應(yīng)用開拓了新的思路。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 馮金瑞;劉立軍;;豬囊尾蚴重組抗原和基因工程疫苗的研究進(jìn)展[J];畜牧獸醫(yī)雜志;2012年02期

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本文編號：2021056