本文選題：HIV + Env��； 參考：《中國藥品生物制品檢定所》2011年碩士論文

【摘要】：自1981年確認(rèn)首例艾滋病以來,艾滋病已成為對(duì)人類健康和社會(huì)穩(wěn)定威脅最大的感染性疾病之一。研制有效的艾滋病疫苗是控制艾滋病流行甚至根除AIDS的理想途徑。選擇合適的抗原作為免疫原以誘導(dǎo)強(qiáng)效、廣譜的中和抗體一直是疫苗研究的重要的基礎(chǔ)。HIV-1膜抗原位于HIV的表面,是中和抗體主要作用靶位。但天然HIV-1膜蛋白免疫原性低,很難誘導(dǎo)出強(qiáng)效廣譜的中和抗體,因而需要對(duì)其進(jìn)行優(yōu)化改造以提高中和表位的免疫原性,增強(qiáng)其誘導(dǎo)中和抗體的能力。本研究以一株CRF07_BC亞型的假病毒株S939為原始株,對(duì)其膜區(qū)進(jìn)行改造以暴露某些保守中和表位,通過單抗、陽性血清的中和試驗(yàn)來驗(yàn)證改造效果,最終獲得一株對(duì)中和性單克隆抗體和HIV-1感染陽性血清均敏感、中和特性強(qiáng)的假病毒,為候選疫苗的抗原選擇和優(yōu)化提供依據(jù)。 本課題根據(jù)已知的單抗識(shí)別表位、已報(bào)道的能增強(qiáng)病毒對(duì)單抗敏感性的位點(diǎn)和N-連接糖基化位點(diǎn)為依據(jù),對(duì)S939 Env設(shè)計(jì)突變,通過環(huán)形誘變,DpnI酶篩選的方式得到突變體,酶切初步鑒定,測序確定突變結(jié)果。將含有突變env的真核表達(dá)質(zhì)粒與HIV-1骨架質(zhì)粒pSG3△env共轉(zhuǎn)染293FT細(xì)胞,收集上清獲得假病毒,通過假病毒單輪感染TZM-bl細(xì)胞檢測假病毒滴度和突變位點(diǎn)對(duì)假病毒感染能力的影響。用HIV-1中和性單抗、HIV-1感染的陽性血清來檢測改造位點(diǎn)對(duì)假病毒中和特性的影響。 單抗2F5、4E10識(shí)別gp41近膜區(qū)(MPER)上的線性表位,在S939上對(duì)該表位進(jìn)行聯(lián)合改造,改造后的假病毒FE相比原始株S939感染力無顯著變化。FE對(duì)單抗2F5敏感性較S939提高11倍以上,對(duì)單抗4E10敏感性無明顯變化。在FE上進(jìn)行單抗2G12表位的改造(I295N、N334S和D386N)獲得假病毒株FE-2G12,其感染力較FE略微增強(qiáng)。2G12表位的改造沒有使假病毒FE-2G12對(duì)單抗2G12敏感,反而降低了對(duì)單抗b12和部分陽性血清的敏感性。 在FE上進(jìn)行了8個(gè)已報(bào)道的增強(qiáng)病毒中和活性位點(diǎn)的改造,其中6個(gè)位點(diǎn)的突變I309L、L669S、G458A、T569A、I675V和D180N顯著降低了病毒的感染力,因而不能進(jìn)行其對(duì)病毒中和特性影響的評(píng)價(jià)。其余兩個(gè)突變中,S365A增強(qiáng)了病毒的感染力,F22L對(duì)病毒感染力無影響,這兩個(gè)突變均沒有增強(qiáng)病毒的中和活性。 以FE為模板,對(duì)25個(gè)N-連接的糖基化位點(diǎn)進(jìn)行單獨(dú)刪除,共獲得25個(gè)含有單個(gè)糖基化刪除的Env表達(dá)質(zhì)粒,并與pSG3△env質(zhì)粒共轉(zhuǎn)染293FT細(xì)胞收獲假病毒。對(duì)這25個(gè)假病毒感染力的檢測發(fā)現(xiàn),其中5個(gè)分布在V1/V2,C1/C2區(qū)糖基化位點(diǎn)的刪除嚴(yán)重影響了病毒的感染力,含有這5個(gè)刪除位點(diǎn)的假病毒檢測不到其感染能力。分布在免疫反應(yīng)靜默面(immunologically-silent face domains)V4、C4和V5區(qū)域中的糖基化位點(diǎn)的刪除(除了N392 (V4))對(duì)假病毒的感染性有一定的增強(qiáng)。其他糖基化位點(diǎn)的刪除均不同程度的減弱了假病毒的感染能力。在假病毒中和特性方面,糖基化位點(diǎn)N197 (C2),N301 (V3),N442 (C4)和N625(gp41)的刪除使假病毒對(duì)部分HIV-1陽性血清、抗CD4結(jié)合位點(diǎn)抗體b12和抗gp41抗體2F5和4E10更加敏感。