發(fā)布時間：2018-06-18 05:33

  本文選題：日本腦炎病毒 + 復(fù)制子��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2009年碩士論文

【摘要】： 黃病毒屬包括70多種病毒,其中絕大多數(shù)是節(jié)肢動物傳播的人類病原。日本腦炎(JE)是一種蚊蟲傳播的病毒疾病,年發(fā)病率約為十萬分之一,病死率約為5-40%,另有20-40%會引起嚴重的神經(jīng)性疾病和后遺癥。我國除新疆,西藏,青海外均有發(fā)病,現(xiàn)在每年仍有幾千例病人,對人民的健康造成極大的危害,被衛(wèi)生部列為乙類傳染病。 黃病毒基因組有一個顯著特性,其RNA具有自主復(fù)制能力、同時具有感染性,裸RNA進入敏感細胞后,能夠大量自主復(fù)制,并且進一步翻譯成相應(yīng)的病毒蛋白,在胞內(nèi)包裝成為完整的病毒顆粒。黃病毒復(fù)制子通常指將基因組上的結(jié)構(gòu)基因部分切割下來,而保留完整的非結(jié)構(gòu)蛋白基因,這樣亞基因組RNA保留了自主復(fù)制能力,其不但能夠表達非結(jié)構(gòu)蛋白,而且能夠表達保留的結(jié)構(gòu)蛋白和/或添加的外源基因。理論上講,這種亞基因組的復(fù)制能力等同于完整的基因組,正鏈RNA呈指數(shù)級增加。如果在這種復(fù)制子中插入外源基因,由于復(fù)制子RNA在細胞胞漿內(nèi)大量擴增,就使得外源基因得到有效表達,因此,復(fù)制子RNA可作為表達外源基因的良好載體。目前,黃病毒中的KUN、DEN和YFV復(fù)制子已經(jīng)被報道可以作為發(fā)展新型疫苗的潛在的有用的工具。而在本研究中,我們希望以JEV SA14-14-2株為基礎(chǔ),構(gòu)建JEV亞基因組復(fù)制子,并進一步建立JEV復(fù)制子載體系統(tǒng),用于更多的疫苗研究。 我們先后構(gòu)建了pMW-G2R~pMW-G4R系列JEV復(fù)制子。這些復(fù)制子的主要區(qū)別在于結(jié)構(gòu)蛋白編碼序列刪除的方式不同,MCS,FMDV-2A等序列的添加。復(fù)制子的穩(wěn)定性是構(gòu)建復(fù)制子的一個主要問題,我們先后選用了不同的質(zhì)粒載體用于作為復(fù)制子的骨架載體,為了促進復(fù)制子的穩(wěn)定性,最終我們使用了一個極低拷貝但穩(wěn)定性很好的細菌質(zhì)粒載體pMW-118。另外,我們?yōu)榱撕罄m(xù)實驗操作的方便,以及提高復(fù)制子載體的表達效力,在復(fù)制子載體上添加了CMV啟動子序列,構(gòu)建了pCMW-2M復(fù)制子載體,多次測序證明JEV復(fù)制子pCMW-2M于30℃在大腸桿菌Top10繁殖過程中十分穩(wěn)定,沒有突變、插入或缺失。 我們通過間接免疫熒光實驗(IFA)分析了JEV復(fù)制子轉(zhuǎn)染BHK-21細胞病毒蛋白的表達�？乖饕ㄎ辉诎麧{。這些抗原陽性的細胞同對照細胞形狀、大小一致。在JEV抗原陽性的細胞沒有觀察到任何明顯的細胞病變效應(yīng)(CPE)。pMW-G2R~pMW-G4R和pCMW-2M復(fù)制子轉(zhuǎn)染細胞IFA的陽性率差別很大,從1%~30%不等,表明C蛋白的完整性、CMV啟動子的存在及非結(jié)構(gòu)蛋白基因的正確表達對復(fù)制子載體表達病毒蛋白的能力影響至關(guān)重要。 我們分別將綠色熒光蛋白(EGFP)和熒光素酶(Luc)報告基因插入復(fù)制子載體pCMW-2M。p2MEGFP轉(zhuǎn)染BHK-21細胞24小時后開始觀察EGFP的表達。陽性細胞的形狀、大小同對照BHK-21細胞沒有差別,同時也沒有觀察到細胞病變效應(yīng)。EGFP陽性細胞在轉(zhuǎn)染后24小時很少,在轉(zhuǎn)染后72小時明顯。與觀察EGFP表達類似,熒光素酶基因的表達在轉(zhuǎn)染后7天仍然能夠檢測到,熒光素酶信號在轉(zhuǎn)染后24小時到96小時呈指數(shù)增長。這些結(jié)果表明了插入了外源基因的JEV復(fù)制子能夠有效地表達外源基因。 我們通過免疫小鼠來研究JEV復(fù)制子pCMW-2M和pCMW-G2R的免疫原性。按每只小鼠50μg質(zhì)粒pCMW-2M劑量肌肉注射6周齡的雌BALB/c小鼠,3次免疫后誘導(dǎo)的抗JEV的抗體滴度為1:1270、中和抗體滴度為1:3(250%噬斑減少實驗),略高于pCMW-G2R組。pCMW-2M組的T淋巴細胞刺激指數(shù)(SI)為2.8,略高于pCMW-G2R組。這些結(jié)果證明了構(gòu)建的JEV復(fù)制子載體pCMW-2M及pCMW-G2R在小鼠體內(nèi)可以誘導(dǎo)針對JEV的體液免疫和細胞免疫。另外,pCMW-2M及pCMW-G2R免疫小鼠血清對JEV病毒攻擊具有較好的保護作用,可達到約70%存活。炭疽桿菌(Bacillus anthracis)是一種能形成芽胞的革蘭氏陽性菌,由它感染引起的炭疽病是嚴重危害人類的一種致死性的重大傳染病,同時它又是一種潛在的生物戰(zhàn)劑和生物恐怖試劑。PA是當(dāng)前應(yīng)用的疫苗主要成分,是引起機體免疫應(yīng)答的最有效的免疫原。為了發(fā)展安全、有效的炭疽疫苗,DNA疫苗是一個重要方向。