本文關(guān)鍵詞：乙型腦炎病毒強(qiáng)弱毒毒力差異的分子機(jī)制研究　出處：《中國農(nóng)業(yè)科學(xué)院》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 乙型腦炎病毒 感染性克隆 E蛋白 神經(jīng)毒力

【摘要】：乙型腦炎病毒(Japanese encephalitis virus,JEV)是黃病毒科黃病毒屬成員,通過侵害中樞神經(jīng)系統(tǒng)引發(fā)人和豬、馬、野生水禽等動物的病毒性腦炎,危害嚴(yán)重。因JEV具有神經(jīng)毒力,對減毒活疫苗的安全性擔(dān)憂使其仍不能被廣泛接受。尋找JEV基因組中的毒力位點并探索其減毒機(jī)制是進(jìn)一步研究JEV致病機(jī)理、研制安全有效的新型疫苗的關(guān)鍵。本研究以具有強(qiáng)神經(jīng)毒力的JEV HEN0701株及其細(xì)胞傳代致弱株HEN-10S3為研究對象,利用反向遺傳操作技術(shù),探索導(dǎo)致JEV毒力差異的分子基礎(chǔ)及毒力增強(qiáng)或減弱的分子機(jī)制。JEV反向遺傳操作系統(tǒng)的建立因其cDNA克隆在宿主菌中所具有的“遺傳不穩(wěn)定性”而受到阻礙。為了建立穩(wěn)定的JEV全長感染性cDNA克隆,我們以HEN0701株基因組中極易發(fā)生突變的5’端序列區(qū)段(nt 1-2913)為研究對象,利用軟件尋找影響病毒cDNA穩(wěn)定性的相關(guān)序列,驗證該序列對病毒cDNA克隆穩(wěn)定性的影響,并對其進(jìn)行定點突變和原核啟動子活性的測定。結(jié)果發(fā)現(xiàn),JEV基因組nt 54-120較高的原核啟動子活性和JEV結(jié)構(gòu)基因在宿主菌中的表達(dá)共同導(dǎo)致了JEV cDNA克隆在E.coli中難以穩(wěn)定增殖。于基因組nt 1275與nt 1600之間截斷E基因可以穩(wěn)定cDNA克隆。于nt 90做A-C突變雖可降低原核啟動子活性,但不能穩(wěn)定克隆。室溫(25°C)培養(yǎng)轉(zhuǎn)化菌,nt 54-120的原核啟動子活性有效降低,明顯提高了2913-nt cDNA克隆在宿主菌中的遺傳穩(wěn)定性。因此,低溫培養(yǎng)轉(zhuǎn)化菌是提高黃病毒cDNA克隆在宿主菌中遺傳穩(wěn)定性的有效方法。以低溫培養(yǎng)轉(zhuǎn)化菌為基本前提,我們將HEN-10S3基因組全長分為相互重疊的四段,并在第一段5’端加上NotI酶切位點和T7啟動子序列,在第四段3’末端加上HDV核酶序列和XhoI酶切位點。利用四條片段重疊序列以及pBR322M載體序列中合適的酶切位點,將四條片段于pBR322M中依次拼接成全長cDNA,構(gòu)建HEN-10S3全長cDNA克隆pBAHC。經(jīng)過體外轉(zhuǎn)錄并轉(zhuǎn)染BHK-21細(xì)胞,獲得拯救病毒vAHEN。經(jīng)鑒定,拯救病毒與親本毒在體外生長特性和對小鼠神經(jīng)毒力兩方面都具有相似特性�？寺BAHC具有較好的穩(wěn)定性且易于操作,連同本實驗早期構(gòu)建的HEN0701感染性克隆pJEHEN,為進(jìn)一步深入研究JEV毒力差異提供了關(guān)鍵的技術(shù)平臺。以HEN0701和HEN-10S3全長感染性cDNA克隆為基礎(chǔ),將兩病毒5’-UTR和結(jié)構(gòu)蛋白編碼區(qū)序列(5’UTR-C-PrM-E)互換。分別構(gòu)建了以HEN0701基因組為骨架嵌合克隆JE-H/5’-CPrME(S)和以HEN-10S3基因組為骨架的嵌合克隆JE-S/5’-CPrME(H),并拯救出嵌合病毒vJE-H/5’-CPr ME(S)和vJE-S/5’-CPrME(H)。對3w小鼠的毒力測試結(jié)果顯示,與親本毒相比,嵌合病毒神經(jīng)毒力發(fā)生顯著變化。vJE-S/5’CPr ME(H)毒力明顯上升,表現(xiàn)出強(qiáng)毒特性;而vJE-H/5’CPrME(S)毒力顯著下降,表現(xiàn)出弱毒特性。說明所替換序列對病毒毒力存在重要影響。由于在該序列中僅存在E蛋白第138位氨基酸(E-138 Glu/Arg)一個位點的差異,說明E-138位點可能是影響病毒神經(jīng)毒力的關(guān)鍵位點。利用PCR定點突變技術(shù),將HEN0701 E-138 Glu(E)分別突變?yōu)樗嵝园被酇sp(D),堿性氨基酸Arg(R)和Lys(K)及中性氨基酸Phe(F)、Ala(A)和Gln(Q)。將HEN-10S3 E-138 R突變成E-138E。并拯救突變病毒vHE138D、vHE138R、vHE138K、vHE138F、vHE138A、vHE138Q和vSE138E。突變病毒對3w小鼠的毒力測試結(jié)果顯示,vHE138D和vSE138E具有強(qiáng)神經(jīng)毒力;vHE138R和vHE138K無神經(jīng)毒力;vHE138F、vHE138A和vHE138Q具有一定的神經(jīng)毒力,介于之間。說明E-138確實是影響JEV神經(jīng)毒力的關(guān)鍵位點,且毒力的強(qiáng)弱與該位點的酸堿性質(zhì)有關(guān)。利用生物信息學(xué)軟件分析HEN0701 E蛋白結(jié)構(gòu),發(fā)現(xiàn)E蛋白E-138與E-47位氨基酸在空間結(jié)構(gòu)上相鄰,以氫鍵相連,但連接方式隨毒力不同而不同。為了研究E-47對病毒毒力的影響及其與E-138之間的關(guān)系,將HEN0701 E-47位Asn(N)分別突變?yōu)樗嵝园被酇sp(D)、堿性氨基酸Lys(K)和中性氨基酸Ala(A),拯救出突變病毒vHE47D、vHE47K和vHE47A。其對小鼠的毒力測試結(jié)果顯示,vHE47D具有強(qiáng)神經(jīng)毒力;vHE47K無神經(jīng)毒力;vHE47A神經(jīng)毒力介于之間。說明JEV E蛋白第47位氨基酸是能夠影響病毒神經(jīng)毒力的潛在毒力位點,且病毒毒力與該位點氨基酸的酸堿性質(zhì)有關(guān)。根據(jù)本研究中所構(gòu)建的14種重組病毒毒力差異與兩位突變位點酸堿性質(zhì)之間的關(guān)系,發(fā)現(xiàn)E-47與E-138位氨基酸的酸堿性質(zhì)共同決定了病毒的神經(jīng)毒力,酸性越強(qiáng)則毒力越強(qiáng),當(dāng)兩個位點同時為酸性氨基酸時,病毒毒力最強(qiáng)。綜上所述,本研究通過一系列試驗發(fā)現(xiàn)在低溫培養(yǎng)轉(zhuǎn)化菌可以有效提高黃病毒cDNA克隆在宿主菌中的遺傳穩(wěn)定性,并在此基礎(chǔ)上,成功構(gòu)建了JEV弱毒株HEN-10S3全長cDNA感染性克隆,并拯救出與親本毒特性相似的重組病毒。以此感染性克隆及強(qiáng)毒株HEN0701感染性克隆為工具,通過強(qiáng)、弱毒株間的嵌合和定點突變,首次發(fā)現(xiàn)影響JEV毒力的關(guān)鍵位點除E-138外,還有E-47,且兩位點氨基酸的酸堿性質(zhì)共同決定了JEV神經(jīng)毒力。由于E-138與E-47空間位置相鄰,推測其所在結(jié)構(gòu)可能是影響病毒毒力的關(guān)鍵結(jié)構(gòu)。