N142 (V1)的刪除使假病毒對(duì)單抗b12,2F5和4E10敏感性增強(qiáng),但對(duì)HIV-1陽性血清敏感性沒有影響。N355 (C3)和N463 (V5)的刪除使假病毒對(duì)2F5和4E10敏感性增強(qiáng)。 根據(jù)前幾部分的改造結(jié)果,選擇其中能對(duì)病毒中和活性顯著增強(qiáng)的位點(diǎn),結(jié)合V5(N463、N466)和C3(N339)區(qū)的糖基化位點(diǎn)以及與N442臨近的N448的刪除,在FE上按不同組合進(jìn)行了聯(lián)合改造,共獲得了7株聯(lián)合突變假病毒,其中除197M-4外,均顯著降低了病毒的感染力。對(duì)其中6株假病毒的中和特性分析發(fā)現(xiàn),相比于原始株S939、改造株FE以及單個(gè)糖基化位點(diǎn)的改造,聯(lián)合突變株對(duì)單抗2F5、4E10和b12,以及大部分陽性血清,均顯示出了很強(qiáng)的敏感性。 本實(shí)驗(yàn)通過對(duì)55個(gè)氨基酸位點(diǎn)的突變改造,發(fā)現(xiàn)了一些能夠顯著影響病毒感染能力的位點(diǎn),發(fā)現(xiàn)了一些能顯著增強(qiáng)病毒中和特性的位點(diǎn)。通過聯(lián)合改造,最終獲得了幾株對(duì)HIV-1中和單抗和陽性血清表現(xiàn)出較強(qiáng)中和特性的假病毒株,為HIV-1膜蛋白的優(yōu)化改造以及候選膜抗原的選擇提供了依據(jù)。
[Abstract]:Since the first AIDS was identified in 1981, AIDS has become one of the most infectious diseases that threaten human health and social stability. The development of an effective AIDS vaccine is an ideal way to control the epidemic and even eradicate AIDS. The important basic.HIV-1 membrane antigen is located on the surface of HIV, which is the main target of neutralizing antibody. But the natural HIV-1 membrane protein is low immunogenicity, it is difficult to induce the strong and broad-spectrum neutralization antibody. Therefore, it needs to be optimized to improve the immunogenicity of neutralizing epitopes and enhance the ability to induce neutralization antibody. The strain CRF07_BC subtype S939 was the original strain, and its membrane region was reformed to expose some conservative neutralization epitopes. The effect was verified by neutralization test of mAb and positive sera. Finally, a strain of neutralizing monoclonal antibody and HIV-1 infection positive sera, a pseudo virus with strong neutralization characteristics, was obtained as a candidate vaccine. The original selection and optimization provide the basis.
