【作者】 李亮；

【导师】 曲娟娟；

【作者基本信息】 东北农业大学 ， 微生物学， 2011， 硕士

【摘要】 人/猴嵌合免疫缺陷病毒(simian/human/immunodeficiency virus,SHIV),是通过基因重组技术,用HIV-1(hunman immunodeficiency viruses -1)的囊膜蛋白基因(env)和部分辅助基因与SIV(simian immunodeficiency viruses)相应基因进行置换,获得的嵌合型病毒。SHIV具有亲本HIV-1和SIV毒株相似的生物学特性,可以在非人灵长类(NHP)动物上模拟HIV-1感染,为研究HIV-1致病机制及疫苗开发提供了重要的模型。SHIV的衣壳蛋白p27构成病毒粒子双层壳的内壳,而且与同属病毒的衣壳蛋白具有相同或相似的抗原性,故可作为SHIV抗原检测的标志物。利用p27抗原和及其抗体,可以在细胞和机体水平对SHIV感染NHP模型进行研究,为艾滋病疫苗研发、免疫策略的有效性以及相关基础研究进行评价。因此,鉴定p27抗原表位对于进一步研究其蛋白结构与功能、开发早期诊断试剂具有重要意义。本研究用Protein A Sepharose CL-4B柱子对前期工作中已制备的3株p27蛋白单克隆抗体(monoclonal antibody,MAb)p27-A5、p27-C7和p27-D2进行纯化,通过ELISA相加试验对3株单克隆抗体进行抗原结合位点的结合试验。为了对3株单抗结合的抗原表位进行精确鉴定,将该序列分为相互重叠15bp的3个基因片段,分别命名为p27-a(1bp~255bp)、p27-b(241bp~495bp)、p27-c(481bp~741bp),根据每个片段的序列特征设计引物,并分别在三个基因片段中引入BamH I及Hind III位点。将3个片段进行PCR扩增后酶切,并分别克隆于与经相同酶切处理的表达载体pET-32a中,经酶切和测序正确的重组质粒,将其进行诱导表达,表达产物经SDS-PAGE电泳分析和Western blot鉴定。在初步鉴定后,设计了一套总数为16个覆盖整个免疫优势区域的短肽,分别命名为A1~A8、C1~C8,短肽长度均为16aa,部分重叠,对其基因序列进行了直接合成,在5’端和3’端分别引入BamHⅠ和HindⅢ位点。退火后与表达载体pET32a连接,以载体携带的His标签的融合方式,表达短肽,分别命名为A1至A8及C1至C8,表达产物分别进行SDS-PAGE电泳分析和Western blot鉴定。本研究结果如下:1.应用Protein A Sepharose CL-4B亲和层析法成功纯化3株单克隆抗体,获得了高纯度的IgG类抗体,3株腹水单克隆体抗纯度达95%以上,可以满足作为检测试剂和其他应用研究的要求。2.确定了单抗p27-A5与p27-C7针对相同或位置相近抗原表位,而另一株单抗p27-D2针对与上述2株单抗不同的抗原表位。3.鉴定出以上3株单抗与p27结合的抗原表位,其中,单抗p27-C7及p27-A5所针对的抗原表位位于p27的61aa~76aa,氨基酸序列为61SEGCTPYDINQMLNCV76;单抗p27-D2所针对的抗原表位位于p27的191aa~206aa,氨基酸序列为191EQTDAAVKNWMTQTLL206。综上所述,本研究成功鉴定了3株抗SHIV p27的单抗体所针对的抗原表位。此外,建立了重组蛋白的原核表达,相关检测以及抗体纯化,抗原/抗体免疫检测等技术平台,为研制检测p27的试剂盒和HIV-1疫苗相关研究提供了物质和技术基础。更多还原

【Abstract】 Chimeric simian/human immunodeficiency virus (SHIV) was developed by replacing the simian immunodeficiency virus (SIV) envelope protein gene (env) and auxiliary genes with corresponding genes from human immunodeficiency virus (HIV-1). SHIV has therefore obtained some biological and immunological characteristics from HIV-1, and SHIV-infected macaques have been applied as non-human primate (NHP) models for studies on basic researches and vaccine development of AIDS.The SHIV p27 capsid protein forms the inside core of the double-shell virions, and is one of the major sources of immunogens of SHIV. Therefore, p27 and p27 antibodies are frequently used to monitor the infection of SHIV in vitro and in vivo for studies on vaccine development, concept of immunization strategies and basic researches of AIDS and HIV-1. It is important to develop monoclonal antibodies against p27 and to identify binding epitopes of p27 antibodies for further studies on the capsid and for the development of detection and diagnostic reagents of SHIV.Three p27 monoclonal antibodies (MAbs), p27-A5, p27-C7 and p27-D2, were developed in a previous study of this laboratory. In this study, these mAbs were firstly concentrated and purified by using a Protein A/Sepharose CL-4B chromatography. The binding sites of these three p27 mAbs were then evaluated by synergic ELISA using different combination of these mAbs as the primary detecting antibody. Results suggested that these three mAbs recognized two different binding sites. To locate these binding sites more accurately, the p27 cDNA was subcloned as three individual fragments with a 15-bp overlapping between each others. These fragments were named as p27-a (1bp-255bp), p27-b (241bp-495bp) and p27-c (481bp-741bp). As for the subcloning, p27 fragments were amplified by PRC using primers with BamH I and Hind III restriction sites at each ends respectively for fragments of p27-a,p27-b and p27-c. All these amplified fragments were cloned into a prokaryotic expression vector pET-32a after being registered by corresponding restriction endonucleases and were checked for correct insertion by restriction enzyme analysis and sequencing.P27 polypeptides were expressed by these vectors and the products were confirmed being able to react with p27 mAbs by Western blot. Based on these results, a set of sixteen 16-residue peptides were designed to cover the whole immunopredominant region of p27, which were overlapped at both ends each other and named as A1-A8 and C1-C8. To express these peptides as fusion proteins with a His-tag, corresponding DNA fragments were synthesized with restriction sites BamH and Xho I at the 5’and 3’ends, respectively, and were subcloned into the expression vector pET32. The resultant clones were accordingly termed as pET-32a-A1 to ET-32a-A8 and pET-32a-C1to pET32a-C8. This set of p27 fusion peptides expressed by these plasmids in E. coli was further checked by SDS-PAGE and Western blot and were used to react with the three p27 mAbs for locating binding sites of these antibodies.The following results were obtained in this study:1. Three anti-p27 monoclonal antibodies were purified and concentrated, which are ready for future experiments and studies.2. It was identified that mAbs p27-A5 and p27-C7 recognized the identical or similar epitope, and p27-D2 bound a different one at a relatively separated location in p27. 3. The detailed binding sites of these three p27 mAbs were further identified, of which mAbs p27-C7 and p27-A5 reacted to the p27 residues 61-76 with the sequence of 61SEGCTPYDINQMLNCV76, and the mAb p27-D2 targeted to residues located at 191-206 with the sequence of 191EQTDAAVKNWMTQTLL206.In conclusion, the reaction epitops of three p27 mAbs in the target protein were identified in this study. Furthermore, several experimental techniques and protocols were established, which include cloning and expression of target gene fragments in E. coli protein detection, antibody purification and detection of antigen/antibody interactions. The immunologically characterized p27 mAbs will be used to develop detection kits for SHIV infection and replication, which are important assays for AIDS related studies.更多还原