發(fā)布時間：2018-06-29 17:42

  本文選題：HbcAg + EV71　； 參考：《北京工業(yè)大學》2014年博士論文

【摘要】：腸道病毒71型（enterovirus71, EV71）與柯薩奇A16病毒是引起手足口�。╤and foot and mouth disease, HFMD）的主要病原體。近年來手足口病在亞太地區(qū)的多次爆發(fā)，使當?shù)厍嗌倌陜和纳眢w健康受到嚴重威脅。雖然手足口病發(fā)病后病情較溫和且具有一定的自愈性，但如任由其發(fā)展也將導致嚴重的神經(jīng)系統(tǒng)并發(fā)癥，更為嚴重者可導致死亡。我國衛(wèi)生部于2008年將手足口病列為法定報告的丙類傳染病。迄今為止，防治手足口病的相關(guān)疫苗仍未上市。因此，研發(fā)廣譜高效的手足口病疫苗意義重大。 EV71病毒是一種非包膜的RNA病毒，隸屬于小RNA病毒科（Picornaradae）家族，其顆粒的直徑大小約30nm左右。EV71病毒的顆粒呈二十面體立體對稱結(jié)構(gòu)；其病毒衣殼由60組多聚蛋白所組成，每組多聚蛋白均含有4種衣殼蛋白（VP1-VP4）。有證據(jù)顯示VP1-VP3蛋白位于正二十面體核衣殼的外表面，而VP4蛋白位于核衣殼的內(nèi)部與RNA核心緊密連接。VP4蛋白長約70個氨基酸，N端豆蔻�；�。晶體結(jié)構(gòu)X光衍射圖分析表明：成熟的EV71病毒顆粒其立體結(jié)構(gòu)與其他腸道病毒極其相似。 本文的研究首先將EV71病毒C4亞型VP4蛋白N端前20個氨基酸的基因融合入乙肝病毒核心抗原（HbcAg）基因中，融合后的重組基因克隆至pET-22b (+)載體中。利用大腸桿菌表達系統(tǒng)分別表達HBc-N149蛋白以及HBc-N149-VP4-N20蛋白。IPTG誘導目的蛋白表達后利用Western-blot檢測蛋白表達情況。誘導表達后的目的蛋白利用鎳柱進行純化。電鏡觀察結(jié)果顯示，HBc-N149蛋白與HBc-N149-VP4-N20蛋白均可有效形成病毒樣顆粒，且直徑約為25-30nm。這一結(jié)果表明，將EV71病毒VP4蛋白N端前20個氨基酸插入HbcAg蛋白后并未改變其自組裝形成病毒樣顆粒的特性。 為了研究重組HbcAg蛋白是否能產(chǎn)生針對VP4-N20的抗體，將純化后的HBc-N149蛋白以及HBc-N149-VP4-N20蛋白通過肌肉注射的方式免疫雌性BALB/c小鼠。陰性對照組免疫PBS溶液。實驗動物免疫后收集血清進行血清學相關(guān)檢測。實驗結(jié)果顯示，重組HbcAg蛋白可以產(chǎn)生針對VP4-N20的抗體。 為了驗證包含有VP4-N20的重組HbcAg蛋白是否能產(chǎn)生針對EV71病毒的中和抗體，利用實驗動物免疫后收集的血清進行體外中和實驗。實驗結(jié)果顯示，HBc-N149-VP4-N20蛋白免疫組血清可以中和EV71病毒；HBc-N149蛋白免疫組血清未能中和EV71病毒。這一結(jié)果表明含有VP4-N20的重組HbcAg蛋白免疫實驗動物后產(chǎn)生的抗體具有針對EV71病毒的中和活性。 為了進一步驗證抗HBc-N149-VP4-N20蛋白的血清是否能為實驗小鼠提供針對EV71病毒的保護能力，進行了小鼠體內(nèi)攻毒實驗。實驗動物選擇1日齡BALB/c乳鼠。實驗中選用針對乳鼠具有較高毒力的EV71病毒BrCr-TR株，通過腹腔注射病毒-血清混合物（血清分別選用免疫HBc-N149-VP4-N20蛋白的血清以及免疫HBc-N149蛋白的血清）、病毒-PBS溶液混合物。實驗進行至第7天時，病毒-HBc-N149蛋白血清組乳鼠、病毒-PBS溶液組乳鼠開始出現(xiàn)行動遲緩、四肢乏力、四肢癱瘓等病癥，有甚者出現(xiàn)死亡癥狀；第16天即實驗結(jié)束時，病毒-HBc-N149蛋白血清組乳鼠存活率為40%，病毒-PBS溶液組乳鼠存活率為20%。反之，，病毒-HBc-N149-VP4-N20蛋白血清組乳鼠存活率為90%。這一結(jié)果表明，重組HbcAg顆粒即HBc-N149-VP4-N20蛋白的免疫血清可以針對EV71病毒為新生乳鼠提供保護效力。 進一步的研究中，利用肽庫篩查實驗確定了VP4-N20蛋白產(chǎn)生中和抗體的抗原決定簇表位。實驗中肽庫的設(shè)計原則為，分別從VP4-N20蛋白N端以及C端每次減少兩個氨基酸。實驗結(jié)果顯示，當VP4-N20蛋白N端減少至第6個氨基酸時，或者C端減少至第10個氨基酸時，針對VP4-N20蛋白的免疫血清將不再和VP4-N20蛋白相結(jié)合。這一實驗結(jié)果表明，VP4-N20蛋白產(chǎn)生中和抗體的抗原決定簇表位為其N端減少6個氨基酸、C端減少10個氨基酸后所剩多肽序列。 本文的研究中，將EV71病毒C4亞型的VP4蛋白N端前20個氨基酸融合入HbcAg中且在大腸桿菌中表達。實驗結(jié)果顯示，融合目的蛋白在表達后可以自組裝形成嵌合式病毒樣顆粒，并且可以誘導產(chǎn)生針對病毒的有效中和抗體。后續(xù)試驗中，通過肽庫篩查確定了這一序列的抗原決定簇位點所在。實驗結(jié)果最終表明，EV71病毒VP4蛋白中和表位的鑒定為研制廣譜抗EV71預防性疫苗提供了新的思路和線索。
[Abstract]:Enterovirus71 (enterovirus71, EV71) and Coxsackie A16 virus are the main pathogens causing hand foot and mouth disease (hand foot and mouth disease, HFMD). In recent years, hand foot and mouth disease (HFMD) has erupted many times in the Asia Pacific region, causing serious threat to the health of young children in the region. Self healing, but the development of it will lead to serious neurological complications, and more serious people can lead to death. In 2008, the Ministry of health of China listed hand foot and mouth disease as a legal report of the class C infectious disease. So far, the related vaccine against hand foot and mouth disease has not been listed. Therefore, the significance of developing a broad spectrum and efficient hand foot and mouth disease vaccine is developed. It's important.
EV71 virus is a non enveloped RNA virus, belonging to the family of small RNA virus family (Picornaradae). The particle size of about 30nm.EV71 virus particles is twenty dimensional symmetry structure; its viral capsid is composed of 60 groups of polyproteins and each group of polyproteins contains 4 kinds of capsid protein (VP1-VP4). Evidence shows VP1-VP3 The protein is located on the outer surface of the nucleocapsid of the positive twenty body, while the VP4 protein is located inside the nucleocapsid and the RNA core closely connected with the.VP4 protein, about 70 amino acids long and N terminal myrisylation. The crystal structure X light diffraction analysis shows that the stereoscopic structure of the mature EV71 virus particles is extremely similar to the other enteroviruses.
This study first fused the 20 amino acids of the EV71 virus C4 subtype VP4 protein into the hepatitis B virus core antigen (HbcAg) gene, and cloned the recombinant gene into the pET-22b (+) vector. The expression system of the Escherichia coli expression system was used to express the HBc-N149 protein and the HBc-N149-VP4-N20 protein.IPTG to induce the expression of the target protein. The protein expression was detected by Western-blot. The purified target protein was purified by the nickel column. The results showed that the HBc-N149 protein and HBc-N149-VP4-N20 protein could effectively form the virus like particles, and the diameter of the protein was about 25-30nm.. The results showed that the 20 amino acids of the N end of the EV71 virus VP4 protein were inserted into HbcAg eggs. The characteristics of self assembling virus like particles did not change after white.
