汉坦病毒核酸疫苗的研究

发布时间：2018-06-22 19:44

本文选题：汉坦病毒 + 核酸疫苗　；参考：《中国医科大学》2004年博士论文

【摘要】： 目的肾综合征出血热(HFRS)是由布尼亚病毒科汉坦病毒属病毒引起的一类全球性自然疫源性疾病，我国是受HFRS病毒危害最严重的国家。由于HFRS发病机制复杂，临床表现多样，病情危重易变，并发症多，目前尚无有效的治疗方法，本病的免疫预防越来越引起人们的重视。基因疫苗不仅能克服目前HFRS病毒灭活疫苗存在的制备方法复杂，免疫力不持久，不易大量生产和保存，有一定的副作用等缺欠，而且能同时激发机体的体液免疫和细胞免疫，可诱导宿主产生抗不同型HFRS病毒感染的保护性免疫，因此探索基因免疫预防肾综合征出血热具有现实意义。鉴于目前基因疫苗普遍存在的一个缺点就是其免疫原性较弱，当前大量的研究工作集中于试图将适当的分子佐剂(细胞因子，共刺激分子等)与抗原基因共同接种动物，以便增加动物对抗原的免疫反应。本课题拟构建汉坦病毒核蛋白(NP)S基因疫苗，然后从细胞因子IL-12，CpG motif及共刺激分子B7-1三个方面来探讨汉坦病毒核蛋白基因疫苗及其分子佐剂的免疫调节作用，为进一步优化汉坦病毒基因疫苗奠定基础。方法常规PCR法扩增S基因，构建重组质粒pTARGET-hanS，经酶切及测序鉴定，再用电穿孔法将重组质粒转染Vero-E6细胞进行体外瞬间表达。用EcoR Ⅰ／Sal Ⅰ酶切已成功构建并可在体外表达的pTARGET-hanS，将S基因片段定向克隆入EcoR Ⅰ／Xho Ⅰ酶切后的pcDNA3.1+，构建重组质粒pcDNA3.1+S。同时将该S基因片段定向插入用同样酶酶切的pCA14，构建pCA14-S，酶切鉴定后，用Bgl Ⅱ切下含启动子的S基因片段，再将该片段插入BglⅡ酶切后的pcDNA3.1+B7，构建双启动子共表达载体砰DNA3 .1+S+B7。peDNA3 .1+S(155)的构建及鉴定与peDNA3 .1+S的构建及鉴定大致相同，所不同的是通过设计PCR引物将CpG motif引人载体中。按分子克隆手册所述方法大量提取所需质粒，用分光光度计测定 A260/A280比值以确定核酸样品的纯度，A260确定样品的浓度，用灭菌的 PBS调整浓度至1 .om扩耐，作为DNA免疫的注射样品，进行动物接种。6 ，SW龄的BABC/C小鼠，随机分成6组，分别接种PcDNA3 .1+(对照)， pcDNA3·1+S，peDNA3.l+S(155)，peDNA3.1+S+peIL一12(体外混合)， peDNA3.1+S+peDNA3.l+B7(体外混合)和peDNA3.l+S+B7。每隔2 周加强免疫1次，共免疫3次。在每次接种前3天都用布比卡因预处理，以提高肌细胞对外源DNA的摄取。在免疫后的sd、10d、17d、35d和42d收集免疫鼠的血清和脾细胞，分别用EUSA法检测血清特异性抗体的变化，MTr 法检测T细胞增殖反应，以及用EllSAkit检测免疫鼠脾细胞上清液中细胞因子IL一4和IFN一，的动态变化，从而了解免疫鼠的体液免疫反应和细胞免疫反应。结果 1.PCR扩增的1.3kb的S基因片段核昔酸序列与genbank中的汉坦病毒76一118株的S基因核昔酸序列符合率为97%一98%，而且前670bp中无一个基因点突变。分别在转染重组质粒pTARGET一hans和pTARGET- hans(155)的细胞中观察到特异性荧光颗粒，即重组质粒pTARGET一hans 和pTARGET一hans(155)在体外均进行了瞬间表达。为了动物接种，又成功地构建了peDNA3 .1+S、peDNA3 .1+S(155)和双启动子共表达载体 peDNA3 .1+S+B7。2.在免疫效果检测中，初次免疫后17d，peDNA3.1+S 接种组的小鼠血清抗体水平明显增加，在初次免疫后35d，即末次免疫后 7d，血清抗体达到较高水平，随后进人平台期并维持一定时间。而空载体 peDNA3.1+接种组的小鼠血清抗体始终在较低水平波动。细胞因子IL一4 的产生与抗体的动态变化基本相对应，即在加强免疫后可持续维持在较高水平;IFN一，的产生尽管在初次免疫后lod下降到较低水平，但经加强免疫后IFN一，的产生又回升到了较高水平。3.不同的佐剂对免疫效果的影响不同:接种peDNA3 .1+S(155)质粒的免疫鼠，，其血清抗体水平较接种 peDNA3.1+S免疫鼠的血清抗体水平明显升高;在整个过程中，细胞因子 IFN一，的产生水平普遍比peDNA3 .1+S接种组的要高，IL一4的产生除了在初次免疫后sd获得了较高水平外，其他时间与peDNA3 .1+S单独接种组的相差不大;而IL一12基因佐剂的接种明显地抑制了血清抗体的产生水平，其抗体水平在整个过程中均较peDNA3 .1+S单独接种组的要低，几一12基因佐剂与核蛋白基因疫苗的共注射，可明显地促进IFN一，的产生，抑制IL一4的产生;双启动子共表达载体peDNA3 .1+S+B7接种组的小鼠抗体产生水平较PcDNA3 .1+S与peDNA3 .1+B7在体外混合后接种组的明显增高。我们通过在体外用NP重组抗原再次刺激免疫鼠的脾细胞来测定T细胞的增殖反应，从而评价其细胞免疫水平。对照组peDNA3 .1+接种组鼠的脾细胞的增殖指数为较低的0.797，而实验组PcDNA3.1+S、PcD- NA3 .1+S(155)、peIL一12协同免疫组、peDNA3.l+B7协同免疫组以及 peDNA3 .1+S+B7接种组的脾细胞的增殖指数分别为较高的1 .43、1 .67、 1.86、1.盯和一55，尤其是peDNA3.l+S(155)和PcIL一12协同免疫组的脾细胞的增殖指数较peDNA3 .1+S单独接种组的明显升高。结论 NP的基因免疫可同时启动机体的Thl和ThZ依赖性免疫
[Abstract]:objective
