EIAV減毒疫苗誘導(dǎo)體液免疫反應(yīng)時相及膜抗原改造研究
發(fā)布時間：2018-10-23 18:04
【摘要】： 馬傳染性貧血病毒(EIAV)是與HIV同科同屬的馬屬動物慢病毒,其病毒形態(tài)、基因組結(jié)構(gòu),病毒編碼蛋白的結(jié)構(gòu)和功能及抗原特性等方面與HIV極為相似。中國EIAV減毒活疫苗是目前世界上唯一大規(guī)模應(yīng)用的慢病毒疫苗。因此深入探索該疫苗的免疫保護機制將為HIV疫苗的研制提供有益的借鑒。 本課題利用研究室前期工作中構(gòu)建的EIAV感染性分子克隆pLGFD3制備減毒疫苗pLGFD3-V,免疫動物后,定期采集血樣本,通過ELISA方法檢測各個時點血清中針對EIAV膜蛋白(Env)Gp90,和基質(zhì)蛋白p26結(jié)合抗體的滴度;同時,采用細胞蝕斑染色方法檢測中和抗體的滴度,分析EIAV減毒疫苗誘導(dǎo)的體液免疫反應(yīng)的成熟時相。結(jié)果顯示,EIAV減毒疫苗免疫馬后,針對Env Gp90和Gag p26蛋白均產(chǎn)生了特異性的體液免疫反應(yīng),Gp90結(jié)合抗體在2個半月左右達峰值(1:320,1:320),隨后盡管有輕微的波動,但一直維持在較高的水平直至免疫后5至6個月,隨后開始輕微回落,在免疫后32個月時仍能檢測到穩(wěn)定存在。而p26結(jié)合抗體則存續(xù)時間較短,在免疫后1-2個月達峰值(1:320,1:40),隨后呈明顯回落,在4個月時回落至檢測水平以下。EIAV減毒疫苗誘導(dǎo)的特異性中和抗體的水平同樣在2個月左右達峰值(約1:2800—1:3000),在整個觀測期內(nèi)持續(xù)穩(wěn)定存在。上述結(jié)果證實,EIAV減毒疫苗免疫后,能誘導(dǎo)產(chǎn)生特異性的Gp90,p26結(jié)合抗體以及中和抗體,而且GpgO特異性結(jié)合抗體和中和抗體可持續(xù)穩(wěn)定存在。 為了增強減毒疫苗誘導(dǎo)中和抗體的免疫原性,我們嘗試對env基因V3,V4區(qū)以及連接片層上糖基化位點進行了缺失型改造,探索上述糖基化位點的缺失對體液免疫反應(yīng)的影響。首先構(gòu)建了上述Env區(qū)糖基化位點缺失的的DNA疫苗(pDRVI—none)和痘苗載體疫苗(rTTV—none),以減毒疫苗膜蛋白的DNA疫苗(pDRVI—env)及痘苗載體活疫苗(rTTV—env),以及空白的DNA疫苗/痘苗病毒載體(pDRVI—sv1.0/rTTV—7521)為對照,采用DNA初免/痘苗加強的策略,分別免疫小鼠。分組如下(1)pDRVI—env/rTTV—env,(2)pDRVI—none/rTTV—none,(3)pDRVI—sv1.0/rTTV—7521和(4)生理鹽水。免疫2個月后,采用與上述相同的實驗方法,檢測各組基因工程疫苗誘導(dǎo)的針對gp90的結(jié)合抗體和中和抗體水平。結(jié)果顯示,和減毒疫苗膜蛋白未經(jīng)改造組相比,糖基化位點的缺失對膜蛋白誘導(dǎo)結(jié)合抗體的能力沒有形成顯著的差異,但降低了膜蛋白誘導(dǎo)中和抗體的頻率和中和滴度。說明減毒疫苗膜蛋白在V3,V4區(qū)所分別保留的199位,217位糖基化位點對于有效中和抗體的誘導(dǎo)是必須的。今后應(yīng)該探索新的策略,增強疫苗誘導(dǎo)體液免疫反應(yīng)的免疫原性。
[Abstract]:Equine infectious anemia virus (EIAV) is a kind of equine lentivirus belonging to the same family as HIV. Its morphology, genome structure, structure and function of virus coding protein and antigenic characteristics of the virus are very similar to HIV. Chinese EIAV attenuated live vaccine is the only large-scale application of lentivirus vaccine in the world. Therefore, further exploring the immune protection mechanism of the vaccine will provide useful reference for the development of HIV vaccine. In this study, the attenuated vaccine pLGFD3-V, immunized animals were prepared by cloning pLGFD3 from EIAV infectious molecules constructed in the previous work of the laboratory, and blood samples were collected regularly. The titer of EIAV membrane protein (Env) Gp90, and matrix protein p26 binding antibody was detected by ELISA method, and the neutralization antibody titer was detected by cell plaque staining. The mature phase of humoral immune response induced by EIAV attenuated vaccine was analyzed. The results showed that EIAV attenuated vaccine produced a specific humoral immune response against both Env Gp90 and Gag p26 protein, and the Gp90 binding antibody reached its peak in about two and a half months (1: 320min: 1: 320), although there was a slight fluctuation. But it remained at a high level until 5 to 6 months after immunization, followed by a slight drop, and a steady presence was detected 32 months after immunization. On the other hand, the survival time of p26 binding antibody was shorter, reaching its peak at 1-2 months after immunization (1: 320 or 1: 40), and then decreased significantly. The level of specific neutralizing antibody induced by EIAV attenuated vaccine also reached its peak at about 2 months (about 1: 2800-1: 3000) and remained stable throughout the observation period. The above results confirmed that EIAV attenuated vaccine could induce the production of specific Gp90,p26 binding antibody and neutralizing antibody, and the existence of GpgO specific binding antibody and neutralizing antibody could be sustained and stable. In order to enhance the immunogenicity of neutralizing antibody induced by attenuated vaccine, we attempted to modify the deletion type of the V3V4 region of env gene and the glycosylation site on the connecting lamellar, and to explore the effect of the above glycosylation sites on humoral immune response. First of all, DNA vaccine (pDRVI-none) and vaccinia vector vaccine (rTTV-none) with missing glycosylation sites in Env region were constructed for attenuated DNA vaccine (pDRVI-env) and vaccinia vector live vaccine (rTTV-env), as well as blank DNA vaccine / vaccinia vaccine. Seedling virus vector (pDRVI-sv1.0/rTTV-7521) was used as control. Mice were immunized with DNA / vaccinia. The groups were as follows: (1) pDRVI-env/rTTV-env, (2) pDRVI-none/rTTV-none, (3) pDRVI-sv1.0/rTTV-7521 and (4) normal saline. After 2 months of immunization, the levels of binding and neutralizing antibodies against gp90 induced by each group of genetically engineered vaccines were detected by the same experimental method as those mentioned above. The results showed that the absence of glycosylation sites had no significant difference in the ability of membrane protein to induce antibody, but decreased the frequency and titer of membrane protein induced neutralizing antibody. The results showed that the 199 and 217 glycosylation sites of attenuated vaccine membrane proteins in V3 / V4 region were necessary for the induction of effective neutralizing antibodies. New strategies should be explored to enhance the immunogenicity of humoral immune response induced by vaccine.