我們將PA抗原的第四結(jié)構(gòu)域基因插入到JEV復(fù)制子pCMW-2M,這個插入了PA4基因的JEV復(fù)制子以DNA形式免疫小鼠后誘導(dǎo)了良好的體液免疫和細胞免疫反應(yīng),免疫3次后抗體滴度達到1:36200,T淋巴細胞刺激指數(shù)為2.6,這是國內(nèi)外首次對構(gòu)建表達炭疽PA的JEV復(fù)制型DNA載體進行的初步探索。 另外,我們還表達并純化了黃熱病毒YFV囊膜蛋白(E蛋白)結(jié)構(gòu)域Ⅲ,研究其作為亞單位疫苗預(yù)防YFV、JEV感染的可能性。YFDⅢ蛋白在大腸桿菌高效可溶性表達,表達量約占菌體蛋白的50%。純化的YFDⅢ蛋白免疫新西蘭兔和BALB/C鼠。Western Blot分析及ELISA表明純化后的表達產(chǎn)物具有良好的抗原性和免疫原性。利用純化的YFDⅢ蛋白免疫新西蘭兔,獲得了高達1:4×105滴度的抗YFV抗體和1:2×104滴度的抗JEV抗體;利用純化的YFDⅢ蛋白免疫BALB/C鼠,獲得了1:7×104滴度的抗YFV抗體和1:2×103滴度的抗JEV抗體。表明能產(chǎn)生對抗YFV和JEV的抗體,抗原性及免疫原性較好。同時抗YFV免疫血清對乳鼠YFV攻毒的保護作用實驗結(jié)果表明YFDⅢ蛋白免疫小鼠血清對YFV病毒攻擊有較好的保護作用,這些結(jié)果提示YFDⅢ蛋白有可能具有開發(fā)研制成亞單位疫苗的潛能,這是國內(nèi)外首次利用大腸桿菌高效可溶性表達了YFDⅢ蛋白并證明其具有較好的免疫原性和保護效力。 綜上所述,我們成功構(gòu)建了新型JEV復(fù)制子載體,進行了國內(nèi)外首次基于JEV減毒活疫苗株SA14-14-2的復(fù)制子載體的構(gòu)建、表達自身和外源蛋白能力、免疫原性等方面的系統(tǒng)研究,證明JEV復(fù)制子具有作為遞送外源基因的良好載體的潛力,為研究JEV的結(jié)構(gòu)蛋白與非結(jié)構(gòu)蛋白在病毒復(fù)制、包裝中的作用積累了重要資料,為進一步研究JEV非結(jié)構(gòu)蛋白(主要是NS1蛋白)的免疫原性及保護效力提供了幫助,為研制新型表達炭疽PA的JEV復(fù)制型DNA載體甚至雙價或多價復(fù)制型DNA載體奠定了基礎(chǔ),同時也為利用JEV復(fù)制子載體作為疫苗載體進一步研制其它新型DNA疫苗建立了一個有價值的技術(shù)平臺。
[Abstract]:The genus yellows includes more than 70 viruses, most of which are the human pathogens that are transmitted by arthropods. Japanese encephalitis (JE) is a mosquito borne virus disease with an annual incidence of about 1/100000, the mortality rate is about 5-40%, and 20-40% can cause severe neurological diseases and sequelae in China, except in Xinjiang, Tibet, and Qinghai, Every year, thousands of patients are still suffering great harm to the health of the people. They are classified as class B infectious diseases by the Ministry of health.
The genome of the yellow virus has a significant characteristic. The RNA has the ability to replicate autonomously, and has the infection. After the naked RNA enters the sensitive cell, it can be replicated in a large amount, and further translated into the corresponding virus protein and packaged into a complete virus particle in the cell. The yellow disease virus replicon usually refers to the structural gene part on the genome. The subgenome RNA preserves the intact non structural protein gene so that the subgenomic DNA preserves the independent replication ability, which not only expresses the unstructured protein, but also expresses the retained structural protein and / or the added exogenous gene. In theory, the