乙型脑炎病毒强弱毒毒力差异的分子机制研究

发布时间：2018-01-02 07:13

  本文关键词：乙型脑炎病毒强弱毒毒力差异的分子机制研究　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

  更多相关文章： 乙型脑炎病毒 感染性克隆 E蛋白 神经毒力

【摘要】：乙型脑炎病毒(Japanese encephalitis virus,JEV)是黄病毒科黄病毒属成员,通过侵害中枢神经系统引发人和猪、马、野生水禽等动物的病毒性脑炎,危害严重。因JEV具有神经毒力,对减毒活疫苗的安全性担忧使其仍不能被广泛接受。寻找JEV基因组中的毒力位点并探索其减毒机制是进一步研究JEV致病机理、研制安全有效的新型疫苗的关键。本研究以具有强神经毒力的JEV HEN0701株及其细胞传代致弱株HEN-10S3为研究对象,利用反向遗传操作技术,探索导致JEV毒力差异的分子基础及毒力增强或减弱的分子机制。JEV反向遗传操作系统的建立因其cDNA克隆在宿主菌中所具有的“遗传不稳定性”而受到阻碍。为了建立稳定的JEV全长感染性cDNA克隆,我们以HEN0701株基因组中极易发生突变的5’端序列区段(nt 1-2913)为研究对象,利用软件寻找影响病毒cDNA稳定性的相关序列,验证该序列对病毒cDNA克隆稳定性的影响,并对其进行定点突变和原核启动子活性的测定。结果发现,JEV基因组nt 54-120较高的原核启动子活性和JEV结构基因在宿主菌中的表达共同导致了JEV cDNA克隆在E.coli中难以稳定增殖。于基因组nt 1275与nt 1600之间截断E基因可以稳定cDNA克隆。于nt 90做A-C突变虽可降低原核启动子活性,但不能稳定克隆。室温(25°C)培养转化菌,nt 54-120的原核启动子活性有效降低,明显提高了2913-nt cDNA克隆在宿主菌中的遗传稳定性。因此,低温培养转化菌是提高黄病毒cDNA克隆在宿主菌中遗传稳定性的有效方法。以低温培养转化菌为基本前提,我们将HEN-10S3基因组全长分为相互重叠的四段,并在第一段5’端加上NotI酶切位点和T7启动子序列,在第四段3’末端加上HDV核酶序列和XhoI酶切位点。利用四条片段重叠序列以及pBR322M载体序列中合适的酶切位点,将四条片段于pBR322M中依次拼接成全长cDNA,构建HEN-10S3全长cDNA克隆pBAHC。经过体外转录并转染BHK-21细胞,获得拯救病毒vAHEN。经鉴定,拯救病毒与亲本毒在体外生长特性和对小鼠神经毒力两方面都具有相似特性。克隆pBAHC具有较好的稳定性且易于操作,连同本实验早期构建的HEN0701感染性克隆pJEHEN,为进一步深入研究JEV毒力差异提供了关键的技术平台。以HEN0701和HEN-10S3全长感染性cDNA克隆为基础,将两病毒5’-UTR和结构蛋白编码区序列(5’UTR-C-PrM-E)互换。分别构建了以HEN0701基因组为骨架嵌合克隆JE-H/5’-CPrME(S)和以HEN-10S3基因组为骨架的嵌合克隆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)。对3w小鼠的毒力测试结果显示,与亲本毒相比,嵌合病毒神经毒力发生显著变化。vJE-S/5’CPr ME(H)毒力明显上升,表现出强毒特性;而vJE-H/5’CPrME(S)毒力显著下降,表现出弱毒特性。说明所替换序列对病毒毒力存在重要影响。由于在该序列中仅存在E蛋白第138位氨基酸(E-138 Glu/Arg)一个位点的差异,说明E-138位点可能是影响病毒神经毒力的关键位点。利用PCR定点突变技术,将HEN0701 E-138 Glu(E)分别突变为酸性氨基酸Asp(D),碱性氨基酸Arg(R)和Lys(K)及中性氨基酸Phe(F)、Ala(A)和Gln(Q)。将HEN-10S3 E-138 R突变成E-138E。并拯救突变病毒vHE138D、vHE138R、vHE138K、vHE138F、vHE138A、vHE138Q和vSE138E。突变病毒对3w小鼠的毒力测试结果显示,vHE138D和vSE138E具有强神经毒力;vHE138R和vHE138K无神经毒力;vHE138F、vHE138A和vHE138Q具有一定的神经毒力,介于之间。说明E-138确实是影响JEV神经毒力的关键位点,且毒力的强弱与该位点的酸碱性质有关。利用生物信息学软件分析HEN0701 E蛋白结构,发现E蛋白E-138与E-47位氨基酸在空间结构上相邻,以氢键相连,但连接方式随毒力不同而不同。为了研究E-47对病毒毒力的影响及其与E-138之间的关系,将HEN0701 E-47位Asn(N)分别突变为酸性氨基酸Asp(D)、碱性氨基酸Lys(K)和中性氨基酸Ala(A),拯救出突变病毒vHE47D、vHE47K和vHE47A。其对小鼠的毒力测试结果显示,vHE47D具有强神经毒力;vHE47K无神经毒力;vHE47A神经毒力介于之间。说明JEV E蛋白第47位氨基酸是能够影响病毒神经毒力的潜在毒力位点,且病毒毒力与该位点氨基酸的酸碱性质有关。根据本研究中所构建的14种重组病毒毒力差异与两位突变位点酸碱性质之间的关系,发现E-47与E-138位氨基酸的酸碱性质共同决定了病毒的神经毒力,酸性越强则毒力越强,当两个位点同时为酸性氨基酸时,病毒毒力最强。综上所述,本研究通过一系列试验发现在低温培养转化菌可以有效提高黄病毒cDNA克隆在宿主菌中的遗传稳定性,并在此基础上,成功构建了JEV弱毒株HEN-10S3全长cDNA感染性克隆,并拯救出与亲本毒特性相似的重组病毒。以此感染性克隆及强毒株HEN0701感染性克隆为工具,通过强、弱毒株间的嵌合和定点突变,首次发现影响JEV毒力的关键位点除E-138外,还有E-47,且两位点氨基酸的酸碱性质共同决定了JEV神经毒力。由于E-138与E-47空间位置相邻,推测其所在结构可能是影响病毒毒力的关键结构。
[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.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65
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本文编号：1368195