HIV復合表位核酸和痘苗病毒載體疫苗構建及免疫原性研究
發(fā)布時間：2018-09-11 11:02
【摘要】：鑒于當前獲得性免疫缺陷綜合征（Acquired immunodeficiency syndrome, AIDS）的流行態(tài)勢和預防治療現(xiàn)狀，研發(fā)安全有效的艾滋病預防/治療性疫苗刻不容緩。近些年來，很多科學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)，開展艾滋病疫苗的免疫原性研究。 本研究在本研究室多年來篩選獲得的HIV復合多表位基因（MEGNp24）的基礎上，在其上游引入CpG基序和CTB基因作為佐劑，構建了一種重組核酸疫苗pVL-CCMp24，將重組DNA質(zhì)粒轉染293T細胞，從RT-PCR和間接免疫熒光檢測結果可知，重組質(zhì)粒編碼的目的基因CCMp24在哺乳動物細胞內(nèi)得到正確轉錄和表達。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HIV特異性抗體、Th1型和Th2型細胞因子、T細胞亞型、淋巴細胞增殖等免疫指標的檢測結果表明，重組DNA候選疫苗pVL-CCMp24可以誘導機體產(chǎn)生一定程度的細胞和體液免疫應答。 基于多價疫苗的設計理念，本研究首先構建了一種含三個表達盒的痘苗病毒穿梭載體pSTKE，含有三個獨立的外源基因表達盒，分別以VTT及其基因缺失突變株作為原始病毒，以增強型綠色熒光蛋白(EGFP)、紅色熒光蛋白（RFP）和藍色熒光蛋白（BFP）作為報告基因，分別對三個表達盒的功能進行驗證。通過噬斑篩選，獲得兩株重組痘苗病毒(rVTT-EGFP、rdVTT-EGFP)，利用PCR、實時熒光定量PCR、Western blot方法對重組病毒以及對外源基因的表達量和遺傳穩(wěn)定性進行分析。結果表明，成功構建了三基因表達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，三個表達盒之間沒有相互影響，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的第一個表達盒內(nèi)即MCS1，由特異性引物擴增獲得的含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的第三個表達盒MCS3中，重組質(zhì)粒pCCMp24-LEL鑒定正確后，轉染dVTT感染的BHK-21細胞，通過同源重組、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檢測結果表明，CCMp24融合基因在痘苗病毒感染的BHK-21細胞中得到高效轉錄和表達。 痘苗病毒在長期的實踐應用中具有毒副作用，存在安全隱患。本研究在缺失病毒毒力相關基因的痘苗病毒的基礎上，通過痘苗病毒穿梭載體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鼻腔感染和顱內(nèi)注射感染途徑感染BALB/c小鼠，通過監(jiān)測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價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)測結果表明，重組病毒rddVTT-CCMp24的毒力較野生型VTT有很大程度的減弱，提高了進一步研究或應用的安全性。說明缺失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病毒的毒力沒有顯著影響。 在了解其毒力的基礎上，以BALB/c小鼠為模型開展了重組病毒rddVTT-CCMp24的免疫原性分析。按照既定的免疫程序免疫后，通過對抗體、細胞因子及T細胞亞型等各項指標的檢測，結果顯示，rddVTT-CCMp24可誘導機體產(chǎn)生分泌IFN-γ的T細胞數(shù)量顯著增加，CD4+和CD8+T細胞數(shù)增加，脾細胞體外接受HIV特異性抗原刺激下增殖能力顯著提高。HIV特異性抗體檢測結果顯示，可誘導較高的HIV特異性抗體水平，，且誘導IL-2和IL-4細胞因子的分泌。綜合結果可知，重組病毒可激發(fā)機體細胞和體液免疫應答。 同時，采用DNA/rddVTT-CCMp24prime-boost免疫策略在小鼠模型上檢測免疫效果，結果相對于DNA疫苗或rddVTT-CCMp24疫苗分別單獨免疫，可更好的刺激分泌IFN-γ產(chǎn)生T細胞的分化和增殖，IFN-γ ELISPOT檢測結果顯示，HIV特異性的分泌IFN-γ的記憶性T細胞數(shù)量顯著高于單獨免疫組。表明，prime-boost免疫策略誘導較強的免疫應答和免疫記憶形成，為下一步的實驗研究提供了數(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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392
本文編號：2236511
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392
【引證文獻】
相關期刊論文 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;金寧一;;HIV復合表位DNA重組體和痘苗病毒疫苗聯(lián)合免疫實驗研究[J];中國科學:生命科學;2013年05期
本文編號：2236511
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