Hemorrhagic fever with renal syndrome (HFRS) is a kind of global natural epidemic disease caused by the virus of khira virus, which is caused by the virus of cohantavirus. Our country is the most seriously harming country of HFRS virus. Because of the complicated pathogenesis of HFRS, the clinical manifestations are diverse, the condition is critical and the complication is many. There is no effective treatment method at present, and the immune preconditioning of this disease is not yet available. More and more people pay attention to it. The genetic vaccine can not only overcome the complex preparation methods of the existing HFRS virus inactivated vaccine, but it is not easy to produce and preserve the immune system, it has some side effects, but also can stimulate the body's humoral immunity and cell immunity at the same time. It can induce the host to produce anti HFRS virus. Therefore, it is of practical significance to explore gene immunization for the prevention of hemorrhagic fever with renal syndrome. In view of the weakness of the immunogenicity of the current gene vaccine, a large number of research efforts are focused on the common inoculation of the appropriate molecular adjuvant (cytokines, CO stimulators, etc.) with the antigen genes. In order to increase the immune response of animals to antigen, we intend to construct a hantavirus nucleoprotein (NP) S gene vaccine, and then discuss the immunization of Hantaan virus nucleoprotein gene vaccine and its molecular adjuvant from the three aspects of cytokine IL-12, CpG motif and co stimulator B7-1, in order to further optimize the gene pestilence of hantavirus. The foundation of the seedling lay.
Method
The S gene was amplified by the conventional PCR method, and the recombinant plasmid pTARGET-hanS was constructed. The recombinant plasmid was transfected into Vero-E6 cells for instant expression in vitro by enzyme digestion and sequencing. The recombinant plasmid was successfully constructed and expressed in vitro by EcoR I / Sal I enzyme. The fragment of S gene fragment was cloned into EcoR I / Xho I enzyme. PcDNA3.1+, the recombinant plasmid pcDNA3.1+S. was constructed and the S gene fragment was inserted into the same enzyme cut pCA14 to construct pCA14-S. After the enzyme digestion, the S gene fragment containing the promoter was cut by Bgl II, and then the fragment was inserted into the pcDNA3.1+B7 after the Bgl II enzyme cut to construct a co expression vector of the double promoter.
Construction and identification of bang DNA3.1+S+B7.peDNA3.1+S (155) and peDNA3.1+S
The construction and identification are basically the same. The difference is that the CpG motif is introduced by designing PCR primers.
