HIV细胞免疫抗原的基因改造、表达及免疫效果研究

发布时间：2018-01-28 05:56

本文关键词： Ⅰ型人类免疫缺陷病毒 DNA疫苗复制型痘苗病毒共有序列密码子优化表达细胞免疫　出处：《中国疾病预防控制中心》2010年博士论文　论文类型：学位论文

【摘要】： 自1981年确认第一例艾滋病人以来,艾滋病一直以惊人的速度在全球蔓延,严重地威胁着人类的健康。20多年来,由于艾滋病病毒(HIV)的特殊性以及人们对HIV感染与免疫保护机理认识的不足,艾滋病疫苗至今尚未研制成功。目前,成功的疫苗多是以诱发产生中和抗体为其主要保护机理的,而像HIV这样的病毒感染,除广谱中和抗体外,广谱的细胞免疫对提高疫苗保护效率也可能是至关重要的。从具有广谱交叉活性的细胞免疫抗原入手,进行免疫抗原改造、多种抗原联合使用及不同疫苗“初免-加强”联合免疫已成为HIV细胞免疫疫苗研发的重要策略。本论文选择HIV-1 B/C亚型的5个主要细胞免疫抗原gag、pol、rev、tat、nef,对它们的基因序列、密码子偏性和表达结构进行人工修饰与改造,通过比较各候选基因优化前后的表达水平及细胞免疫效果,以期获得表达水平高、细胞免疫谱宽、免疫反应强的HIV候选细胞免疫抗原基因及表达结构。为了到达上述研究目的,本论文进行了四个方面的研究工作：1. HIV B/C亚型5个细胞免疫抗原的选择与密码子优化；2. HIV DNA疫苗和复制型痘苗病毒载体疫苗的构建及鉴定；3.密码子优化前后及不同表达结构的目的基因表达水平比较；4.密码子优化前后及不同表达结构的疫苗在小鼠体内免疫效果比较。主要结果如下：第一,选择了HIV-1 BVC亚型5个以细胞免疫为主的抗原,进行了基因序列优化及表达结构改造。根据11株中国大陆HIV-1 BVC亚型的全长基因组的氨基酸共有序列,按照哺乳动物细胞偏好的优势密码子设计合成了hgag、hpol、hRTN (rev、tat1、nef融合结构)、hrev、htat、hnef6个基因。经酶切及测序鉴定,改造的6个基因均与设计相符。同时为了便于比较,克隆了cn54株4个野生型基因,包括gagpol、gag、pol、RTN(rev、tat1、nef融合结构)。第二,分别构建了以质粒DNA (pVRC)和复制型天坛株痘苗病毒(rVV)为载体的两大类HIV-1疫苗。其中,包括6个基因优化的DNA疫苗pVRC-hgag、pVRC-hpol、pVRC-hRTN、pVRC-hrev、pVRC-htat、pVRC-hnef,6个基因优化的复制型痘苗病毒载体疫苗RVJ1175hgag、RVJ1175hpol、RVJ1175hRTN、RVJ1175hrev、RVJ1175htat、RVJ1175hnef。同时为了便于比较,构建了4个野生型基因的DNA疫苗pVRC-gagpol、pVRC-gag、pVRC-pol、pVRC-RTN和4个野生型基因的复制型痘苗病毒载体疫苗RVJ1175gagpol、RVJ1175gag、RVJ1175pol、RVJ1175RTN。各疫苗经酶切和测序鉴定均与设计相符。第三,密码子优化后的疫苗可以提高gag、pol、rev、tat、nef等目的基因表达水平,单独基因结构表达水平较融合基因结构高。采用间接免疫荧光(IF)、Western blot (WB)和流式细胞仪(FCM)三种方法检测了各目的基因优化前后的表达水平。结果表明,优化前后各目的基因均能够在这两类载体中有效表达,DNA疫苗的表达水平均强于重组痘苗病毒疫苗；密码子优化后Gag、Pol蛋白的表达均有提高,Pol蛋白提高的较为明显；单独pol基因比gagpol天然结构表达水平高,单独gag与gagpol中的gag表达水平相近；rev、tat、nef基因优化后单独基因结构要略高于优化后融合结构(hRTN),且二者均高于未优化的融合结构(RTN)。第四,密码子优化后可明显提高DNA疫苗中Gag、Pol、Rev、Tat、Nef蛋白在小鼠体内的细胞免疫效果,单独结构的Rev、Tat、Nef优于融合结构RTN。利用ELIspot及ICS等方法完成了各目的基因改造前后在DNA疫苗和重组痘苗病毒疫苗小鼠中免疫效果的评价。结果显示,利用优化前后各目的基因构建的这两种疫苗均能够刺激小鼠产生一定的细胞免疫应答,DNA疫苗的免疫效果均好于重组痘苗病毒疫苗；重组痘苗病毒疫苗中各个基因密码子改造前后的细胞免疫反应没有统计学差异；DNA疫苗中,gag、pol基因优化之后的免疫反应均明显提高。rev、tat、nef基因中,除优化后单独hrev的免疫反应明显高于优化后融合hRTN外,其余两个单独基因结构与优化的融合结构(hRTN)的免疫反应相差不大,但二者略高于未优化的融合结构(RTN)。本研究还对HIV-1 con-B Rev肽库进行筛选,确定了2条未见报道的Rev特异性T细胞表位肽的序列为6006:STYLGRPAEPVPLQL、6007:GRPAEPVPLQLPPLE。验证了Gag和Pol特异性肽刺激的细胞免疫应答是由CD8+T细胞介导的,Tat和Nef特异性肽刺激的细胞免疫应答是由CD4+T细胞介导的。上述结果表明,对HIV-1主要细胞免疫抗原进行序列、密码子、结构的人工修饰与改造后,可以在一定程度上提高各目的基因表达水平及细胞免疫效果。在DNA疫苗中,密码子优化及单独基因结构均能提高表达水平及细胞免疫效果,故可选用这样的基因作为靶抗原。在重组痘苗病毒疫苗中,由于痘苗病毒载体对密码子兼容性很广,密码子优化对于表达水平及细胞免疫效果影响不明显,且融合基因结构中相应的基因可诱导出与单独基因结构相近的细胞免疫反应,故可选择密码子优化及融合结构的基因作为免疫原。本研究为进一步确定HIV-1疫苗中有效的交叉保护性细胞免疫抗原、研究不同抗原在DNA载体和痘苗病毒载体中的免疫原性奠定了实验基础,为进一步研究DNA疫苗和重组痘苗病毒疫苗联合免疫提供了实验依据。
[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.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392.1

【共引文献】