subgenome is reproduced with the complete genome, and the positive chain RNA is exponentially. If the extraneous gene is inserted in this replicon, the extraneous gene is effectively expressed because the replicon RNA is amplified in the cytoplasm. Therefore, the replicon RNA can be used as a good carrier for the expression of foreign genes. At present, the KUN, DEN and YFV replicas in the yellow virus have been reported to be used as a new type of vaccine. Potential useful tools. In this study, we hope to build the JEV subgenome replicon on the basis of the JEV SA14-14-2 strain and further establish a JEV replicator system for more vaccine research.
We have successively constructed the pMW-G2R~pMW-G4R series JEV replicon. The main difference between these replicas lies in the different ways of deletion of the structural protein coding sequence, the addition of MCS, FMDV-2A and other sequences. The stability of the replicon is a major problem for the construction of the replicon. We have selected the different plasmid vectors for the replicon bone. In order to promote the stability of the replicator, in order to promote the stability of the replicator, we finally used a very low copy but stable bacterial plasmid carrier pMW-118.. We added the CMV promoter sequence on the replicator for the convenience of the follow-up experiments and the enhancement of the expression effectiveness of the replicator carrier, and constructed the pCMW-2M replication subcarrier. Repeated sequencing showed that the JEV replicon pCMW-2M was stable at 30 C at the temperature of Escherichia coli Top10, without mutation, insertion or deletion.
We analyzed the expression of BHK-21 cell virus protein transfected by JEV replicator by indirect immunofluorescence assay (IFA). The antigen was mainly located in the cytoplasm. These antigen positive cells were in the same shape as the control cells. No obvious cytopathic effect (CPE).PMW-G2R~pMW-G4R and pCMW-2M were observed in the JEV antigen positive cells. The positive rate of the replicon transfected cell IFA is very different, which is different from 1%~30%, indicating the integrity of the C protein, the existence of the CMV promoter and the correct expression of the non structural protein gene, which are very important to the ability of the replicator to express the virus protein.
