本文關(guān)鍵詞： Ⅰ型人類免疫缺陷病毒 DNA疫苗 復(fù)制型痘苗病毒 共有序列 密碼子優(yōu)化 表達(dá) 細(xì)胞免疫　出處：《中國疾病預(yù)防控制中心》2010年博士論文　論文類型：學(xué)位論文

【摘要】： 自1981年確認(rèn)第一例艾滋病人以來,艾滋病一直以驚人的速度在全球蔓延,嚴(yán)重地威脅著人類的健康。20多年來,由于艾滋病病毒(HIV)的特殊性以及人們對(duì)HIV感染與免疫保護(hù)機(jī)理認(rèn)識(shí)的不足,艾滋病疫苗至今尚未研制成功。目前,成功的疫苗多是以誘發(fā)產(chǎn)生中和抗體為其主要保護(hù)機(jī)理的,而像HIV這樣的病毒感染,除廣譜中和抗體外,廣譜的細(xì)胞免疫對(duì)提高疫苗保護(hù)效率也可能是至關(guān)重要的。從具有廣譜交叉活性的細(xì)胞免疫抗原入手,進(jìn)行免疫抗原改造、多種抗原聯(lián)合使用及不同疫苗“初免-加強(qiáng)”聯(lián)合免疫已成為HIV細(xì)胞免疫疫苗研發(fā)的重要策略。 本論文選擇HIV-1 B/C亞型的5個(gè)主要細(xì)胞免疫抗原gag、pol、rev、tat、nef,對(duì)它們的基因序列、密碼子偏性和表達(dá)結(jié)構(gòu)進(jìn)行人工修飾與改造,通過比較各候選基因優(yōu)化前后的表達(dá)水平及細(xì)胞免疫效果,以期獲得表達(dá)水平高、細(xì)胞免疫譜寬、免疫反應(yīng)強(qiáng)的HIV候選細(xì)胞免疫抗原基因及表達(dá)結(jié)構(gòu)。 為了到達(dá)上述研究目的,本論文進(jìn)行了四個(gè)方面的研究工作：1. HIV B/C亞型5個(gè)細(xì)胞免疫抗原的選擇與密碼子優(yōu)化；2. HIV DNA疫苗和復(fù)制型痘苗病毒載體疫苗的構(gòu)建及鑒定；3.密碼子優(yōu)化前后及不同表達(dá)結(jié)構(gòu)的目的基因表達(dá)水平比較；4.密碼子優(yōu)化前后及不同表達(dá)結(jié)構(gòu)的疫苗在小鼠體內(nèi)免疫效果比較。主要結(jié)果如下： 第一,選擇了HIV-1 BVC亞型5個(gè)以細(xì)胞免疫為主的抗原,進(jìn)行了基因序列優(yōu)化及表達(dá)結(jié)構(gòu)改造。根據(jù)11株中國大陸HIV-1 BVC亞型的全長基因組的氨基酸共有序列,按照哺乳動(dòng)物細(xì)胞偏好的優(yōu)勢(shì)密碼子設(shè)計(jì)合成了hgag、hpol、hRTN (rev、tat1、nef融合結(jié)構(gòu))、hrev、htat、hnef6個(gè)基因。經(jīng)酶切及測(cè)序鑒定,改造的6個(gè)基因均與設(shè)計(jì)相符。同時(shí)為了便于比較,克隆了cn54株4個(gè)野生型基因,包括gagpol、gag、pol、RTN(rev、tat1、nef融合結(jié)構(gòu))。 第二,分別構(gòu)建了以質(zhì)粒DNA (pVRC)和復(fù)制型天壇株痘苗病毒(rVV)為載體的兩大類HIV-1疫苗。其中,包括6個(gè)基因優(yōu)化的DNA疫苗pVRC-hgag、pVRC-hpol、pVRC-hRTN、pVRC-hrev、pVRC-htat、pVRC-hnef,6個(gè)基因優(yōu)化的復(fù)制型痘苗病毒載體疫苗RVJ1175hgag、RVJ1175hpol、RVJ1175hRTN、RVJ1175hrev、RVJ1175htat、RVJ1175hnef。同時(shí)為了便于比較,構(gòu)建了4個(gè)野生型基因的DNA疫苗pVRC-gagpol、pVRC-gag、pVRC-pol、pVRC-RTN和4個(gè)野生型基因的復(fù)制型痘苗病毒載體疫苗RVJ1175gagpol、RVJ1175gag、RVJ1175pol、RVJ1175RTN。各疫苗經(jīng)酶切和測(cè)序鑒定均與設(shè)計(jì)相符。 第三,密碼子優(yōu)化后的疫苗可以提高gag、pol、rev、tat、nef等目的基因表達(dá)水平,單獨(dú)基因結(jié)構(gòu)表達(dá)水平較融合基因結(jié)構(gòu)高。采用間接免疫熒光(IF)、Western blot (WB)和流式細(xì)胞儀(FCM)三種方法檢測(cè)了各目的基因優(yōu)化前后的表達(dá)水平。