本文選題：鼠疫菌 + 天然F1抗原��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年碩士論文

【摘要】： 背景:鼠疫是由鼠疫耶爾森氏菌(Yersinia pestis) (以下簡稱鼠疫菌)引起的烈性傳染病,同時(shí)也是一種標(biāo)準(zhǔn)的生物戰(zhàn)劑和生物恐怖劑。鼠疫菌的主要保護(hù)性抗原是F1(莢膜抗原)和LcrV(V-抗原),以保護(hù)性抗原作為亞單位疫苗成為當(dāng)今鼠疫疫苗的重要研究方向。F1抗原由鼠疫菌pMT1質(zhì)粒上的F1操縱子編碼,以多聚體的形式在細(xì)胞表面形成莢膜,F1抗體通過阻斷F1蛋白的抗吞噬作用從而具有免疫保護(hù)作用。由pCD1質(zhì)粒編碼的V抗原(LcrV)是鼠疫菌重要的毒力因子、低鈣反應(yīng)調(diào)節(jié)因子和保護(hù)性抗原,但存在一定的免疫抑制作用:V抗原在體內(nèi)除抑制前炎性細(xì)胞因子(γ-干擾素、腫瘤壞死因子)的釋放外還增加白細(xì)胞介素-10的釋放,從而抑制宿主的天然免疫應(yīng)答。最近在V抗原的研究中發(fā)現(xiàn)產(chǎn)生免疫抑制作用的區(qū)段為271-300氨基酸,從全長V抗原(326個(gè)氨基酸)中去除這個(gè)區(qū)段可有效地降低V抗原的免疫抑制作用而同時(shí)保留其免疫保護(hù)作用。近來我們發(fā)現(xiàn)在云南感染東方型鼠疫菌的病人血清中檢測不到V抗原的抗體,這也決定了采用F1和V抗原聯(lián)合應(yīng)用成為鼠疫疫苗的亞單位疫苗的熱點(diǎn)和方向。 方法:采用玻璃珠處理、飽和硫酸銨沉淀和分子篩過濾相結(jié)合的方案,從鼠疫菌減毒活疫苗株(EV76)的培養(yǎng)物中提取天然表達(dá)的F1抗原。依據(jù)已知的LcrV抗原核苷酸序列,避開LcrV抗原產(chǎn)生免疫抑制作用的區(qū)段設(shè)計(jì)引物,克隆表達(dá)rV270抗原。表達(dá)產(chǎn)物先后經(jīng)過Co2+親和層析和Sephacryl S-200 HR凝膠柱純化,并在純化過程中應(yīng)用凝血酶切除His標(biāo)簽。純化的蛋白用免疫印記法、N-末端測序、質(zhì)譜肽圖、高效液相色譜(HPLC)及紫外光譜掃描方法鑒定其性質(zhì)和純度。使用氫氧化鋁佐劑分別吸附兩種抗原后免疫BALB/c小鼠,使用一次免疫和兩次免疫觀察兩種抗原的單一及聯(lián)合免疫保護(hù)作用,從而得出抗原配伍最佳劑量和聯(lián)合抗原免疫方法對(duì)比結(jié)果。 結(jié)果:本試驗(yàn)建立了一種新型天然鼠疫菌F1抗原的提取與純化方法,成功提取出純度超過98%的天然F1抗原。純化的蛋白通過免疫印記法、N-末端測序、質(zhì)譜肽圖、高效液相色譜(HPLC)及紫外光譜掃描方法鑒定,證明該蛋白正是鼠疫菌F1抗原。通過ELISA檢測發(fā)現(xiàn)純化出的天然F1抗原對(duì)F1抗體檢測的敏感度高于重組rF1抗原。rV270基因成功地克隆到pET24a載體中,并以可溶性方式表達(dá)。應(yīng)用Co2+親和層析柱和Sephacryl S-200 HR凝膠柱結(jié)合凝血蛋白酶切除His標(biāo)簽的方法可得到不含標(biāo)簽的較高純度目的蛋白。動(dòng)物實(shí)驗(yàn)證實(shí)純化的天然F1抗原和重組的rV270抗原單抗原及聯(lián)合抗原免疫均產(chǎn)生較高的抗體滴度和均有良好的免疫保護(hù)作用,且得到免疫方案和抗原免疫優(yōu)化劑量組,為下一步亞單位疫苗研制奠定基礎(chǔ)。 結(jié)論:1.本實(shí)驗(yàn)所提供的鼠疫菌天然F1抗原的提取、純化方法,是先從鼠疫菌培養(yǎng)物中分離培養(yǎng)上清和菌體沉淀,再利用飽和硫酸銨沉淀從培養(yǎng)上清中以及利用玻璃珠從菌體沉淀中分別提取出各部分天然F1抗原粗蛋白,再將各部分天然F1抗原粗蛋白各自通過飽和硫酸銨沉淀、凝膠過濾得到各部分純化的鼠疫菌天然F1抗原,保持了蛋白的天然構(gòu)象,并且避免了有機(jī)溶劑對(duì)蛋白活性的影響和對(duì)環(huán)境的污染。2.為了制備不帶任何載體序列的rV270抗原,采用融合表達(dá)帶His標(biāo)簽的蛋白,然后凝血酶切除標(biāo)簽的策略,使目的蛋白不含有任何載體序列,保持天然的蛋白構(gòu)象和生物學(xué)活性。本研究利用金屬螯合親和層析柱將帶有His標(biāo)簽的目的蛋白與其它大量雜蛋白分離,同時(shí)利用凝血酶在親和層析柱上完成標(biāo)簽的切割,然后再利用分子篩進(jìn)一步對(duì)蛋白進(jìn)行純化得到了高純和度的目的蛋白,為下一步動(dòng)物免疫觀察抗原的免疫保護(hù)效果提供基礎(chǔ)。