We observed the expression of EGFP by inserting the green fluorescent protein (EGFP) and luciferase (Luc) reporter gene into the replicator pCMW-2M.p2MEGFP and transfecting BHK-21 cells for 24 hours. The shape and size of the positive cells were not different from those of the control BHK-21 cells. At the same time, there was no observation that the cell lesion effect of.EGFP positive cells was 24 after transfection. There were few hours, 72 hours after transfection. Similar to the expression of EGFP, the expression of luciferase gene could still be detected at 7 days after transfection, and the luciferase signal increased exponentially at 24 hours to 96 hours after transfection. These results showed that the JEV replica inserted into the foreign gene could effectively express the foreign gene.
We studied the immunogenicity of JEV replicator pCMW-2M and pCMW-G2R by immunizing mice. By intramuscular injection of 6 weeks old female BALB/c mice at the dose of 50 mu g plasmid per mouse, the antibody titer of anti JEV induced by 3 immunity was 1:1270, and the neutralizing antibody titer was 1:3 (250% plaque reduction test), slightly higher than the T lymph nodes in the.PCMW-2M group of the pCMW-G2R group. The cell stimulation index (SI) was 2.8, slightly higher than that in the pCMW-G2R group. These results showed that the constructed JEV replicator, pCMW-2M and pCMW-G2R, could induce humoral and cellular immunity against JEV in mice. In addition, the serum of pCMW-2M and pCMW-G2R immune mice had a good protective effect on the JEV virus attack, and could reach about 70%. Bacillus anthracis is a gram positive bacterium that can form a bud. Anthracnose caused by it is a fatal major infectious disease that seriously endangers human beings. At the same time, it is a potential biological warfare agent and bioterrorism reagent.PA, the main component of the current vaccine, and the most important immune response to the body. Effective immunogen. In order to develop a safe and effective anthrax vaccine, DNA vaccine is an important direction. We insert the fourth domain gene of PA antigen into the JEV replicon pCMW-2M. This JEV replicator, inserted in the PA4 gene, immunized mice in the form of DNA and induced a good body liquid immunity and cellular immune response, and the antibody was immunized for 3 times. Titer reached 1:36200 and T lymphocyte stimulation index was 2.6. This is the first exploration of constructing JEV replication DNA vector expressing anthrax PA for the first time at home and abroad.
In addition, we also expressed and purified the YFV capsule protein (E protein) domain of the yellow fever virus (HDV) III, and studied the possibility of its subunit vaccine as a vaccine to prevent YFV, the possibility of JEV infection, the expression of.YFD III protein in Escherichia coli, and the expression of the YFD III protein of the 50%. purification of the bacterial protein, the.Western Blot analysis of New Zealand rabbits and BALB/C mice. And ELISA showed that the purified expression product had good antigenicity and immunogenicity. The purified YFD III protein was used to immunize New Zealand rabbits to obtain anti YFV antibody and 1:2 x 104 titer with a 1:4 x 105 titer, and the purified YFD III protein was used to immunize BALB/C mice, and the anti YFV antibody and 1:2 x 103 titer of 1:7 x 104 titre were obtained. Anti JEV antibody. It showed that antibodies against YFV and JEV were produced, antigenicity and immunogenicity were better. Meanwhile, the protective effect of anti YFV immunized serum against YFV in milk mice showed that YFD III protein immunized with mice serum had a good protective effect on the attack of YFV virus. These results suggest that YFD III protein may have development and development. The potential of the unit vaccine is the first use of Escherichia coli to efficiently express YFD III protein and prove that it has good immunogenicity and protective effect.
To sum up, we successfully constructed a new JEV replicator, and carried out the construction of the first replicating carrier based on the JEV live attenuated vaccine strain SA14-14-2, expressing the ability of self and exogenous protein, immunogenicity and so on. It proved that the JEV replicator has the potential as a good carrier for delivery of foreign genes. The role of JEV's structural proteins and unstructured proteins in the replication and packaging of the virus has accumulated important information, which helps to further study the immunogenicity and protective effect of the JEV non structural protein (mainly NS1 protein), and provides a new JEV replicative DNA carrier for the expression of anthrax PA and even the bivalent or multivalent replicative DNA carrier. It also provides a valuable technology platform for further development of other DNA vaccines by using JEV replicon vectors as vaccine vectors.
【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)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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