In order to study whether the recombinant HbcAg protein could produce antibodies against VP4-N20, the purified HBc-N149 protein and HBc-N149-VP4-N20 protein were immunized by intramuscular injection of the female BALB/c mice. The negative control group was immunized with PBS solution. The serum was collected from the experimental animal after immunization and the serological correlation was detected. The experimental results showed that the recombinant HbcAg was reconstituted. The protein can produce antibodies against VP4-N20.
In order to verify whether the recombinant HbcAg protein containing VP4-N20 could produce neutralizing antibodies against EV71 virus, the serum collected from the experimental animal immune system was used to neutralize the neutralization test in vitro. The results showed that the serum of HBc-N149-VP4-N20 protein immune group could neutralize the EV71 virus, and the serum of HBc-N149 protein immune group did not neutralize the EV71 virus. The results showed that the recombinant HbcAg protein containing VP4-N20 immunized with experimental animals had neutralizing activity against EV71 virus.
In order to further verify whether the serum of anti HBc-N149-VP4-N20 protein could provide the protective ability of EV71 virus for experimental mice, the experiment was carried out in mice. The experimental animals selected 1 day old BALB/c milk mice. In the experiment, the EV71 virus BrCr-TR strain with high virulence was selected and the virus serum mixture was injected into the abdominal cavity. The serum of the immune HBc-N149-VP4-N20 protein and the serum of the immune HBc-N149 protein, and the mixture of the -PBS solution of the virus, were used in the serum, respectively. When the experiment was carried out for seventh days, the virus -HBc-N149 protein serum group of milk mice, the virus -PBS solution group of milk mice began to appear to be slow in action, fatigue in the extremities, limbs paralysis and so on. At the end of the 16 day, the survival rate of the virus -HBc-N149 protein serum group was 40%, the survival rate of the virus -PBS solution group was 20%. on the contrary, the survival rate of the virus -HBc-N149-VP4-N20 protein serum group was 90%.. The result showed that the immune serum of the recombinant HbcAg granule, HBc-N149-VP4-N20 protein, could be a new milk rat against EV71 virus. Provide protection effectiveness.
In the further study, the peptide epitope of the neutralizing antibody produced by VP4-N20 protein was determined by the peptide library screening test. The design principle of the peptide library in the experiment was to reduce two amino acids each time from the VP4-N20 protein N end and the C terminal respectively. The experimental results showed that when the N end of the VP4-N20 protein was reduced to sixth amino acids, or the C end was reduced to the first. At 10 amino acids, the immune sera against VP4-N20 protein will no longer be combined with VP4-N20 protein. This experimental result shows that the epitope of the antigenic determinant of the neutralizing antibody produced by VP4-N20 protein reduces 6 amino acids for its N terminal, and the C terminal reduces the sequence of polypeptide after 10 amino acids.
In this study, the 20 amino acids of the VP4 protein N terminal of the VP4 protein of the C4 subtype of the virus were fused into HbcAg and expressed in Escherichia coli. The results showed that the fusion target protein could be self assembled to form chimeric virus like particles after the expression, and could induce effective neutralizing antibodies against the virus. In the follow-up test, the peptide library was used. The test results showed that the identification of the neutralization epitopes of the EV71 virus VP4 protein provided new ideas and clues for the development of the broad-spectrum anti EV71 preventive vaccine.
【學位授予單位】：北京工業(yè)大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R512.5

