【作者】 王茜；

【导师】 曾抗； 周再高；

【作者基本信息】 第一军医大学 ， 皮肤性病学， 2006， 硕士

【摘要】 一、研究意义和目的单纯疱疹病毒(herpes simplex virus，HSV)属于疱疹病毒科a病毒亚科，病毒颗粒大小约180纳米。根据抗原性的差别目前把该病毒分为1型和2型。人群中HSV感染较为普遍，人是疱疹病毒的自然宿主。HSV-2型主要与外生殖器感染及新生儿感染有关，可引起生殖器疱疹、新生儿疱疹。通过医学界近年来多方面的研究表明HSV-2与宫颈癌发生有关，并且增加了AIDS的感染机会。据1992年统计，美国每年有1200多万STD新患者，生殖器疱疹病毒感染近20年增加15倍，累计人数超过2000万，跃升为最流行的STD之一。从STD发展趋势看，病毒和衣原体感染引起的STD比传统的性病显得更为重要，称为第二代STD。如果说以前对传统的性病易于治疗的话，那么对日益剧增的第二代STD病原体，尤其是病毒性STD，其危害性不仅表现在诊断、治疗上困难增大，而且并发症和后遗症也更为严重，直接威胁人类的生命健康。生殖器疱疹的临床表现多种多样，可表现为典型的水疱和糜烂，但不典型和亚临床感染者更为多见，后者易被人们所忽视，成为重要的感染源。目前对于单纯疱疹病毒感染的检测手段有限，血清学方法标准的补体结合技术不能鉴别HSV-1和HSV-2，传统的病毒培养法费时费力。我们旨在建立基于HSV型特异的糖蛋白G(gG)的检测HSV血清抗体的蛋白印迹试验疫吸附试验，为临床中单纯疱疹病毒的感染提供更快、更有效及明确的诊断方法。大量研究表明，HSV病毒颗粒中有至少12种胞膜糖蛋白(glycoprotein g)，gB、gC、gD、gE／gI、gG、gH／gL、gK、gM／gN、gJ。其中gG携带有HSV型特异抗原决定簇，gG-2在单纯疱疹2型病毒诊断、分型、疫苗研究中有举足轻重的地位。gG-2由US4编码，基因片段长度为2100bp，可以编码699个氨基酸的蛋白质。本研究分析了单纯疱疹病毒Ⅰ型糖蛋白G-1和单纯疱疹病毒Ⅱ型糖蛋白G-2的B细胞抗原表位，并用信息学软件比较了两者之间的基因差异。研究发现HSV-2的gG-2基因全长中包括一个与HSV-1的gG-1基因高度同源的区域和一个“独特区”。gG-1基因和gG-2基因在“独特区”中的同源性非常低。对gG-2蛋白的B细胞抗原表位预测发现，在gG-2蛋白中存在7段抗原指数以及表面可能性强的肽段相对集中区域。另有研究者发现，HSV-2的gG-2基因序列抗原区域极少突变，即使出现了点突变也不会削弱gG-2的血清学活性。该报告进一步证实gG-2基因可以作为型特异性抗原，同时指出gG-2基因的保守性是作为HSV-2特异疫苗成分的先决条件。因此，本研究将靶蛋白抗原锁定为gG-2的“独特区”(gG-2_T)。对单纯疱疹病毒gG-2_T进行扩增、克隆。在原核系统中表达，并对表达蛋白的蛋白进行纯化，对其抗原活性进行初步研究。二、研究方法和内容1、从临床确诊病人水疱部位取材、分离病毒并在Vero细胞中培养。2、根据GenBank提供的单纯疱疹病毒Ⅱ型糖蛋白G-2的基因组序列，设计扩增引物，上游的引物序列是“5′GAATTCatggcacgacccacggaagacg-3′”；下游的引物序列是5′-CTCGAG cggggttgcgggtccgg-3′。以Vero细胞病毒培养液上清提取的DNA为模板，扩增gG-2基因，构建克隆载体pGEM-T-G2，通过PCR鉴定。3、从GenBank中下载单纯疱疹病毒1型糖蛋白G-1序列，输入LaserGene软件包中的Editseq软件中，截取gG-1基因片段，翻译成氨基酸序列。利用Clustal X软件将扩增的gG-2基因与下载的gG-1基因片段核苷酸和氨基酸序列进行比较。利用Proetean和DNAMAN软件对单纯疱疹病毒1型和2型的糖蛋白G进行抗原表位、α螺旋中心以及疏水区域进行预测，并比较它们之间的差异4、根据序列分析结果，选定gG-2_T为目的序列。根据单纯疱疹病毒333株gG-2基因序列设计引物，在引物两端加上EcoRⅠ／XhoⅠ限制性内切酶位点。以pGEM-T-G2质粒为模板扩增gG-2_T，得到pGEM-T-gG-2_T重组克隆载体。5、将pGEX-4T-1载体与pGEM-T-gG-2_T分别经过EcoRⅠ／XhoⅠ双酶切后连接，构建融合表达载体pGEX-4T-1-gG-2_T，转化宿主菌BL21，通过双酶切，PCR方法鉴定后测序。6、利用IPTG诱导表达，SDS-PAGE电泳及Western-blot分析对表达蛋白进行初步鉴定。7、优化表达条件。将目的蛋白于上清表达，并使用GST亲和纯化试剂盒进行表达蛋白的纯化。8、利用纯化蛋白与单纯疱疹病毒1型、2型病人血清以及正常人血清进行Western blotting、间接EHSA反应。三、研究结果1、Vero细胞变性，病毒成功地在Vero细胞中扩增。2、提取病毒DNA，扩增单纯疱疹病毒2型gG-2基因，gG-2基因全长2100bp，与单纯疱疹病毒2型333株gG-2基因同源性为99.5％。以gG-2基因为模板，扩增并克隆gG-2_T区，gG-2_T区序列大小为591bp，编码197个氨基酸。3、利用生物信息学软件对Poly-Kyte-Doolittl亲水性、Polt-Emini表面可能性、Jameson-Wolf抗原指数三参数综合预测，结果显示，HSV-2-G2蛋白C端的抗原指数较强。4、通过优化表达条件，pGEX-4T-1-gG-2_T可在大肠杆菌BL21中表达融合蛋白，分子量约为46KD。通过超声裂解，表达的融合蛋白以上清的形式溶解在裂解液中。融合蛋白含有GST标签，可以通过GST亲和纯化柱进行纯化。5、通过调整优化条件，获得纯度较高的重组表达蛋白gG-2_T。蛋白的纯化纯度大于95％。6、纯化的目的蛋白能与GST多克隆抗体发生反应，能与单纯疱疹病毒2型病人血清反应，而不与单纯疱疹病毒1型病人血清发应。故通过实验得到我们需要的目的蛋白。四、研究结论鉴于预防、检测和治疗HSV感染的重要性，研制新型特异性诊断试剂盒以及开发疫苗是目前诊断和预防单纯疱疹病毒感染的最可行有效的方法。前者能快速准确诊断、鉴别初次感染和再次感染、用于大规模HSV感染的流行病筛查，后者能使机体在抗HSV感染免疫中，发挥体液免疫和细胞免疫功能来消除HSV感染。HSV-2包膜糖蛋白gG-2基因全长为2100bp，由US4基因编码699个密码子，包括一个与HSV-1包膜糖蛋白gG-1基因高度同源的区域和一个“独特区”。通过对gG-1蛋白和gG-2蛋白的比对以及B细胞抗原表位的预测我们发现，gG-1基因和gG-2基因在“独特区”中的同源性非常低。