本文選題：炭疽 + 鼠疫　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2009年博士論文

【摘要】： 炭疽和鼠疫都屬于自然疫源性烈性傳染病,這兩種疾病的病原體炭疽芽孢桿菌和鼠疫耶爾森菌是極其重要的生物恐怖劑/生物戰(zhàn)劑。美國、前蘇聯(lián)、日本等多個(gè)國家都曾把炭疽桿菌和鼠疫菌制成生物武器, 2001年美國發(fā)生的郵寄炭疽芽孢恐怖襲擊事件再次提醒我們時(shí)刻不可對(duì)生物恐怖掉以輕心,我國存在較大范圍的炭疽和鼠疫的自然疫源地,平戰(zhàn)時(shí)都有可能同時(shí)面對(duì)炭疽和鼠疫的威脅。近年來隨著抗生素的大量使用,多重耐藥菌株的出現(xiàn)使得炭疽和鼠疫的預(yù)防顯得更加重要。目前針對(duì)這兩種疾病國內(nèi)外已上市的疫苗有炭疽減毒活疫苗、炭疽吸附上清疫苗、鼠疫滅活全菌死疫苗、鼠疫減毒活疫苗等,但都存在副作用大等缺點(diǎn)。目前以rPA為基礎(chǔ)構(gòu)建的新型重組亞單位炭疽疫苗,以rF1和rV為基礎(chǔ)構(gòu)建的新型重組亞單位鼠疫疫苗都已進(jìn)入臨床試驗(yàn)階段。炭疽芽孢桿菌和鼠疫耶爾森菌保護(hù)性抗原明確,誘導(dǎo)的免疫以體液免疫為主,研制炭疽、鼠疫基因工程聯(lián)合疫苗可以簡化免疫程序、增加使用者的依從性、降低生產(chǎn)成本并節(jié)約衛(wèi)生資源,將為應(yīng)對(duì)與炭疽和鼠疫相關(guān)的生物戰(zhàn)/生物恐怖、重大傳染病和突發(fā)公共衛(wèi)生事件提供保障。本研究的目的是利用基因工程技術(shù)制備鼠疫保護(hù)性抗原,評(píng)價(jià)炭疽和鼠疫亞單位疫苗聯(lián)合免疫的效果,為炭疽、鼠疫基因工程聯(lián)合疫苗的研制奠定基礎(chǔ)。 大腸桿菌原核表達(dá)系統(tǒng)具有遺傳背景清楚、技術(shù)操作簡便、生產(chǎn)周期短、培養(yǎng)條件簡單等特點(diǎn),是最常用的外源蛋白表達(dá)系統(tǒng)之一。此前本室已成功在大腸桿菌表達(dá)系統(tǒng)中表達(dá)并純化了炭疽保護(hù)性抗原PA�？紤]到F1和V來自于原核細(xì)胞,分子量比較適中,本研究首先進(jìn)行了F1和V抗原在大腸桿菌系統(tǒng)中的高效表達(dá)研究。F1抗原由于疏水性較強(qiáng),因而對(duì)表達(dá)和純化都提出了挑戰(zhàn)。此前研究報(bào)道V抗原多采用融合GST標(biāo)簽親和純化后,再去除標(biāo)簽的方法進(jìn)行純化。我們考慮無標(biāo)簽蛋白與標(biāo)簽蛋白相比雖然純化步驟更為復(fù)雜,但不必考慮標(biāo)簽去除以及成品中是否殘存標(biāo)簽或蛋白酶,更便于進(jìn)行成品的質(zhì)量控制。經(jīng)過多次嘗試,最后選擇使用pET32a(+)載體在大腸桿菌BL21(DE3)中表達(dá)了無標(biāo)簽的F1和V抗原,結(jié)果表明實(shí)現(xiàn)了無標(biāo)簽F1和V的高效表達(dá)。 隨后將搖瓶工藝放大5倍至14L生物反應(yīng)器中,然后又放大5倍至42L生物反應(yīng)器中,成功得到了優(yōu)化的中試發(fā)酵生產(chǎn)工藝。在此基礎(chǔ)上分別進(jìn)行了三次中試發(fā)酵,結(jié)果顯示發(fā)酵工藝穩(wěn)定,具有良好的重復(fù)性。繼而充分利用蛋白質(zhì)的各種性質(zhì)進(jìn)行目的蛋白分離純化。在V抗原純化過程中,首先利用V抗原的疏水性質(zhì)將V抗原從裂菌后的上清中捕獲出來。然后利用V抗原在pH值高于等電點(diǎn)時(shí)帶負(fù)電荷的性質(zhì),將捕獲的V抗原通過弱陰離子柱DEAE層析進(jìn)行進(jìn)一步純化,最后采用凝膠過濾層析純化得到純度95%的V抗原。蛋白免疫印跡結(jié)果顯示純化的V抗原中存在單體和二聚體兩種形式。研究表明,F1抗原在生理?xiàng)l件下易聚集形成多聚體,且多聚體形式的F1抗原與單體F1抗原相比具有更好的免疫原性和保護(hù)效果。本研究首先采用鹽析的方法進(jìn)行F1抗原初步分離提純,隨后利用凝膠過濾層析進(jìn)行進(jìn)一步的精純并交換緩沖液,結(jié)果獲得純度95%的F1抗原。蛋白N端氨基酸序列測定顯示結(jié)果與預(yù)期完全一致。 在此基礎(chǔ)上,將蛋白使用氫氧化鋁佐劑進(jìn)行吸附,在小鼠、豚鼠和兔體內(nèi)進(jìn)行了免疫原性研究,觀察抗原免疫后的體液免疫反應(yīng),并比較聯(lián)合免疫組和單獨(dú)免疫組之間體液免疫反應(yīng)的差異。免疫原性的結(jié)果顯示,在三種動(dòng)物模型上,聯(lián)合免疫組和單獨(dú)免疫組相比具有相似的體液免疫反應(yīng),初次免疫后一至二周后動(dòng)物血清中可檢測到相應(yīng)的抗原特異性IgG抗體,加強(qiáng)免疫后達(dá)到較高水平�？贵w亞型分析結(jié)果顯示,小鼠共免疫rPA、rF1+rV沒有改變抗原特異性的IgG1/IgG2a比值,抗體反應(yīng)趨向于Th2型;小鼠長期免疫原性研究顯示抗體滴度維持超過一年之久,預(yù)示了聯(lián)合疫苗具有良好的長期效果。 在掌握了抗原免疫原性資料后,選擇炭疽敏感動(dòng)物模型兔進(jìn)行了炭疽芽孢攻擊保護(hù)效果的研究,選擇鼠疫敏感動(dòng)物模型小鼠進(jìn)行了鼠疫菌攻擊保護(hù)效果的研究。