本文選題：人免疫缺陷病毒 + 序列分析　； 參考：《北京工業(yè)大學(xué)》2014年碩士論文

【摘要】：獲得性免疫缺陷綜合征（Acquired Immunodeficiency Syndrome, AIDS）由人免疫缺陷病毒（Human Immunodeficiency Virus, HIV）感染引起，已經(jīng)成為世界性健康難題。盡管世界范圍內(nèi)眾多研究者都在致力于艾滋病的預(yù)防和治療工作，但至今仍沒有安全有效的艾滋病疫苗問世。我國(guó)流行的HlV-1是以B',CRF07/08_BC和CRF_01AE3種亞型為主。因此，有必要研制針對(duì)上述不同亞型的疫苗。本課題組前期研究中構(gòu)建了基于B'亞型的多種載體疫苗，通過這些疫苗序貫及重復(fù)免疫能長(zhǎng)期維持高水平特異性免疫反應(yīng)。我們擬利用前期研究積累的經(jīng)驗(yàn)構(gòu)建基于B/C重組型及A/E重組型的多載體疫苗，將不同亞型疫苗聯(lián)合應(yīng)用從而誘導(dǎo)更廣譜的免疫反應(yīng)。本研究主要從B/C重組型HIV-1感染者基因組中擴(kuò)增全長(zhǎng)rev-env基因，對(duì)其進(jìn)行序列測(cè)定及分析，根據(jù)推導(dǎo)的Env氨基酸序列對(duì)重要的功能結(jié)構(gòu)域進(jìn)行深入分析；進(jìn)步將克隆得到的rev-env基因構(gòu)建假病毒，觀察其對(duì)不同中和抗體及不同亞型感染者血漿的中和敏感性，為B/C重組型HIV-1疫苗的抗原設(shè)計(jì)奠定基礎(chǔ)。同時(shí)，將本課題組已經(jīng)獲得的A/E重組型env基因共享序列構(gòu)建DNA疫苗，并在小鼠體內(nèi)初步評(píng)價(jià)該DNA疫苗的免疫原性。 采集來自北京地區(qū)、經(jīng)蛋白印跡（Western Blot）試驗(yàn)確證的HIV-1感染者抗凝靜脈血100份，提取基因組DNA，用巢式PCR法擴(kuò)增gag基因，根據(jù)測(cè)序結(jié)果對(duì)樣品分型。其中12份樣品屬于B/C重組亞型，再進(jìn)步用巢式PCR法擴(kuò)增這12份樣品的rev-env基因片段，，將擴(kuò)增產(chǎn)物連接至pcDNA3.1（+）載體并進(jìn)行序列測(cè)定。結(jié)果從12份樣品中共獲得6個(gè)有完整開放閱讀框的env基因，亞型鑒定全部為CRF_07B/C重組型。氨基酸序列分析發(fā)現(xiàn)這些Env蛋白氨基酸的CD4受體結(jié)合位點(diǎn)高度保守，全部使用CCR5輔助受體。gp120/gp41剪切位點(diǎn)高度保守，所有g(shù)p160前體都能有效剪切。6個(gè)序列都對(duì)2G12和2F5抗體中和不敏感；對(duì)4E10、PG9、PG16、及NIH45-46抗體中和敏感。將克隆到的上述6個(gè)env表達(dá)質(zhì)粒與env缺失的HIV-1骨架質(zhì)粒pSG3△env共轉(zhuǎn)染293T細(xì)胞包裝HIV-1假病毒，成功包裝出3個(gè)HIV-1假病毒。3個(gè)假病毒對(duì)幾種已知中和抗體的敏感性檢測(cè)結(jié)果與預(yù)測(cè)致，都對(duì)PG9和NIH45-46中和敏感，對(duì)2G12中和不敏感。3個(gè)假病毒都能被來自B'、B/C重組型和A/E重組型感染者的血漿中和。 將表達(dá)A/E重組型env的DNA疫苗pVR-mod.AE env分別通過肌肉注射和電穿孔免疫Balb/c小鼠，末次免疫后2周，分別用酶聯(lián)免疫斑點(diǎn)法（Enzyme-linked Immunospot, ELISPOT）和酶聯(lián)免疫吸附法（Enzyme-linkedImmunosorbent Assay, ELISA）檢測(cè)小鼠體內(nèi)的細(xì)胞及體液免疫反應(yīng)。結(jié)果電穿孔免疫誘導(dǎo)的細(xì)胞免疫和體液免疫反應(yīng)水平均高于肌肉注射途徑，同時(shí)，20μg劑量經(jīng)電穿孔途徑與100μg經(jīng)肌肉注射途徑在小鼠體內(nèi)產(chǎn)生的免疫反應(yīng)水平相當(dāng)。表明該DNA疫苗可以有效地誘導(dǎo)env特異性免疫，電穿孔免疫途徑可提高疫苗的免疫應(yīng)答水平。 綜上所述，我們共獲得6株全長(zhǎng)有完整開放閱讀框的B/C重組亞型env，成功構(gòu)建了3株B/C重組型HIV-1假病毒。這3株假病毒對(duì)幾種已知中和抗體都比較敏感，對(duì)2G12抗體不敏感，提示我們?cè)跇?gòu)建該亞型疫苗時(shí)，可增加2G12識(shí)別表位。這些研究結(jié)果將為下步進(jìn)行以B/C重組亞型env為基礎(chǔ)的多載體疫苗研究奠定基礎(chǔ)。我們對(duì)DNA疫苗pVR-mod.AE env的免疫原性初步試驗(yàn)證實(shí)該疫苗在小鼠體內(nèi)能有效誘導(dǎo)細(xì)胞及體液免疫反應(yīng)，為繼續(xù)構(gòu)建表達(dá)該A/E重組型env的多種病毒載體疫苗提供了依據(jù)。
[Abstract]:It has become a worldwide health problem that acquired immunodeficiency syndrome ( AIDS ) is caused by human immunodeficiency virus ( HIV ) infection . Although many researchers in the world are working on the prevention and treatment of AIDS , there is no safe and effective vaccine for AIDS .
The cloned rev - env gene was constructed to construct a pseudovirus , which was used to establish a base for the antigen design of the B / C recombinant HIV - 1 vaccine . At the same time , the DNA vaccine was constructed with the A / E recombinant env gene sharing sequence which was already obtained in the study group , and the immunogenicity of the DNA vaccine was evaluated in vivo .

