發(fā)布時間：2018-06-22 19:44

  本文選題：漢坦病毒 + 核酸疫苗��； 參考：《中國醫(yī)科大學》2004年博士論文

【摘要】： 目的 腎綜合征出血熱(HFRS)是由布尼亞病毒科漢坦病毒屬病毒引起的一類全球性自然疫源性疾病，我國是受HFRS病毒危害最嚴重的國家。由于HFRS發(fā)病機制復雜，臨床表現(xiàn)多樣，病情危重易變，并發(fā)癥多，目前尚無有效的治療方法，本病的免疫預防越來越引起人們的重視�；蛞呙绮粌H能克服目前HFRS病毒滅活疫苗存在的制備方法復雜，免疫力不持久，不易大量生產(chǎn)和保存，有一定的副作用等缺欠，而且能同時激發(fā)機體的體液免疫和細胞免疫，可誘導宿主產(chǎn)生抗不同型HFRS病毒感染的保護性免疫，因此探索基因免疫預防腎綜合征出血熱具有現(xiàn)實意義。鑒于目前基因疫苗普遍存在的一個缺點就是其免疫原性較弱，當前大量的研究工作集中于試圖將適當?shù)姆肿幼魟?細胞因子，共刺激分子等)與抗原基因共同接種動物，以便增加動物對抗原的免疫反應。本課題擬構建漢坦病毒核蛋白(NP)S基因疫苗，然后從細胞因子IL-12，CpG motif及共刺激分子B7-1三個方面來探討漢坦病毒核蛋白基因疫苗及其分子佐劑的免疫調(diào)節(jié)作用，為進一步優(yōu)化漢坦病毒基因疫苗奠定基礎。 方法 常規(guī)PCR法擴增S基因，構建重組質粒pTARGET-hanS，經(jīng)酶切及測序鑒定，再用電穿孔法將重組質粒轉染Vero-E6細胞進行體外瞬間表達。用EcoR Ⅰ／Sal Ⅰ酶切已成功構建并可在體外表達的pTARGET-hanS，將S基因片段定向克隆入EcoR Ⅰ／Xho Ⅰ酶切后的pcDNA3.1+，構建重組質粒pcDNA3.1+S。同時將該S基因片段定向插入用同樣酶酶切的pCA14，構建pCA14-S，酶切鑒定后，用Bgl Ⅱ切下含啟動子的S基因片段，再將該片段插入BglⅡ酶切后的pcDNA3.1+B7，構建雙啟動子共表達載體 砰DNA3 .1+S+B7。peDNA3 .1+S(155)的構建及鑒定與peDNA3 .1+S的 構建及鑒定大致相同，所不同的是通過設計PCR引物將CpG motif引人載 體中。按分子克隆手冊所述方法大量提取所需質粒，用分光光度計測定 A260/A280比值以確定核酸樣品的純度，A260確定樣品的濃度，用滅菌的 PBS調(diào)整濃度至1 .om擴耐，作為DNA免疫的注射樣品，進行動物接種。6 ，SW齡的BABC/C小鼠，隨機分成6組，分別接種PcDNA3 .1+(對照)， pcDNA3·1+S，peDNA3.l+S(155)，peDNA3.1+S+peIL一12(體外混合)， peDNA3.1+S+peDNA3.l+B7(體外混合)和peDNA3.l+S+B7。每隔2 周加強免疫1次，共免疫3次。在每次接種前3天都用布比卡因預處理，以 提高肌細胞對外源DNA的攝取。在免疫后的sd、10d、17d、35d和42d收集 免疫鼠的血清和脾細胞，分別用EUSA法檢測血清特異性抗體的變化，MTr 法檢測T細胞增殖反應，以及用EllSAkit檢測免疫鼠脾細胞上清液中細胞 因子IL一4和IFN一，的動態(tài)變化，從而了解免疫鼠的體液免疫反應和細胞 免疫反應。 結果 1.PCR擴增的1.3kb的S基因片段核昔酸序列與genbank中的漢坦病 毒76一118株的S基因核昔酸序列符合率為97%一98%，而且前670bp中 無一個基因點突變。分別在轉染重組質粒pTARGET一hans和pTARGET- hans(155)的細胞中觀察到特異性熒光顆粒，即重組質粒pTARGET一hans 和pTARGET一hans(155)在體外均進行了瞬間表達。為了動物接種，又成 功地構建了peDNA3 .1+S、peDNA3 .1+S(155)和雙啟動子共表達載體 peDNA3 .1+S+B7。2.在免疫效果檢測中，初次免疫后17d，peDNA3.1+S 接種組的小鼠血清抗體水平明顯增加，在初次免疫后35d，即末次免疫后 7d，血清抗體達到較高水平，隨后進人平臺期并維持一定時間。而空載體 peDNA3.1+接種組的小鼠血清抗體始終在較低水平波動。細胞因子IL一4 的產(chǎn)生與抗體的動態(tài)變化基本相對應，即在加強免疫后可持續(xù)維持在較高 水平;IFN一，的產(chǎn)生盡管在初次免疫后lod下降到較低水平，但經(jīng)加強免 疫后IFN一，的產(chǎn)生又回升到了較高水平。3.不同的佐劑對免疫效果的影 響不同:接種peDNA3 .1+S(155)質粒的免疫鼠，，其血清抗體水平較接種 peDNA3.1+S免疫鼠的血清抗體水平明顯升高;在整個過程中，細胞因子 IFN一，的產(chǎn)生水平普遍比peDNA3 .1+S接種組的要高，IL一4的產(chǎn)生除 了在初次免疫后sd獲得了較高水平外，其他時間與peDNA3 .1+S單獨接 種組的相差不大;而IL一12基因佐劑的接種明顯地抑制了血清抗體的產(chǎn)生 水平，其抗體水平在整個過程中均較peDNA3 .1+S單獨接種組的要低，幾 一12基因佐劑與核蛋白基因疫苗的共注射，可明顯地促進IFN一，的產(chǎn)生， 抑制IL一4的產(chǎn)生;雙啟動子共表達載體peDNA3 .1+S+B7接種組的小鼠 抗體產(chǎn)生水平較PcDNA3 .1+S與peDNA3 .1+B7在體外混合后接種組的 明顯增高。我們通過在體外用NP重組抗原再次刺激免疫鼠的脾細胞來測 定T細胞的增殖反應，從而評價其細胞免疫水平。對照組peDNA3 .1+接 種組鼠的脾細胞的增殖指數(shù)為較低的0.797，而實驗組PcDNA3.1+S、PcD- NA3 .1+S(155)、peIL一12協(xié)同免疫組、peDNA3.l+B7協(xié)同免疫組以及 peDNA3 .1+S+B7接種組的脾細胞的增殖指數(shù)分別為較高的1 .43、1 .67、 1.86、1.盯和一55，尤其是peDNA3.l+S(155)和PcIL一12協(xié)同免疫組的 脾細胞的增殖指數(shù)較peDNA3 .1+S單獨接種組的明顯升高。 結論 NP的基因免疫可同時啟動機體的Thl和ThZ依賴性免疫
[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.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2004
【分類號】：R392

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