HIV-1env基因序列分析及DNA疫苗免疫原性研究

发布时间：2018-06-16 20:00

本文选题：人免疫缺陷病毒 + 序列分析　；参考：《北京工业大学》2014年硕士论文

【摘要】：获得性免疫缺陷综合征（Acquired Immunodeficiency Syndrome, AIDS）由人免疫缺陷病毒（Human Immunodeficiency Virus, HIV）感染引起，已经成为世界性健康难题。尽管世界范围内众多研究者都在致力于艾滋病的预防和治疗工作，但至今仍没有安全有效的艾滋病疫苗问世。我国流行的HlV-1是以B',CRF07/08_BC和CRF_01AE3种亚型为主。因此，有必要研制针对上述不同亚型的疫苗。本课题组前期研究中构建了基于B'亚型的多种载体疫苗，通过这些疫苗序贯及重复免疫能长期维持高水平特异性免疫反应。我们拟利用前期研究积累的经验构建基于B/C重组型及A/E重组型的多载体疫苗，将不同亚型疫苗联合应用从而诱导更广谱的免疫反应。本研究主要从B/C重组型HIV-1感染者基因组中扩增全长rev-env基因，对其进行序列测定及分析，根据推导的Env氨基酸序列对重要的功能结构域进行深入分析；进步将克隆得到的rev-env基因构建假病毒，观察其对不同中和抗体及不同亚型感染者血浆的中和敏感性，为B/C重组型HIV-1疫苗的抗原设计奠定基础。同时，将本课题组已经获得的A/E重组型env基因共享序列构建DNA疫苗，并在小鼠体内初步评价该DNA疫苗的免疫原性。采集来自北京地区、经蛋白印迹（Western Blot）试验确证的HIV-1感染者抗凝静脉血100份，提取基因组DNA，用巢式PCR法扩增gag基因，根据测序结果对样品分型。其中12份样品属于B/C重组亚型，再进步用巢式PCR法扩增这12份样品的rev-env基因片段，，将扩增产物连接至pcDNA3.1（+）载体并进行序列测定。结果从12份样品中共获得6个有完整开放阅读框的env基因，亚型鉴定全部为CRF_07B/C重组型。氨基酸序列分析发现这些Env蛋白氨基酸的CD4受体结合位点高度保守，全部使用CCR5辅助受体。gp120/gp41剪切位点高度保守，所有gp160前体都能有效剪切。6个序列都对2G12和2F5抗体中和不敏感；对4E10、PG9、PG16、及NIH45-46抗体中和敏感。将克隆到的上述6个env表达质粒与env缺失的HIV-1骨架质粒pSG3△env共转染293T细胞包装HIV-1假病毒，成功包装出3个HIV-1假病毒。3个假病毒对几种已知中和抗体的敏感性检测结果与预测致，都对PG9和NIH45-46中和敏感，对2G12中和不敏感。3个假病毒都能被来自B'、B/C重组型和A/E重组型感染者的血浆中和。将表达A/E重组型env的DNA疫苗pVR-mod.AE env分别通过肌肉注射和电穿孔免疫Balb/c小鼠，末次免疫后2周，分别用酶联免疫斑点法（Enzyme-linked Immunospot, ELISPOT）和酶联免疫吸附法（Enzyme-linkedImmunosorbent Assay, ELISA）检测小鼠体内的细胞及体液免疫反应。结果电穿孔免疫诱导的细胞免疫和体液免疫反应水平均高于肌肉注射途径，同时，20μg剂量经电穿孔途径与100μg经肌肉注射途径在小鼠体内产生的免疫反应水平相当。表明该DNA疫苗可以有效地诱导env特异性免疫，电穿孔免疫途径可提高疫苗的免疫应答水平。综上所述，我们共获得6株全长有完整开放阅读框的B/C重组亚型env，成功构建了3株B/C重组型HIV-1假病毒。这3株假病毒对几种已知中和抗体都比较敏感，对2G12抗体不敏感，提示我们在构建该亚型疫苗时，可增加2G12识别表位。这些研究结果将为下步进行以B/C重组亚型env为基础的多载体疫苗研究奠定基础。我们对DNA疫苗pVR-mod.AE env的免疫原性初步试验证实该疫苗在小鼠体内能有效诱导细胞及体液免疫反应，为继续构建表达该A/E重组型env的多种病毒载体疫苗提供了依据。
[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 .
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91

【二级参考文献】