The gag gene was amplified by nested PCR . The amplified products were amplified by nested PCR and sequenced . The results showed that 12 samples belonged to the B / C recombination subtype , and the amplified products were ligated to pcDNA3.1 ( + ) vector and sequenced . The results showed that the CD4 receptor binding sites of these 12 samples were highly conserved and all of gp160 precursors were highly conserved . All of the 6 sequences were insensitive to the 2G12 and 2F5 antibodies .
Three pseudoviruses were sensitive to PG9 and NIH45 - 46 and were insensitive to PG9 and NIH45 - 46 . Three pseudoviruses could be neutralized by plasma from B ' , B / C recombinant and A / E recombinant infection .

The results showed that the immune response of mice immunized by electroporation was higher than that of intramuscular injection , and the level of immune response induced by electroporation was higher than that of 100 渭g by intramuscular injection .

In conclusion , we have successfully constructed three B / C recombinant HIV - 1 pseudoviruses with a complete open reading frame of B / C . These 3 pseudoviruses are sensitive to several known neutralizing antibodies and are not sensitive to the 2gl 2 antibody . These results will provide a basis for the study of the recombinant vaccine . The results show that the vaccine can induce cellular and humoral immune responses effectively in mice , and provide a basis for the continued construction of a variety of viral vector vaccines expressing the A / E recombinant env .
【學(xué)位授予單位】：北京工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.91

【二級(jí)參考文獻(xiàn)】

相關(guān)期刊論文 前2條

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