Grabowsk等人利用噬菌体展示技术发现两个位于gG-2“独特区”内的免疫显位在氨基酸残基的351-427、525-587内，并通过试验证明这两段氨基酸残基只与HSV-2型单纯疱疹病毒反应而不予HSV-1型单纯疱疹病毒反应。Ikoma等利用杆状病毒表达系统表达gG-2(281aa～594aa)，检测HSV-2型感染病人血清特异性抗体，特异性和敏感性达到95.5％。由此说明这两段区域的肽段适合用作临床抗体检测，尤其适合用作HSV-2分型诊断。研究发现，HSV-2的gG-2基因序列抗原区域极少突变，即使出现了点突变也不会削弱gG-2的血清学活性。该报告进一步证实gG-2基因可以作为型特异性抗原，同时指出gG-2基因的保守性是作为HSV-2特异疫苗成分的先决条件。本文采用分子克隆及基因重组技术成功构建出HSV-2型gG-2_T(285～482aa)基因片段，利用原核表达载体表达出含有gG-2_T的融合蛋白，通过Western-Blot免疫印迹检测表达蛋白能与GST多克隆抗体特异性结合。纯化的蛋白即为我们需要的目的蛋白。间接ELISA证明表达的蛋白能够与HSV-2阳性病人反应而不与正常人血清和HSV-1病人血清反应，表明原核表达的蛋白具有一定的免疫学活性。以上研究为单纯疱疹病毒2型感染的型特异性诊断试剂盒的研发，生殖器疱疹筛查、型特异性疫苗研究寻找更好的靶位点提供参考。更多还原

【Abstract】 Significance and ObjectiveHerpes simplex virus (HSV) belongs to Herpesviridae a subtype, its plasmid is 180 nanometers. According to antigenic difference, the virus has type land type 2. HSV infection is widespread in the crowd, because people are only physio-host of herpes simplex virus. Herpes simplex virus type 2 mainly concerned with infection of anoperineogenital and newborn, and it can induce genital herpes and newborn infant herpes. By medical science study for many years, HSV infection is related with uterine cervix cancer in woman patients, and increases chance of AIDS infection. Disease incidence of genital herpes is second only to gonorrhea and syphilis, occupying NO. 3, and is the first in sexually transmitted disease caused by virus. Genital herpes clinical manifestation is multiformity, it possibly is typical blister and anabrosis, but non-typical and subclinical infection is more visible which is easily ignored by people. Therefore today medical science workers think highly of the genital herpes again. Now, Test facility of herpes simplex virus infection is limited, for example that complement fixation of serology classic method can not identify HSV-1 and HSV-2. Orthodox virus cultivation is time-consuming and exhausted. So in our study we aim at founding Herpeselect HSV-2 enzyme-linked immunosorbent assay (ELISA) and Western blot on account of type-specific HSV-2 glycoprotein G to detect HSV serum antibody, and these methods will be fast and effective diagnostic method.Many study and researches manifest that HSV viral particle has at least 12 glycoprotein, these are gB、gC、gD、gE/gI、gG、gH/gL、gK、gM/gN、gJ. Among the total, gG take along type-specific antigen determinant, gG-2 takes a big role in diagnosis, typing and vaccine study of herpes simplex virus type 2. US4 encode gG-2, and gG-2 gene fragment length is about 2100bp, can encode 699 amino acids. We analyze B cell antigen epitope between gG-1 and gG-2 of HSV, and compare their gene difference. Gene total length of HSV-2 gG-2 has high congelleric domains with HSV-1 gG-1 and unique domain. There is very low homology in unique domain between gG-1 gene and gG-2 gene. There are 7 antigen index number and surface possible fortis peptide section relative concentrate district. Other researchers find that gene antigen district