結(jié)果顯示聯(lián)合免疫后,兔可以抵抗皮下注射80LD50的毒力炭疽芽孢攻擊,而佐劑對(duì)照組的兔在6天內(nèi)全部死亡。小鼠模型結(jié)果顯示聯(lián)合免疫后,小鼠可抵抗皮下攻擊高達(dá)60,000MLD鼠疫菌,佐劑對(duì)照組則于4天內(nèi)全部死亡。在兩種動(dòng)物模型中,聯(lián)合免疫組生存率/生存時(shí)間不低于單獨(dú)免疫組。 本研究利用基因工程技術(shù)制備了鼠疫的保護(hù)性抗原F1和V,與炭疽保護(hù)性抗原PA聯(lián)合后,使用氫氧化鋁佐劑進(jìn)行吸附,在小鼠、豚鼠、兔模型上初步評(píng)價(jià)了抗原聯(lián)合免疫的免疫效果和保護(hù)效果。結(jié)果表明聯(lián)合免疫后可分別對(duì)毒力鼠疫耶爾森菌和炭疽芽孢攻擊產(chǎn)生有效保護(hù)。此外,免疫后產(chǎn)生的抗體滴度在小鼠體內(nèi)維持超過一年時(shí)間,預(yù)示聯(lián)合疫苗具有良好的長期效果。
[Abstract]:Anthrax and plague are all natural foci of infectious diseases. The pathogens of the two diseases, Bacillus anthracis and Jerson, are extremely important bioterrorism agents / biological agents. The United States, the former Soviet Union, Japan and other countries have made the Bacillus anthracis and Yersinia pestis into biological weapons, and the anthrax spores were mailed in the United States in 2001. The terrorist attacks remined us once again that we could not take care of biological terror at all times. There is a wide range of natural foci of anthrax and plague in our country. It is possible to face the threat of anthrax and plague at the same time in peacetime. In recent years, with the extensive use of antibiotics, the emergence of multiple resistant strains has made the prevention of anthrax and plague appear. More important. At present, the vaccine has been listed at home and abroad of these two diseases, including anthracnose attenuated live vaccine, anthrax adsorbed supernatant vaccine, plague inactivated whole bacteria vaccine, plague attenuated live vaccine and so on, but there are many disadvantages such as large side effects. The new recombinant subunit anthrax vaccine based on rPA is based on rF1 and rV. The new recombinant subunit plague vaccine has entered the clinical trial stage. The protective antigen of Bacillus anthracis and Yersinia pestis is clear, the induced immunity is mainly humoral immunity, and anthrax is developed. The combined vaccine of Yersinia pestis gene engineering can simplify the immune procedure, increase the compliance of the users, reduce the cost of production and save the health resources. The aim of this study is to prepare the plague protective antigen by genetic engineering technology, to evaluate the effect of combined immunization with anthrax and the plague subunit vaccine, and to develop a joint vaccine for anthrax and plague gene engineering. Lay the foundation.