【參考文獻】

相關(guān)期刊論文 前8條

1 陳偉;王明麗;;腸道病毒71型感染研究進展[J];中國熱帶醫(yī)學;2009年02期

2 吳靜;雷楗勇;張蓮芬;花慧;金堅;;改善稀有密碼子和氨基酸殘基限制提高重組人ADAM15去整合素結(jié)構(gòu)域蛋白表達水平[J];微生物學報;2008年08期

3 李振國;徐明波;牛罡;陳遙;姚文兵;;在大腸桿菌周質(zhì)表達重組蛋白的研究進展[J];藥物生物技術(shù);2011年01期

4 楊朝輝;尹少甫;張宗久;許文波;;柯薩奇病毒A組16型[J];中國疫苗和免疫;2009年01期

5 朵建英;王衛(wèi);馬春梅;郝翊;徐艷峰;許文波;董小平;王健偉;魏強;秦川;;EV71小鼠適應株制備及體內(nèi)外感染特點[J];中國實驗動物學報;2010年05期

6 黃貴彪,萬宗舉,李榮成;巴維信甲型肝炎滅活疫苗在兒童與成人中應用的安全性和免疫原性研究[J];中華流行病學雜志;2000年04期

7 陳曙霞,彭旭,劉晶星,丁智勇;苦參對感染柯薩奇B3病毒乳鼠搏動心肌細胞的保護作用[J];中華實驗和臨床病毒學雜志;2000年02期

8 梁爭論;;腸道病毒71型疫苗候選株和疫苗標準的研究[J];中國病毒病雜志;2011年01期

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