人类疱疹病毒6型U94 ORF在大肠杆菌中的表达、纯化、多克隆抗体的制备及其在血清学检测中的应用

发布时间：2018-01-19 15:31

本文关键词： 人类疱疹病毒6型 U94 细胞病变效应套式聚合酶链反应序列分析重组基因表达血清学检测多克隆抗体　出处：《南京医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 人类疱疹病毒6型(Human HerpesvirUS 6，HHV-6)属于疱疹病毒β亚科，最初分离于淋巴增生和免疫抑制的病人，对CD4~+的T淋巴细胞具有亲嗜性。HH-6是引起婴幼儿急疹(exanthem subitum，ES)的病原，初次感染后，HHV-6可持续存在于单核巨噬细胞中，在机体免疫低下时可被反复激活。HHV-6与多种临床疾病相关，如脑膜脑炎、传染性单核细胞增多症、慢性淋巴结病、暴发性肝炎、自身免疫性疾病、慢性疲劳综合症、多发性硬化症、器官移植及骨髓移后的移植物抗宿主反应。 HHV-6可分为A、B二个亚型，二者具有相似的基因结构，全长161kbp含有112个开放读码框。U94 ORF是HHV-6独特于其他疱疹病毒的序列，在IE期表达，其编码产物RepH6与腺病毒伴随病毒AAV-2rep的基因产物Rep68／78类似。RepH6在病毒DNA复制、基因调节及阻止细胞间转化和抑制由HIV-1长末端重复序列(longterminalrepeat，LTR)引发的转录中起作用。稳定转染了U94 ORF基因的淋巴细胞对HHV-6易感，但HHV-6的复制受限。U94 mRNA是在正常人PBMC中唯一被检出的UHV-6转录本，提示它的表达可能与控制病毒潜伏及增殖性感染相关。U94基因产物能建立和维持HHV-6在细胞中的潜伏感染。在HHV-6的裂解性复制阶段U94低水平表达，而在HHV-6潜伏状态下它是一主要转录本。本研究用PCR方法扩增U94 ORF并将其克隆至质粒pGEX-6p-1，构建原核表达质粒pGEX-6p-1-U94，转化大肠杆菌进行表达并纯化；以纯化的蛋白为抗原免疫新西兰兔制备多克隆抗体并鉴定；初步应用纯化的蛋白来进行血清学检测。主要研究内容和结果如下：一、HHV-6的培养、鉴定 HHV-6 GS标准株接种于PHA预刺激的人脐带血单个核细胞(CBMCs)培养，10～12天时，镜检出现明显细胞病变效应(CPE)，以培养细胞的核酸为模板，通过特异性引物，套式PCR扩增得到287bp的外引物扩增产物及163bp的内引物扩增产物；内引物扩增产物不能被HindⅢ单酶切；内外引物扩增产物的测序结果分别与Genbank中HHV-6A相应序列比对，同源性为100％。二、HHV-6 U94 ORF的克隆、表达和纯化本研究通过PCR技术扩增了U94 ORF，应用基因重组技术构建含有U94 ORF的重组质粒pGEX-6p-1-U94，并在大肠杆菌中进行了诱导表达，同时还进行了IPTG最佳诱导浓度与最佳诱导时间的筛选试验，确定IPTG的最佳浓度为1mmol／L，最佳诱导培养时间为5h。之后应用亲和层析方法对表达蛋白进行了纯化，紫外分光光度计结果表明纯化蛋白含量为0.1mg／ml。重组质粒经PCR、酶切鉴定、核酸序列测定和分析后转化大肠杆菌(E.coli)Rosatta，诱导表达融合蛋白，表达的重组蛋白经GST柱亲和层析纯化，SDS-PAGE和Western blot结果显示表达的蛋白分子量约为40 Kd。以纯化的蛋白作为抗原，用ELISA法检测不同人群血清中的抗体，，结果显示免疫抑制人群与健康对照人群在抗体阳性率方面存在显著统计学差异，阳性率分别为87.5％和53.3％(P＜0.01)，而神经胶质瘤病人与健康对照人群在抗体阳性率方面无显著统计学差异，阳性率分别为56.3％和53.3％(P＞0.05)，提示免疫系统于自然状态下即对HHV-6发生免疫应答，免疫抑制病人强度更高。三、RepH6蛋白多克隆抗体的制备与鉴定用重组RepH6蛋白接种新西兰兔，制备抗RepH6蛋白多克隆抗体，经过基础免疫和加强免疫后，分离免疫血清，得到血清后，以饱和硫酸铵沉淀法(SAS)纯化，ELISA检测结果表明，血清抗体效价在1：10000以上。
[Abstract]:Human herpes virus type 6 (Human HerpesvirUS 6, HHV-6) belongs to the beta herpesvirus subfamily, was originally isolated from lymphoid hyperplasia and immunosuppressive patients of CD4~+ T lymphocytes with ecotropic.HH-6 is caused by the infant (exanthem subitum, ES) for emergency treatment of the pathogen infection after initial HHV-6, sustainable exist in macrophages in the low in immunity can be repeated activation of.HHV-6 is related to many diseases, such as encephalitis, infectious mononucleosis, chronic lymph node disease, fulminant hepatitis, autoimmune disease, chronic fatigue syndrome, multiple sclerosis, organ transplantation and bone marrow transplantation after graft-versus-host the reaction.
HHV-6 can be divided into A, B two subtypes, two have similar gene structure, the full-length 161kbp contains 112 open reading frames.U94 ORF HHV-6 sequence is unique from the rest of the herpes simplex virus, expressed in the IE phase, copy the RepH6 encoding product and adeno-associated virus AAV-2rep gene product Rep68 / 78 similar.RepH6 in the DNA virus, gene regulation and prevent cell transformation and inhibition by HIV-1 long terminal repeat (longterminalrepeat, LTR) plays a role in transcription by stable transfection of ORF gene. U94 lymphocytes susceptible to HHV-6, but HHV-6.U94 mRNA is the only restricted replication was detected UHV-6 transcripts in normal human PBMC in May indicated that the expression and control of virus latent infection and proliferation related.U94 gene products can establish and maintain HHV-6 cells in the latent infection. The HHV-6 lytic replication stage U94 low expression in HHV-6 It is a major transcript under the latent state.