of HSV-2 gG-2 happen mutation seldom. Even if gene mutation appears, that also can not weaken serology activity. They also confirm that gG-2 gene can be good type-specific antigen, and gG-2 gene’s conservatism is prerequisite of HSV-2 idio-vaccine, thus, we make unique domain of gG-2 gene as target protein antigen, amplifying and cloning it, then expressing it in prokaryotic system and cleansing, at last, we get antigen activity of interest protein studied initially.Method and Content1、Drawing and separating the materials from blister, which patients are clinical definite as HSV-2, and cultivating virus in Vero cell.2、According to genome series of HSV-2 glycoprotein G provided from GenBank, to design amplification primer, the upper stream primer series is "5’- GAATTCatggcacgacccacggaagacg -3’"; The down stream primer series is 5’- CTCGAG cggggttgcgggtccgg -3’. Making DNA extracted from supernatant of virus culture fluid, we amplify gG-2 gene and construct cloning vector pGEM-T-G2, utilizing PCR to identify it.3、Rrecording HSV-1 glycoprotein G-1 series from GenBank to input Editseq software of the LaserGene package. Cutting gG-1 gene fragment and translating it into amino acid sequence. To utilize Clustal X software, we compare nucleotide and amino acid sequence between amplified gG-2 gene and cut gG-2 gene. To utilize Proetean and DNAMAN software, we also make a forecast about epitope,αcoiling center and hydrophobic region, and compare difference of them.4、According to sequence analysis results, we select unique domain (gG-2_T) of gG-2 gene as purpose order. On the basis of gene order of herpes simplex virus 333 stock, we design its primer and add EcoRⅠ/XhoⅠrestriction enzyme situs in both side primer. We make pGEM-T-G2 plasmid formwork to amplify gG-2_T, getting pGEM-T-gG-2_T recombinant cloning vector.5、Connecting pGEX-4T-1 carrier and pGEM-T-gG-2_T after they are both enz-cut by EcoRⅠ/XhoⅠto construct fusion expression vector pGEX-4T-1-gG-2_T to convert it into parasitifer BL21. By enz-cutting and PCR, they will be accredited and sequenced.6、We induce to express pGEX-4T-1-gG-2_T by utilizing IPTG and initially accredit express protein by SDS-PAGE electrophoresis and Western blot.7、We optimize expression condition and express interest protein in supernatant and purify expression protein by using GST affinity purification kit.8、We make Western-blot and indirect ELISA by utilizing pure protein to respectively detect serums of HSV-1, HSV-2 and health adult.Finding1、Vero cell degeneration, the virus amplify in Vero cell successfully.2、Extracting virus DNA, amplifying herpes simplex virus type 2 gG-2 gene, gG-2 gene length is about 2100bp, the homology is 99.45% between it and gG-2 gene of herpes simplex virus type 2333 stock. Making gG-2 gene as template to amplify and clone gG-2_T, series length of gG-2_T is 591bp, encoding 