本文關(guān)鍵詞： 人類皰疹病毒6型 U94 細(xì)胞病變效應(yīng) 套式聚合酶鏈反應(yīng) 序列分析 重組基因 表達(dá) 血清學(xué)檢測 多克隆抗體　出處：《南京醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 人類皰疹病毒6型(Human HerpesvirUS 6，HHV-6)屬于皰疹病毒β亞科，最初分離于淋巴增生和免疫抑制的病人，對CD4~+的T淋巴細(xì)胞具有親嗜性。HH-6是引起嬰幼兒急疹(exanthem subitum，ES)的病原，初次感染后，HHV-6可持續(xù)存在于單核巨噬細(xì)胞中，在機(jī)體免疫低下時可被反復(fù)激活。HHV-6與多種臨床疾病相關(guān)，如腦膜腦炎、傳染性單核細(xì)胞增多癥、慢性淋巴結(jié)病、暴發(fā)性肝炎、自身免疫性疾病、慢性疲勞綜合癥、多發(fā)性硬化癥、器官移植及骨髓移后的移植物抗宿主反應(yīng)。 HHV-6可分為A、B二個亞型，二者具有相似的基因結(jié)構(gòu)，全長161kbp含有112個開放讀碼框。U94 ORF是HHV-6獨(dú)特于其他皰疹病毒的序列，在IE期表達(dá)，其編碼產(chǎn)物RepH6與腺病毒伴隨病毒AAV-2rep的基因產(chǎn)物Rep68／78類似。RepH6在病毒DNA復(fù)制、基因調(diào)節(jié)及阻止細(xì)胞間轉(zhuǎn)化和抑制由HIV-1長末端重復(fù)序列(longterminalrepeat，LTR)引發(fā)的轉(zhuǎn)錄中起作用。穩(wěn)定轉(zhuǎn)染了U94 ORF基因的淋巴細(xì)胞對HHV-6易感，但HHV-6的復(fù)制受限。U94 mRNA是在正常人PBMC中唯一被檢出的UHV-6轉(zhuǎn)錄本，提示它的表達(dá)可能與控制病毒潛伏及增殖性感染相關(guān)。U94基因產(chǎn)物能建立和維持HHV-6在細(xì)胞中的潛伏感染。在HHV-6的裂解性復(fù)制階段U94低水平表達(dá)，而在HHV-6潛伏狀態(tài)下它是一主要轉(zhuǎn)錄本。 本研究用PCR方法擴(kuò)增U94 ORF并將其克隆至質(zhì)粒pGEX-6p-1，構(gòu)建原核表達(dá)質(zhì)粒pGEX-6p-1-U94，轉(zhuǎn)化大腸桿菌進(jìn)行表達(dá)并純化；以純化的蛋白為抗原免疫新西蘭兔制備多克隆抗體并鑒定；初步應(yīng)用純化的蛋白來進(jìn)行血清學(xué)檢測。主要研究內(nèi)容和結(jié)果如下： 一、HHV-6的培養(yǎng)、鑒定 HHV-6 GS標(biāo)準(zhǔn)株接種于PHA預(yù)刺激的人臍帶血單個核細(xì)胞(CBMCs)培養(yǎng)，10～12天時，鏡檢出現(xiàn)明顯細(xì)胞病變效應(yīng)(CPE)，以培養(yǎng)細(xì)胞的核酸為模板，通過特異性引物，套式PCR擴(kuò)增得到287bp的外引物擴(kuò)增產(chǎn)物及163bp的內(nèi)引物擴(kuò)增產(chǎn)物；內(nèi)引物擴(kuò)增產(chǎn)物不能被HindⅢ單酶切；內(nèi)外引物擴(kuò)增產(chǎn)物的測序結(jié)果分別與Genbank中HHV-6A相應(yīng)序列比對，同源性為100％。 二、HHV-6 U94 ORF的克隆、表達(dá)和純化 本研究通過PCR技術(shù)擴(kuò)增了U94 ORF，應(yīng)用基因重組技術(shù)構(gòu)建含有U94 ORF的重組質(zhì)粒pGEX-6p-1-U94，并在大腸桿菌中進(jìn)行了誘導(dǎo)表達(dá)，同時還進(jìn)行了IPTG最佳誘導(dǎo)濃度與最佳誘導(dǎo)時間的篩選試驗(yàn)，確定IPTG的最佳濃度為1mmol／L，最佳誘導(dǎo)培養(yǎng)時間為5h。之后應(yīng)用親和層析方法對表達(dá)蛋白進(jìn)行了純化，紫外分光光度計結(jié)果表明純化蛋白含量為0.1mg／ml。重組質(zhì)粒經(jīng)PCR、酶切鑒定、核酸序列測定和分析后轉(zhuǎn)化大腸桿菌(E.coli)Rosatta，誘導(dǎo)表達(dá)融合蛋白，表達(dá)的重組蛋白經(jīng)GST柱親和層析純化，SDS-PAGE和Western blot結(jié)果顯示表達(dá)的蛋白分子量約為40 Kd。以純化的蛋白作為抗原，用ELISA法檢測不同人群血清中的抗體，，結(jié)果顯示免疫抑制人群與健康對照人群在抗體陽性率方面存在顯著統(tǒng)計學(xué)差異，陽性率分別為87.5％和53.3％(P＜0.01)，而神經(jīng)膠質(zhì)瘤病人與健康對照人群在抗體陽性率方面無顯著統(tǒng)計學(xué)差異，陽性率分別為56.3％和53.3％(P＞0.05)，提示免疫系統(tǒng)于自然狀態(tài)下即對HHV-6發(fā)生免疫應(yīng)答，免疫抑制病人強(qiáng)度更高。 三、RepH6蛋白多克隆抗體的制備與鑒定 用重組RepH6蛋白接種新西蘭兔，制備抗RepH6蛋白多克隆抗體，經(jīng)過基礎(chǔ)免疫和加強(qiáng)免疫后，分離免疫血清，得到血清后，以飽和硫酸銨沉淀法(SAS)純化，ELISA檢測結(jié)果表明，血清抗體效價在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:.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392;R446.6

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