In this study, U94 ORF was amplified by PCR and cloned into plasmid pGEX-6p-1 to construct prokaryotic expression plasmid pGEX-6p-1-U94 and transformed into Escherichia coli for expression and purification; using the purified protein as antigen to immunize New Zealand rabbits to prepare polyclonal antibody and identification of purified protein; preliminary application of serological detection. The main research contents and results the following:
1. Culture and identification of HHV-6
HHV-6 GS standard strains were inoculated on PHA pre stimulation of human umbilical cord blood mononuclear cells (CBMCs) cultured for 10 ~ 12 days, microscopic examination of significant cytopathic effects (CPE) in cultured cells, DNA as template, the specific primers and nested PCR amplification primers in 287bp outer primers amplified and 163bp amplification products; inner primers amplified products cannot be Hind III single enzyme digestion and sequencing of amplified products of primer; Genbank HHV-6A results were compared with the corresponding sequences, the homology was 100%.
Cloning, expression and purification of two, HHV-6 U94 ORF
In this study, U94 ORF was amplified by PCR technique, the recombinant plasmid pGEX-6p-1-U94 containing U94 ORF by gene recombination technique and expressed in Escherichia coli, but also the best IPTG induced screening test concentration and the best induction time, to determine the optimal concentration of IPTG was 1mmol / L, the best induction time for 5h. after the protein was purified by affinity chromatography method, the results showed that the purified protein was 0.1mg / ml. recombinant plasmid PCR by UV spectrophotometry, enzyme digestion, DNA sequencing and analysis after transformation of Escherichia coli (E.coli) Rosatta, induce the expression of the fusion protein, recombinant protein expression by GST column affinity chromatography, SDS-PAGE and Western blot showed that the molecular weight of the expressed protein was about 40 Kd. with the purified protein as antigen, the antibody detected by ELISA in sera of different people, The results showed that immunosuppressive patients and healthy volunteers. There was significant difference in the positive rate of antibody positive rates were 87.5% and 53.3% (P < 0.01), and glioma patients and healthy controls in the positive rate of antibody had no significant difference. The positive rates were 56.3% and 53.3% (P > 0.05). Indicating that the immune system in the natural state of the HHV-6 immune response, immune suppression in patients with higher strength.
Three, preparation and identification of RepH6 protein polyclonal antibody
The recombinant RepH6 protein was used to inoculate New Zealand rabbits, and the polyclonal antibody against RepH6 protein was prepared. After immunization and immunization, the immune serum was separated and the serum was purified by saturated ammonium sulfate precipitation (SAS). The results of ELISA showed that the titer of serum antibody was above 10000 1:.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;R446.6

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