The prokaryotic expression system of Escherichia coli has the characteristics of clear genetic background, simple operation, short production cycle, simple culture conditions, and is one of the most commonly used foreign protein expression systems. This room has successfully expressed and purified the protective antigen PA. of anthrax in the Escherichia coli expression system, considering that F1 and V are derived from the prokaryotic cells. The high expression of F1 and V antigen in the Escherichia coli system was first carried out in this study. The.F1 antigen was highly hydrophobic, so the expression and purification were challenged. The previous study reported that the V antigen was purified by fusion GST label affinity purification and then the label was removed. We consider no label. Although the purification step is more complex than the protein label protein, it is not necessary to consider the label removal and the remaining labels or proteases in the finished product. The pET32a (+) carrier is selected to express the unlabeled F1 and V antigen in the Escherichia coli BL21 (DE3) after several attempts. The results show that it is realized. The efficient expression of unlabeled F1 and V.
Then the shake flask process was amplified by 5 times to the 14L bioreactor, and then amplified by 5 times to the 42L bioreactor, the optimized fermentation process was successfully obtained. On this basis, three pilot fermentation processes were carried out respectively. The results showed that the fermentation process was stable and had good refolding. Then the various properties of protein were fully utilized. V antigen was purified by using the hydrophobic properties of V antigen to capture the V antigen from the supernatant of the cracked bacteria in the process of purification of the antigen. Then the V antigen was purified by the DEAE layer of the weak anion column and purified by the V antigen at the pH value higher than the isoelectric point. Finally, the gel filtration was used. The purity of 95% V antigen was purified by chromatography. The results of protein immunoblotting showed that there were two forms of monomer and two polymer in the purified V antigen. The study showed that the F1 antigen was easily aggregated under physiological conditions, and the F1 antigen of the polymer form had better immunogenicity and protective effect compared with the monomer F1 antigen. The method of salting out was used to separate and purify the F1 antigen preliminarily, then further purified and exchanged by gel filtration chromatography. The results obtained the purity of 95% F1 antigen. The results of the amino acid sequence of protein N were in full agreement with the expected results.
On this basis, the protein was adsorbed with aluminum hydroxide adjuvant, and the immunogenicity was studied in mice, guinea pigs and rabbits. The humoral immune response after antigen immunization was observed and the difference of humoral immune response between the combined immune group and the individual immune group was compared. The results of immunogenicity showed that the immunogenicity was combined with the three animal models. The pestilence group had a similar humoral immune response compared with the individual immune group. The corresponding antigen specific IgG antibody could be detected in the animal serum for one to two weeks after the initial immunization, and the immunization reached a high level. The antibody subtype analysis showed that the mice were immunized with rPA and rF1+rV did not change the specific IgG1/IgG2a ratio of the antigen, and the antibody was not changed. The response tended to be Th2 type. Long term immunogenicity study showed that the antibody titer lasted for more than a year, indicating that the combined vaccine had good long-term effects.
After mastering the antigen immunogenicity data, the rabbit model of anthrax sensitive animal model was selected to study the protective effect of the anthrax spore attack. The mouse pestis sensitive animal model mice were selected to carry out the study on the protection effect of the Yersinia pestis attack. The results showed that the rabbit could resist the attack of the 80LD50 anthracnose spore under the subcutaneous injection. The rabbits in the control group died within 6 days. The mice model showed that the mice could resist the subcutaneous attack as high as 60000MLD pestis, and the adjuvant control group died in 4 days after the combined immunization. In the two animal models, the survival rate / survival time of the combined immune group was not lower than that of the single immune group.
In this study, the protective antigen F1 and V of plague were prepared by genetic engineering. After combined with the protective antigen PA of anthrax, the adsorption was carried out by aluminum hydroxide adjuvant. In mice, guinea pigs and rabbits, the immune effect and protective effect of antigen combined immunization were preliminarily evaluated. The results showed that the combined immunization could be used for the virulence of Yersinia pestis, Jerson. Bacteria and anthrax spore attacks produce effective protection. In addition, the antibody titer produced after immunization is maintained in mice for more than one year, indicating that the combined vaccine has a good long-term effect.

【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】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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相關(guān)期刊論文 前2條

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