According to the known epitopes of the known monoclonal antibody, it has been reported that the virus can enhance the sensitivity of the virus to the loci of the monoclonal antibody and the glycosylation site of the N- connection. The mutation is designed for the S939 Env, the mutation is obtained through the ring mutation and the DpnI enzyme screening. The mutation results are determined by the enzyme digestion and the mutation results are sequenced. The eukaryotic expression plasmid containing the mutant env is expressed. The HIV-1 cytoskeleton plasmid pSG3 delta env was co transfected with 293FT cells, and the pseudo virus was collected from the supernatant. The pseudo virus titer and the mutation site were detected by the pseudo virus single wheel infection. The effect of the HIV-1 neutralization monoclonal antibody and the positive serum of HIV-1 infection on the neutralization characteristics of the pseudo virus was detected by the HIV-1 neutralization monoclonal antibody and the positive serum of HIV-1 infection.
The monoclonal antibody 2F5,4E10 identified the linear epitopes on the gp41 near membrane region (MPER), and reformed the epitopes on the S939. The transformed pseudo virus FE had no significant changes in the S939 infection force compared with the original strain.FE, and the sensitivity of.FE to the monopagion 2F5 was 11 times higher than that of S939, and the sensitivity of the monoclonal antibody 4E10 was not changed. 334S and D386N) obtained the pseudo virus strain FE-2G12, and its infection force was slightly enhanced by a slight enhancement of the.2G12 epitopes, which did not make the pseudo virus FE-2G12 sensitive to the monoclonal antibody 2G12, but reduced the sensitivity to the monoclonal antibody B12 and the partial positive serum.
8 reported enhanced viral and active sites were reported on FE, of which mutations I309L, L669S, G458A, T569A, I675V and D180N significantly reduced the virus's infectivity and therefore failed to evaluate the effect of the virus neutralization. In the other two mutations, S365A enhanced the virus infection and F22L to the virus. The two mutations did not enhance the neutralizing activity of the virus.
The glycosylation sites of 25 N- connections were deleted by FE as a template, and 25 Env expressing plasmids containing single glycosylation were obtained, and pSG3 delta env plasmids were co transfected with 293FT cells to harvest the pseudo virus. The detection of the 25 pseudo virus infection forces was found, of which 5 were distributed in V1/V2, C1/C2 region glycosylation sites were deleted seriously. The infectivity of the virus, which contains the 5 deletion sites, is not detected. The deletion of the glycosylation sites in the immune response (immunologically-silent face domains) V4, C4 and V5 regions (except for N392 (V4)) has a certain enhancement in the susceptibility to the pseudo virus. The deletion of the glycosylation site N197 (C2), N301 (V3), N442 (C4) and N625 (gp41) in the same degree of neutralization of the pseudo virus makes the false virus to some HIV-1 positive serum, the anti CD4 binding site antibody B12 and the gp41 antibody and more sensitive. 10 sensitivity increased, but had no effect on the sensitivity of HIV-1 positive serum. The deletion of.N355 (C3) and N463 (V5) increased the sensitivity of the virus to 2F5 and 4E10.
According to the transformation results of the previous parts, we selected 7 sites with significant enhancement of virus neutralization activity, combined with the glycosylation sites of V5 (N463, N466) and C3 (N339) region and the deletion of N448 near N442. The combined transformation was carried out on FE according to different combinations, and a total of 7 joint mutant pseudo viruses were obtained, all of which were significantly reduced except 197M-4. The neutralization characteristics of 6 strains of the virus were found to be more sensitive than the original strain S939, the transformation strain FE and the modification of the single glycosylation site. The combined mutant strain showed strong sensitivity to the mAb 2F5,4E10 and B12, as well as most of the positive serum.
In this experiment, we found some sites that could significantly affect the ability of virus infection by changing the 55 amino acid sites, and found some sites that could significantly enhance the neutralization characteristics of the virus. Through the combined transformation, several strains of the HIV-1 neutralization and positive sera were obtained, which were HIV-1. It provides a basis for optimization of membrane proteins and selection of candidate membrane antigens.

【學(xué)位授予單位】：中國藥品生物制品檢定所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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相關(guān)期刊論文 前2條

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本文編號(hào)：1803816