A large number of plasmid vectors were extracted according to the molecular cloning manual, and determined by spectrophotometer.
A260/A280 ratio to determine the purity of nucleic acid samples, A260 determines the concentration of the sample, and sterilize the sample.
PBS adjusted the concentration to 1.Om for tolerance, as an injection sample for DNA immunization, and.6 for animal inoculation.
SW age BABC/C mice were randomly divided into 6 groups, inoculated with PcDNA3.1+ (control) respectively.
PcDNA3. 1+S, peDNA3.l+S (155), peDNA3.1+S+peIL 12 (mixed in vitro).
PeDNA3.1+S+peDNA3.l+B7 (in vitro mixing) and peDNA3.l+S+B7. are 2
The mice were immunized 1 times, immunized 3 times, and pretreated with bupivacaine 3 days before each inoculation.
Increase the uptake of exogenous DNA by myocytes. Collection of SD, 10d, 17D, 35d and 42d after immunization
The serum and spleen cells of the immunized mice were detected by EUSA method, respectively. MTr
The proliferation of T cells was detected by the method, and the cells in the supernatant of spleen cells were detected by EllSAkit.
The dynamic changes of factor IL 4 and IFN 1 can help us to understand humoral immune response and cells in immune mice.
Immune response.
Result
1.PCR amplified 1.3kb gene fragment of S gene and Hantaan disease in GenBank
The sequence coincidence rate of S gene nucleoxy acid in 76, 118, and 97% strains was 97% 1 98%, and the former was in the middle.
There was no point mutation in transfected plasmid pTARGET 1 Hans and pTARGET-.
Specific fluorescent particles were observed in cells of Hans (155), that is, recombinant plasmid pTARGET Hans.
Both pTARGET and Hans (155) were expressed instantaneously in vitro.
The co expression vectors of peDNA3.1+S, peDNA3.1+S (155) and double promoters were constructed.
PeDNA3.1+S+B7.2. in immunization test, after initial immunization 17D, peDNA3.1+S
The serum antibody level of mice in inoculation group increased significantly, after 35d after initial immunization.
7d, the serum antibody reached a high level, and then entered the platform stage and maintained for a certain time.
The serum antibody level of peDNA3.1+ inoculation group was always at a low level. Cytokine IL 4
The production is basically corresponding to the dynamic change of antibody, that is to say, after immunization, it is maintained at a higher level.
The level of production of IFN 1, though decreased to a lower level after initial immunization, was enhanced by LOD.
The emergence of IFN 1 after infection increased to a higher level. The effect of.3.'s different adjuvants on immune effects.
The antibody level of vaccinated mice inoculated with peDNA3.1+S (155) plasmid was different from that of vaccinated mice.
The serum antibody level of peDNA3.1+S immunized mice increased significantly.
The production level of IFN 1 is generally higher than that of peDNA3.1+S inoculation group, and the production of IL 4 is higher than that of the inoculation group.
After the initial immunization, the SD level was higher than that of other peDNA3.1+S.
There was little difference between the groups, but the inoculation of IL 12 gene adjuvant significantly inhibited the production of serum antibodies.
The antibody level was lower in the whole process than in the peDNA3.1+S inoculation group.
Co injection of a 12 gene adjuvant and nucleoprotein gene vaccine can significantly promote the production of IFN 1.
Inhibition of IL 4 production; double promoter co expression vector peDNA3.1+S+B7 inoculation group mice
The level of antibody production was mixed with PcDNA3.1+S and peDNA3.1+B7 in vitro.
In vitro, we stimulated the spleen cells of immunized mice again by using NP recombinant antigen in vitro.
The proliferative response of T cells was determined to evaluate their cellular immunity. The control group received peDNA3.1+.
The proliferation index of spleen cells in the group of rats was 0.797 lower than that in the experimental group PcDNA3.1+S, PcD-
NA3.1+S (155), peIL 12 + immunization group, peDNA3.l+B7 co immunization group and
The proliferation index of spleen cells in peDNA3.1+S+B7 inoculation group was 1.43,1.67 higher.
1.86,1. stared at one, 55, especially peDNA3.l+S (155) and PcIL 12 plus immunization group.
The proliferation index of splenocytes was significantly higher than that of peDNA3.1+S alone inoculation group.
conclusion
NP gene immunization can simultaneously initiate Thl and ThZ dependent immunity in the body.
【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2004
【分类号】：R392

【参考文献】