3.動(dòng)物實(shí)驗(yàn)實(shí)驗(yàn)采用不同劑量組的抗原免疫,旨在尋找最佳劑量配伍來免疫動(dòng)物從而刺激機(jī)體產(chǎn)生更高的抗體滴度,增強(qiáng)免疫保護(hù)作用和延長免疫時(shí)間;使用單抗原和聯(lián)合抗原免疫,目的是比較單抗原和聯(lián)合抗原免疫效果,結(jié)果證實(shí)聯(lián)合抗原免疫較單抗原免疫能產(chǎn)生更高的抗體滴度;使用一次免疫方案和初次免疫后加強(qiáng)免疫的兩次免疫方案對(duì)比,得出兩次免疫較一次免疫能產(chǎn)生更高的抗體滴度且產(chǎn)生抗體滴度時(shí)間更長。從這三方面結(jié)論得到免疫方案及抗原免疫優(yōu)化劑量組,為下一步亞單位疫苗的研制奠定基礎(chǔ)。
[Abstract]:Background: plague is a strong infectious disease caused by the Yersinia pestis (Yersinia pestis). It is also a standard biological warfare agent and bioterrorism agent. The main protective antigen of Yersinia pestis is F1 (capsule antigen) and LcrV (V- antigen), and the protective antigen is used as subunit vaccine to become the plague vaccine. The important research direction.F1 antigen is encoded by the F1 operon on the pMT1 plasmid of Yersinia pestis, which forms a capsule on the cell surface in the form of polymer. The F1 antibody has the immune protection by blocking the anti phagocytosis of the F1 protein. The V antigen (LcrV) encoded by the pCD1 plasmid is an important virulence factor of the Yersinia pestis, and the low calcium response regulator and the regulation factor. Protective antigen, but there is a certain immune inhibitory effect: V antigen in the body in addition to inhibiting the release of pro-inflammatory cytokines (interferon, tumor necrosis factor) in addition to increase the release of interleukin -10, thus inhibiting the host's natural immune response. Recently, the area of the immunosuppressive effect of the V antigen was found to be 271-300. The removal of this section from the full length V antigen (326 amino acids) can effectively reduce the immunosuppressive effect of V antigen and retain its protective effect. Recently, we found that the antibodies against V antigen in the sera of Yunnan infected with the Oriental Yersinia pestis have been found to be combined with F1 and V antigens. The hotspots and directions of subunit vaccines for vaccination.