結(jié)果表明,優(yōu)化前后各目的基因均能夠在這兩類載體中有效表達(dá),DNA疫苗的表達(dá)水平均強(qiáng)于重組痘苗病毒疫苗；密碼子優(yōu)化后Gag、Pol蛋白的表達(dá)均有提高,Pol蛋白提高的較為明顯；單獨(dú)pol基因比gagpol天然結(jié)構(gòu)表達(dá)水平高,單獨(dú)gag與gagpol中的gag表達(dá)水平相近；rev、tat、nef基因優(yōu)化后單獨(dú)基因結(jié)構(gòu)要略高于優(yōu)化后融合結(jié)構(gòu)(hRTN),且二者均高于未優(yōu)化的融合結(jié)構(gòu)(RTN)。 第四,密碼子優(yōu)化后可明顯提高DNA疫苗中Gag、Pol、Rev、Tat、Nef蛋白在小鼠體內(nèi)的細(xì)胞免疫效果,單獨(dú)結(jié)構(gòu)的Rev、Tat、Nef優(yōu)于融合結(jié)構(gòu)RTN。利用ELIspot及ICS等方法完成了各目的基因改造前后在DNA疫苗和重組痘苗病毒疫苗小鼠中免疫效果的評(píng)價(jià)。結(jié)果顯示,利用優(yōu)化前后各目的基因構(gòu)建的這兩種疫苗均能夠刺激小鼠產(chǎn)生一定的細(xì)胞免疫應(yīng)答,DNA疫苗的免疫效果均好于重組痘苗病毒疫苗；重組痘苗病毒疫苗中各個(gè)基因密碼子改造前后的細(xì)胞免疫反應(yīng)沒有統(tǒng)計(jì)學(xué)差異；DNA疫苗中,gag、pol基因優(yōu)化之后的免疫反應(yīng)均明顯提高。rev、tat、nef基因中,除優(yōu)化后單獨(dú)hrev的免疫反應(yīng)明顯高于優(yōu)化后融合hRTN外,其余兩個(gè)單獨(dú)基因結(jié)構(gòu)與優(yōu)化的融合結(jié)構(gòu)(hRTN)的免疫反應(yīng)相差不大,但二者略高于未優(yōu)化的融合結(jié)構(gòu)(RTN)。 本研究還對(duì)HIV-1 con-B Rev肽庫進(jìn)行篩選,確定了2條未見報(bào)道的Rev特異性T細(xì)胞表位肽的序列為6006:STYLGRPAEPVPLQL、6007:GRPAEPVPLQLPPLE。驗(yàn)證了Gag和Pol特異性肽刺激的細(xì)胞免疫應(yīng)答是由CD8+T細(xì)胞介導(dǎo)的,Tat和Nef特異性肽刺激的細(xì)胞免疫應(yīng)答是由CD4+T細(xì)胞介導(dǎo)的。 上述結(jié)果表明,對(duì)HIV-1主要細(xì)胞免疫抗原進(jìn)行序列、密碼子、結(jié)構(gòu)的人工修飾與改造后,可以在一定程度上提高各目的基因表達(dá)水平及細(xì)胞免疫效果。在DNA疫苗中,密碼子優(yōu)化及單獨(dú)基因結(jié)構(gòu)均能提高表達(dá)水平及細(xì)胞免疫效果,故可選用這樣的基因作為靶抗原。在重組痘苗病毒疫苗中,由于痘苗病毒載體對(duì)密碼子兼容性很廣,密碼子優(yōu)化對(duì)于表達(dá)水平及細(xì)胞免疫效果影響不明顯,且融合基因結(jié)構(gòu)中相應(yīng)的基因可誘導(dǎo)出與單獨(dú)基因結(jié)構(gòu)相近的細(xì)胞免疫反應(yīng),故可選擇密碼子優(yōu)化及融合結(jié)構(gòu)的基因作為免疫原。本研究為進(jìn)一步確定HIV-1疫苗中有效的交叉保護(hù)性細(xì)胞免疫抗原、研究不同抗原在DNA載體和痘苗病毒載體中的免疫原性奠定了實(shí)驗(yàn)基礎(chǔ),為進(jìn)一步研究DNA疫苗和重組痘苗病毒疫苗聯(lián)合免疫提供了實(shí)驗(yàn)依據(jù)。
[Abstract]:Since 1981 confirmed the first cases of AIDS, AIDS has been spreading at an alarming rate in the world, a serious threat to human health.20 for many years, because the AIDS virus (HIV) and the particularity of lack of awareness of HIV infection and immune protection mechanism, AIDS vaccine has not yet been developed. At present, the success of the vaccine is to induce neutralizing antibody is the main mechanism of