【摘要】：鑒于當(dāng)前獲得性免疫缺陷綜合征（Acquired immunodeficiency syndrome, AIDS）的流行態(tài)勢(shì)和預(yù)防治療現(xiàn)狀，研發(fā)安全有效的艾滋病預(yù)防/治療性疫苗刻不容緩。近些年來(lái)，很多科學(xué)家利用不同的疫苗載體和形式、不同的人免疫缺陷病毒（Human immunodeficiency virus，HIV）免疫原、不同的接種或免疫途徑進(jìn)行了大量的有益的嘗試。本研究選擇DNA質(zhì)粒和痘苗病毒天壇株作為免疫原遞送系統(tǒng)，開(kāi)展艾滋病疫苗的免疫原性研究。 本研究在本研究室多年來(lái)篩選獲得的HIV復(fù)合多表位基因（MEGNp24）的基礎(chǔ)上，在其上游引入CpG基序和CTB基因作為佐劑，構(gòu)建了一種重組核酸疫苗pVL-CCMp24，將重組DNA質(zhì)粒轉(zhuǎn)染293T細(xì)胞，從RT-PCR和間接免疫熒光檢測(cè)結(jié)果可知，重組質(zhì)粒編碼的目的基因CCMp24在哺乳動(dòng)物細(xì)胞內(nèi)得到正確轉(zhuǎn)錄和表達(dá)。肌肉注射途徑免疫BALB/c小鼠，分析重組DNA疫苗的免疫原性，由對(duì)HIV特異性抗體、Th1型和Th2型細(xì)胞因子、T細(xì)胞亞型、淋巴細(xì)胞增殖等免疫指標(biāo)的檢測(cè)結(jié)果表明，重組DNA候選疫苗pVL-CCMp24可以誘導(dǎo)機(jī)體產(chǎn)生一定程度的細(xì)胞和體液免疫應(yīng)答。 基于多價(jià)疫苗的設(shè)計(jì)理念，本研究首先構(gòu)建了一種含三個(gè)表達(dá)盒的痘苗病毒穿梭載體pSTKE，含有三個(gè)獨(dú)立的外源基因表達(dá)盒，分別以VTT及其基因缺失突變株作為原始病毒，以增強(qiáng)型綠色熒光蛋白(EGFP)、紅色熒光蛋白（RFP）和藍(lán)色熒光蛋白（BFP）作為報(bào)告基因，分別對(duì)三個(gè)表達(dá)盒的功能進(jìn)行驗(yàn)證。通過(guò)噬斑篩選，獲得兩株重組痘苗病毒(rVTT-EGFP、rdVTT-EGFP)，利用PCR、實(shí)時(shí)熒光定量PCR、Western blot方法對(duì)重組病毒以及對(duì)外源基因的表達(dá)量和遺傳穩(wěn)定性進(jìn)行分析。結(jié)果表明，成功構(gòu)建了三基因表達(dá)盒痘苗病毒穿梭載體，成功篩選出兩株重組痘苗病毒，EGFP基因在痘苗病毒內(nèi)得到高水平表達(dá)，三個(gè)表達(dá)盒之間沒(méi)有相互影響，而且重組病毒具有良好的遺傳穩(wěn)定性。 將重組核酸疫苗pVL-CCMp24上的目的免疫原基因CCMp24連接到痘苗病毒穿梭載體pSTKE的第一個(gè)表達(dá)盒內(nèi)即MCS1，由特異性引物擴(kuò)增獲得的含Loxp基因序列的EGFP基因（EGFP基因兩端的Loxp基因方向相同）克隆到pSTKE的第三個(gè)表達(dá)盒MCS3中，重組質(zhì)粒pCCMp24-LEL鑒定正確后，轉(zhuǎn)染dVTT感染的BHK-21細(xì)胞，通過(guò)同源重組、病毒蝕斑篩選獲得重組CCMp24和EGFP的E3L及TK基因缺失的痘苗病毒rddVTT-CCMp24G，然后利用Cre/Loxp重組系統(tǒng)將EGFP基因敲除，獲得僅表達(dá)目的免疫原基因的雙基因缺失重組痘苗病毒rddVTT-CCMp24。RT-PCR、間接免疫熒光、Western Blot檢測(cè)結(jié)果表明，CCMp24融合基因在痘苗病毒感染的BHK-21細(xì)胞中得到高效轉(zhuǎn)錄和表達(dá)。 痘苗病毒在長(zhǎng)期的實(shí)踐應(yīng)用中具有毒副作用，存在安全隱患。本研究在缺失病毒毒力相關(guān)基因的痘苗病毒的基礎(chǔ)上，通過(guò)痘苗病毒穿梭載體pSTKE攜帶的目的免疫原基因?qū)K基因缺失，篩選的基因缺失型重組痘苗病毒通過(guò)鼻腔感染和顱內(nèi)注射感染途徑感染BALB/c小鼠，通過(guò)監(jiān)測(cè)感染小鼠的體征變化、體重變化、病毒感染后的死亡率等方面評(píng)價(jià)E3L基因和TK基因缺失的重組痘苗病毒在小鼠體內(nèi)的毒性。監(jiān)測(cè)結(jié)果表明，重組病毒rddVTT-CCMp24的毒力較野生型VTT有很大程度的減弱，提高了進(jìn)一步研究或應(yīng)用的安全性。