Methods: using the scheme of glass bead treatment, saturated ammonium sulfate precipitation and molecular sieve filtration, the naturally expressed F1 antigen was extracted from the culture of Yersinia pestis live attenuated vaccine strain (EV76). According to the known nucleotide sequence of LcrV antigen nucleotides, the primers were designed to avoid the immune inhibition effect of LcrV antigen, and the expression of rV270 antigen was cloned. The products were purified by Co2+ affinity chromatography and Sephacryl S-200 HR gel column, and the His tags were excised by thrombin in the purification process. The purified protein was determined by immunoimprinting, N- terminal sequencing, mass spectrometry peptide, high performance liquid chromatography (HPLC) and UV spectrum scanning method to determine its properties and purity. The BALB/c mice were immunized with two antigens. The single immunization and two immunization were used to observe the single and combined protective effects of two antigens. The results were compared with the best antigenic dosage and the combined antigen immunization.
Results: a new method for the extraction and purification of a new type of natural Yersinia pestis F1 antigen was established, and the natural F1 antigen with purity of more than 98% was successfully extracted. The purified protein was identified by immunoimprinting, N- terminal sequencing, mass spectrometry peptide, HPLC and UV spectral scanning, which proved that the protein was the F1 antigen of Yersinia pestis. ELISA detection showed that the sensitivity of the purified natural F1 antigen to F1 antibody was higher than that of the recombinant rF1 antigen.RV270 gene, which was successfully cloned into the pET24a vector and expressed in a soluble way. The method of using Co2+ affinity chromatography column and Sephacryl S-200 HR gel column combined with coagulation protease to excision the His label could be obtained without the label. The animal experiments confirmed that the purified natural F1 antigen, the recombinant rV270 antigen single antigen and the combined antigen immunization produced a high antibody titer and good immune protection, and the immunization scheme and the immune optimal dose group were obtained, which laid the foundation for the development of the next subunit vaccine.
Conclusion: 1. the extraction and purification of the natural F1 antigen of Yersinia pestis were isolated and cultured first from the culture of Yersinia pestis, and then using the saturated ammonium sulfate precipitation from the culture supernatant and the use of glass beads to extract the natural F1 antigen of each part of the natural antigen, and then the natural F1 of each part. The antigen crude protein was precipitated by saturated ammonium sulfate, and the natural F1 antigen of Yersinia pestis was purified by gel filtration. The natural conformation of the protein was preserved, and the effect of organic solvents on the activity of protein and the environmental pollution.2. were avoided to prepare the rV270 antigen without any carrier sequence, and the fusion expression with the His label was used. In this study, the target protein with the His label was separated from a large number of other proteins with a metal chelating affinity chromatography column, and the tag was cut on the affinity chromatography column by thrombin. And then the protein was further purified by molecular sieve to obtain the high purity and degree target protein, which provided the basis for the immune protection effect of the next animal immune observation antigen in the.3. animal experiment experiment using the antigen immunization of different dose groups, aiming at finding the best dose to match the immune animals to stimulate the organism to produce higher production. The titer of antibody, enhanced immune protection and prolongation of immune time; immunization with single antigen and combined antigen was used to compare the immune effect of single antigen and combined antigen. The results confirmed that the combined antigen immunization produced a higher antibody titer than the single antigen immunization, and the two immunization was enhanced after the first immunization program and the first immunization. The comparison of the scheme shows that the two immunization can produce a higher antibody titer and produce a longer antibody titer. From these three aspects, the immune scheme and the optimal dose group of the antigen immunization are obtained, which lays the foundation for the development of the next subunit vaccine.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 汪潔英;魏東;王國治;;鼠疫疫苗的研究進(jìn)展[J];微生物學(xué)免疫學(xué)進(jìn)展;2011年02期

相關(guān)碩士學(xué)位論文 前1條

1 汪潔英;鼠疫雙組分疫苗的免疫學(xué)評(píng)價(jià)[D];福建醫(yī)科大學(xué);2011年

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