protection, such as HIV and virus infection, in addition to broadly neutralizing antibodies, immune cells to improve the efficiency of broad-spectrum vaccine protection may also be crucial. Starting from the cellular immune antigen with broad-spectrum activity, immune antigen modification, combined the use of a variety of different antigen and vaccine "prime boost immunization" has become an important strategy for HIV cellular immune vaccine.
This paper selects 5 main cellular immune antigen gag, HIV-1 subtype B/C pol, rev, tat, Nef, on their gene sequences, codon bias and expression of structure modification and optimization, the expression level and cellular immune effects before and after each candidate gene optimization, in order to obtain the high expression level immune cells, spectral width, structure and expression of HIV antigen gene candidate immune reaction.
In order to achieve the purpose of the study, this paper studies four aspects: the selection and optimization of codon 1. HIV subtype B/C 5 antigen; 2. HIV DNA vaccine and replicating vaccinia virus vector vaccine construction and identification; 3. codon optimized gene expression level before and after different expression structure the 4. codon; and different expression structure before and after optimization of vaccine immunogenicity in mice. The main results are as follows:
First, choose the HIV-1 BVC subtype in 5 cellular immune antigen, optimized gene sequence and expression of structure transformation. According to the amino acids of 11 strains of HIV-1, the full-length genome Chinese subtype BVC consensus sequence, according to the advantage of codon preference of mammalian cells hgag, synthesized HPOL, hRTN (Rev TAT1, Nef, hrev, htat, fusion structure), hnef6 gene. After enzyme digestion and sequencing, 6 gene transformation were consistent with the design. At the same time, for comparison, 4 strains of cloned cn54 wild type gene, including gagpol, gag, pol, RTN (Rev, TAT1, Nef fusion structure).