[Abstract]:Japanese encephalitis virus (Japanese encephalitis, virus, JEV) is a member of flavivirus, causing human and pig, through damaging the central nervous system, viral encephalitis, wild waterfowl and other animal serious harm. Because JEV has nerve toxicity, the live attenuated vaccine safety concerns that they are still not widely accepted find virulence sites in the genome of JEV and explore the mechanism of attenuation is to further study the pathogenic mechanism of JEV, the key to develop a new safe and effective vaccines. In this study, with strong virulence strain HEN0701 and JEV neural cells attenuated strain HEN-10S3 as the research object, using reverse genetics technology, to explore the molecular mechanism of.JEV lead to the establishment of reverse genetic operating system and molecular basis of virulence of JEV virulence differences enhanced or reduced due to its cDNA cloning is in host the "genetic instability" hampered In order to establish a stable JEV full-length infectious cDNA clone, we using the genome of HEN0701 mutation occurred easily in the 5 'end sequence segment (NT 1-2913) as the research object, using the software for the related sequence of the virus cDNA stability, influence the stability of the test sequence of cDNA virus and its clone, and prokaryotic promoter activity by site directed mutagenesis. The results showed that the prokaryotic expression of JEV gene promoter activity and structure of JEV genomic nt of 54-120 was higher in the host bacteria caused the JEV cDNA clone to stable proliferation in E.coli. On the base of NT 1275 and NT 1600 for the group between the truncated E gene can be stable cDNA clones in NT 90. A-C mutation can reduce the prokaryotic promoter activity, but not stable clone. At room temperature (25 degrees C) culture transformation bacteria, 54-120 NT prokaryotic promoter activity is reduced effectively, improves the 2913-nt cDNA clone in the night The genetic stability of the main bacteria. Therefore, low temperature culture transformed bacteria is an effective method to improve the flavivirus cDNA clone in host genetic stability. In low temperature culture transformed bacteria as the basic premise, we will HEN-10S3 genome is divided into four segments overlapping, and cutting sites and T7 promoter sequence in the first section of the 5 'end with NotI enzyme, in the fourth paragraph of the 3' end with HDV ribozyme sequence and XhoI restriction sites. The appropriate enzyme using four fragments overlapping sequences and pBR322M vector sequence in the cleavage site, four fragments in pBR322M were spliced into a full-length cDNA, constructed the HEN-10S3 full-length cDNA clone pBAHC. by in vitro transcription and transfected into BHK-21 cell rescue vAHEN. virus was identified, and the rescued virus parent virus growth characteristics in vitro and in two mice neurovirulence have similar characteristics. Cloning of pBAHC has good stability and easy operation For this experiment, together with the early construction of HEN0701 infectious clone pJEHEN, provides a key platform for further research on JEV. HEN0701 and HEN-10S3 in virulence of full-length infectious cDNA clone based, two -UTR and 5 'virus structural protein encoding region sequence (5 UTR-C-PrM-E) were constructed by HEN0701 exchange. The genome for skeleton chimeric clone JE-H/5' -CPrME (S) and the HEN-10S3 genome for the skeleton of the chimeric clone JE-S/5 '-CPrME (H), and rescued the chimeric virus vJE-H/5' -CPr ME vJE-S/5 '(S) and -CPrME (H). The virulence test results of 3W mice showed that compared with the parental virus, chimeric virus neurovirulence significantly changed.VJE-S/5 CPr ME (H) was significantly increased, showing strong toxic properties; and vJE-H/5 CPrME (S) was decreased significantly, showing attenuated characteristics. The replacement sequence has important influence on the virulence of the virus. Because the sequence is only E protein of 138th amino acid (E-138 Glu/Arg) between a site, suggesting that the E-138 site may affect the key sites of neurovirulence. By using the technology of PCR HEN0701 E-138 Glu point mutation (E) were mutated to acidic amino acid Asp (D), alkaline amino acid Arg (R) and Lys (K) and neutral amino acids (F), Phe Ala (A) and Gln (Q). The HEN-10S3 E-138 mutation from R to E-138E. and save the mutant virus vHE138D, vHE138R, vHE138K, vHE138F, vHE138A, vHE138Q and vSE138E. mutations in 3W mice toxicity test results showed that vHE138D and vSE138E have strong nerves vHE138R and vHE138K had no neurological toxicity; toxicity; vHE138F, vHE138A and vHE138Q have certain toxicity, nerve between. E-138 really is a key site of JEV nerve toxicity, and acid-base properties and virulence of the site. The use of biological information HEN0701 analysis of E protein structure software, found that the E protein E-138 and E-47 amino acids in the space structure of adjacent, linked by hydrogen bond, but the connection with the virulence of different. In order to study the effect of E-47 on the virulence of the virus and its relationship with E-138, HEN0701 E-47 Asn (N) were mutated to acidic amino acid Asp (D) Lys (K), basic amino acids and neutral amino acid Ala (A), to rescue the mutant virus vHE47D, vHE47K and vHE47A. in the mouse virulence test results show that vHE47D has strong nerve toxicity; vHE47K nerve toxicity; vHE47A neural toxicity. The results showed that JEV E protein is forty-seventh amino acids are able to influence the potential the virulence locus neurovirulence, the acid-base properties and virulence of the virus and the sites of amino acids. According to the 14 kinds of recombinant virus virulence differences constructed in this study and two mutation sites of acid-base properties off The Department found, E-47 and acid-base properties of E-138 amino acids determines the virulence of the virus of the nerve, the more acid is more virulent, when two loci and acidic amino acids, the strongest virulence of the virus. In summary, this study through a series of tests found that the genetic stability of bacteria cultivation can effectively improve the flavivirus cDNA clone in host bacteria at low temperature, and on this basis, the successful construction of JEV attenuated strain HEN-10S3 of infectious full-length cDNA clone, and rescue the recombinant virus similar to parent virus characteristics. By infection of HEN0701 clone and virulent infectious clone as a tool, through the strong, weak mutation strains of chimeric and point, found for the first time key virulence sites of JEV except E-138 and E-47, and two amino acid-base properties determined JEV neurovirulence. Because E-138 and adjacent spatial positions of E-47, speculated that the Structure may be the key structure that affects virulence of the virus.

【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】：2016
【分類號】：S852.65
，

本文編號：1368195