說(shuō)明缺失E3L基因和TK基因可以使痘苗病毒致弱，且外源基因的引入對(duì)病毒的毒力沒(méi)有顯著影響。 在了解其毒力的基礎(chǔ)上，以BALB/c小鼠為模型開(kāi)展了重組病毒rddVTT-CCMp24的免疫原性分析。按照既定的免疫程序免疫后，通過(guò)對(duì)抗體、細(xì)胞因子及T細(xì)胞亞型等各項(xiàng)指標(biāo)的檢測(cè)，結(jié)果顯示，rddVTT-CCMp24可誘導(dǎo)機(jī)體產(chǎn)生分泌IFN-γ的T細(xì)胞數(shù)量顯著增加，CD4+和CD8+T細(xì)胞數(shù)增加，脾細(xì)胞體外接受HIV特異性抗原刺激下增殖能力顯著提高。HIV特異性抗體檢測(cè)結(jié)果顯示，可誘導(dǎo)較高的HIV特異性抗體水平，，且誘導(dǎo)IL-2和IL-4細(xì)胞因子的分泌。綜合結(jié)果可知，重組病毒可激發(fā)機(jī)體細(xì)胞和體液免疫應(yīng)答。 同時(shí)，采用DNA/rddVTT-CCMp24prime-boost免疫策略在小鼠模型上檢測(cè)免疫效果，結(jié)果相對(duì)于DNA疫苗或rddVTT-CCMp24疫苗分別單獨(dú)免疫，可更好的刺激分泌IFN-γ產(chǎn)生T細(xì)胞的分化和增殖，IFN-γ ELISPOT檢測(cè)結(jié)果顯示，HIV特異性的分泌IFN-γ的記憶性T細(xì)胞數(shù)量顯著高于單獨(dú)免疫組。表明，prime-boost免疫策略誘導(dǎo)較強(qiáng)的免疫應(yīng)答和免疫記憶形成，為下一步的實(shí)驗(yàn)研究提供了數(shù)據(jù)支持。
[Abstract]:In view of the current epidemic situation of acquired immunodeficiency syndrome (AIDS) and the current status of prevention and treatment, it is urgent to develop a safe and effective AIDS prevention / treatment vaccine. In this study, DNA plasmid and vaccinia virus Tiantan strain were selected as the immunogen delivery system to study the immunogenicity of AIDS vaccine.
In this study, a recombinant nucleic acid vaccine pVL-CCMp24 was constructed by introducing CpG motif and CTB gene as adjuvant on the basis of the HIV multiple epitope gene (MEGNp24) screened by our laboratory for many years. The recombinant DNA plasmid was transfected into 293T cells. The results of RT-PCR and indirect immunofluorescence showed that the recombinant plasmid encoded the target gene. The gene CCMp24 was correctly transcribed and expressed in mammalian cells. BALB/c mice were immunized by intramuscular injection, and the immunogenicity of the recombinant DNA vaccine was analyzed. The results of immune indices such as specific antibodies to HIV, Th1 and Th2 cytokines, T cell subtypes, lymphocyte proliferation and so on showed that the recombinant DNA vaccine pVL-CCMp24 was feasible. To induce a certain degree of cellular and humoral immune response.