【學位授予單位】：中國疾病預(yù)防控制中心
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392
本文編號：2290055
[Abstract]:Equine infectious anemia virus (EIAV) is a kind of equine lentivirus belonging to the same family as HIV. Its morphology, genome structure, structure and function of virus coding protein and antigenic characteristics of the virus are very similar to HIV. Chinese EIAV attenuated live vaccine is the only large-scale application of lentivirus vaccine in the world. Therefore, further exploring the immune protection mechanism of the vaccine will provide useful reference for the development of HIV vaccine. In this study, the attenuated vaccine pLGFD3-V, immunized animals were prepared by cloning pLGFD3 from EIAV infectious molecules constructed in the previous work of the laboratory, and blood samples were collected regularly. The titer of EIAV membrane protein (Env) Gp90, and matrix protein p26 binding antibody was detected by ELISA method, and the neutralization antibody titer was detected by cell plaque staining. The mature phase of humoral immune response induced by EIAV attenuated vaccine was analyzed. The results showed that EIAV attenuated vaccine produced a specific humoral immune response against both Env Gp90 and Gag p26 protein, and the Gp90 binding antibody reached its peak in about two and a half months (1: 320min: 1: 320), although there was a slight fluctuation. But it remained at a high level until 5 to 6 months after immunization, followed by a slight drop, and a steady presence was detected 32 months after immunization. On the other hand, the survival time of p26 binding antibody was shorter, reaching its peak at 1-2 months after immunization (1: 320 or 1: 40), and then decreased significantly. The level of specific neutralizing antibody induced by EIAV attenuated vaccine also reached its peak at about 2 months (about 1: 2800-1: 3000) and remained stable throughout the observation period. The above results confirmed that EIAV attenuated vaccine could induce the production of specific Gp90,p26 binding antibody and neutralizing antibody, and the existence of GpgO specific binding antibody and neutralizing antibody could be sustained and stable. In order to enhance the immunogenicity of neutralizing antibody induced by attenuated vaccine, we attempted to modify the deletion type of the V3V4 region of env gene and the glycosylation site on the connecting lamellar, and to explore the effect of the above glycosylation sites on humoral immune response. First of all, DNA vaccine (pDRVI-none) and vaccinia vector vaccine (rTTV-none) with missing glycosylation sites in Env region were constructed for attenuated DNA vaccine (pDRVI-env) and vaccinia vector live vaccine (rTTV-env), as well as blank DNA vaccine / vaccinia vaccine. Seedling virus vector (pDRVI-sv1.0/rTTV-7521) was used as control. Mice were immunized with DNA / vaccinia. The groups were as follows: (1) pDRVI-env/rTTV-env, (2) pDRVI-none/rTTV-none, (3) pDRVI-sv1.0/rTTV-7521 and (4) normal saline. After 2 months of immunization, the levels of binding and neutralizing antibodies against gp90 induced by each group of genetically engineered vaccines were detected by the same experimental method as those mentioned above. The results showed that the absence of glycosylation sites had no significant difference in the ability of membrane protein to induce antibody, but decreased the frequency and titer of membrane protein induced neutralizing antibody. The results showed that the 199 and 217 glycosylation sites of attenuated vaccine membrane proteins in V3 / V4 region were necessary for the induction of effective neutralizing antibodies. New strategies should be explored to enhance the immunogenicity of humoral immune response induced by vaccine.
【學位授予單位】：中國疾病預(yù)防控制中心
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392
【參考文獻】
相關(guān)期刊論文 前1條
1 張曉燕;李紅梅;梁華;沈_";馬燕;相文華;沈榮顯;邵一鳴;;EIAV減毒疫苗誘導(dǎo)的特異性細胞免疫應(yīng)答[J];細胞與分子免疫學雜志;2006年01期
，本文編號：2290055
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