Second, were constructed with the plasmid DNA (pVRC) and replicating vaccinia virus Tiantan strain (rVV) of two kinds of HIV-1 vaccine. Among them, 6 genes including the optimization of DNA vaccine pVRC-hgag, pVRC-hpol, pVRC-hRTN, pVRC-hrev, pVRC-htat, pVRC-hnef, 6 gene optimization replicating vaccinia virus vector vaccine RVJ1175hgag RVJ1175hpol, RVJ1175hRTN, RVJ1175hrev, RVJ1175htat, RVJ1175hnef., and for comparison, to construct the DNA vaccine pVRC-gagpol, 4 wild type pVRC-gag gene, pVRC-pol, replicating vaccinia virus vector vaccine RVJ1175gagpol, pVRC-RTN and 4 wild type gene RVJ1175gag, RVJ1175pol, RVJ1175RTN. of the vaccine by enzyme digestion and sequencing were consistent with the design.
Third, codon optimized vaccine can improve gag, pol, rev, tat, the expression level of nef gene, the expression of individual genes structure level is high. The fusion gene structure by indirect immunofluorescence (IF), Western blot (WB) and flow cytometry (FCM) method to detect the expression level of three before and after each gene optimization. The results show that before and after optimization of each target gene can be expressed efficiently in all the two kinds of vector, the expression level of DNA vaccine was better than recombinant vaccinia virus vaccine; codon optimized Gag, showed the expression of Pol protein increased, Pol protein increased significantly; the expression level of pol gene alone gagpol higher than the natural structure, separate gag and gagpol expression levels of gag in Rev, tat, Nef are similar; gene optimized single gene structure is slightly higher than the optimized fusion structure (hRTN), and two of them were higher than non optimized fusion (RTN).
Fourth, codon optimized DNA vaccine can significantly improve Gag, Pol, Rev, Tat, Nef protein in the cellular immune effect in mice, separate structure of the Rev, Tat, Nef is better than RTN. evaluation fusion structure using ELIspot and ICS methods before and after the completion of the transformation of the target gene in immune DNA vaccine and recombinant vaccinia virus the effect of the vaccine in mice. The results showed that the use of these two kinds of vaccines before and after optimization of the target gene construct can stimulate mice to produce cellular immune responses, immune effect of DNA vaccine was better than recombinant vaccinia virus vaccine; no significant difference before and after each gene codon of the cellular immune response to recombinant vaccinia virus vaccine; DNA vaccine, gag, immune response after optimization of the pol gene were significantly increased by.Rev, tat, nef gene, in addition to the immune response after optimization of hrev alone was significantly higher than that of the optimized fusion hRTN, The immune responses of the remaining two individual gene structures and the optimized fusion structure (hRTN) were small, but the two were slightly higher than that of the non optimized fusion structure (RTN).
The study of HIV-1 con-B Rev peptide library screening, to determine the sequence of Rev specific T cell epitope peptide 2 has not been reported for 6006:STYLGRPAEPVPLQL, 6007:GRPAEPVPLQLPPLE. examined the cellular immune responses of Gag and Pol specific peptide stimulation is mediated by CD8+T cells, the cellular immune response to Tat and Nef specific peptide stimulation is mediated by CD4+T cells.
The results show that the sequence of HIV-1, cellular immune antigen codon modification and transformation of structure, can improve the gene expression level and immune effect in a certain extent. In the DNA vaccine, codon optimization and single gene structure can increase the expression levels and cellular immune effects, so it can be use this gene as a target antigen. The recombinant vaccinia virus vaccine, because vaccinia virus vector of codon compatibility is very wide, codon optimization is not obvious influenced the expression level and immune response and cellular immune response, fusion gene structure of the corresponding gene can be induced and individual genes with similar structures, so it can be selected the codon optimized gene and fusion structure as an immunogen. This study was to further determine the cross protective antigen of effective HIV-1 vaccine research, different resistance In the original DNA vector and the recombinant vaccinia virus in the immunogenicity of the experimental basis, provide experimental basis for further research of DNA vaccine and immunization of recombinant vaccinia virus vaccine.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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本文編號(hào)：1469963