Based on the design concept of multivalent vaccine, a vaccinia virus shuttle vector pSTKE containing three expression cassettes was constructed, which contained three independent exogenous gene expression cassettes. VTT and its deleted mutants were used as the original viruses, and enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP) and blue fluorescent protein (B) were used as the primary viruses. Two recombinant vaccinia virus strains (rVTT-EGFP, rdVTT-EGFP) were obtained by plaque screening. The recombinant virus was successfully constructed by PCR, real-time fluorescence quantitative PCR and Western blot. Two recombinant vaccinia viruses were successfully screened out by shuttle vector of three gene expression cassettes. EGFP gene was highly expressed in vaccinia viruses. There was no interaction between the three expression cassettes, and the recombinant virus had good genetic stability.
The target immunogen gene CCMp24 on the recombinant nucleic acid vaccine pVL-CCMp24 was linked to the first expression cassette of vaccinia virus shuttle vector pSTKE, namely, MCS1. The EGFP gene containing Loxp gene sequence (the direction of the Loxp gene at both ends of the EGFP gene) was cloned into the third expression cassette MCS3 of pSTKE by specific primer amplification. CMp24-LEL was identified correctly and transfected into BHK-21 cells infected with dVTT. Recombinant CCMp24, E3L of EGFP and rddVTT-CCMp24G of vaccinia virus with TK gene deletion were obtained by homologous recombination and virus plaque screening. Then, the EGFP gene was knocked out by Cre/Loxp recombinant system, and the recombinant vaccinia virus RDD with double gene deletion was obtained. The results of VTT-CCMp24.RT-PCR, indirect immunofluorescence and Western Blot showed that CCMp24 fusion gene was highly transcribed and expressed in vaccinia virus-infected BHK-21 cells.
Vaccinia virus has toxic side effects and potential safety hazards in its long-term practical application. Based on vaccinia virus with virulence-related genes deleted, TK gene was deleted by the target immunogen gene carried by vaccinia virus shuttle vector pSTKE, and the recombinant vaccinia virus with deleted genes was screened for nose infection and intracranial infection. The toxicity of recombinant vaccinia virus with deletion of E3L gene and TK gene was evaluated in BALB/c mice infected by injection. The results showed that the virulence of recombinant virus rddVTT-CCMp24 was much weaker than that of wild type VTT. The results showed that deletion of E3L gene and TK gene could weaken vaccinia virus, and the introduction of exogenous gene had no significant effect on virulence of the virus.
The immunogenicity of recombinant virus rddVTT-CCMp24 was analyzed in BALB/c mice on the basis of its virulence. The results showed that rddVTT-CCMp24 could induce the production of IFN-gamma-secreting T cells in vivo after immunization according to the established immune program, and the detection of antibodies, cytokines and T cell subtypes. The results showed that the recombinant virus could induce high levels of HIV-specific antibodies and secretion of IL-2 and IL-4 cytokines. The results showed that the recombinant virus could stimulate cellular and humoral immunity. Answer.
At the same time, DNA/rddVTT-CCMp24 prime-boost immunization strategy was used to detect the immune effect in mice model. Results Compared with DNA vaccine or rddVTT-CCMp24 vaccine alone, it could stimulate the differentiation and proliferation of T cells secreting IFN-gamma better. The results of IFN-gamma ELISPOT showed that the memory T cells secreting IFN-gamma were specific to HIV. The results showed that prime-boost strategy could induce stronger immune response and immune memory formation, which provided data support for further experimental study.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉存霞;杜壽文;李昌;王宇航;王茂鵬;李沂;尹榮蘭;李霄;任大勇;秦艷青;任靜強(qiáng);金寧一;;HIV復(fù)合表位DNA重組體和痘苗病毒疫苗聯(lián)合免疫實(shí)驗(yàn)研究[J];中國(guó)科學(xué):生命科學(xué);2013年05期

本文編號(hào)：2236511