197 amino acids.3、We synthetically predict glycoprotein G of HSV-1 and HSV-2 according to Poly-Kyte-Doolittl hydrophilicity, Polt-Emini surface possibility and Jameson-Wolf antigen index number by bioinformatics software analysis.4、We optimize expression condition to express pGEX-4T-1-gG-2_T into fusion protein in Escherichia coli BL21, which protein molecular mass is about 46KD. By supersound spallation, fusion protein is dissolved in lysate as supernatant. Fusion protein has GST label, can pass GST affinity purification pillar to be purified.5、By setting condition optimized, we reduce protein polluted effectively. Content of purification protein is GST 0.85mg/ml and HSV2-G-2 unique domain 0.49mg/ml. purity coefficient of interest protein exceeds 95%.6、Interest protein purified take reaction with GST polyclonal antibody, also make reaction with patients serum of herpes simplex virus type 2, but not react with HSV-1 patients serum. So we get interest protein that we need.ConclusionManufacturing new type specificity diagnostic kit and developing vaccine are best effective method to diagnose and prevent herpes simplex virus infection now respecting significance of preventing, detecting and treating HSV infection. The former can diagnose and identify primary infection and reinfection, at the same time can make large scale epidemic screening. The latter can bring into full play humoral immunity and cellular immune function to wipe out HSV infection in reject HSV infection immunity. HSV-2 gG-2 gene fragment length is about 2100bp, can encoded 699 codons by US4, including a unique domain and a height congelleric area with HSV-1 envelope glycoprotein gG-1. By comparing gG-1protein and gG-2 protein and making a forecast to B cell epitope of it, we find there is very low homology in unique domain between gG-1 gene and gG-2 gene. Grabowsk found tow immunizing sites in amino acid residue 351～427、525～587 in gG-2 "unique domain" by bacterial invader technique. He also authenticated the two amino acid residue just react with HSV-2 not HSV-1 in test. Ikoma expressed gG-2 (281aa-594aa) by using granulosis virus expression system, and got interest protein to detect HSV-2 infection patiens serological specific antibody, the specificity and sensitivity is 95.5%. The study illustrated that the tow district peptides are fit for clinical antibody detection, especially fitting for typing diagnosis.A lot of study and research find that gene antigen district of HSV-2 gG-2 happen mutation seldom. Even if gene mutation appears, that also can not weaken serology activity. This report also confirmed that gG-2 gene can be typical specific antigen, and conservatism of gG-2 gene is the prerequisite as HSV-2 specific vaccine.We construct HSV-2 gG-2 unique domain gene fragment (285～482aa) successfully by molecular cloning and gene recombination technology, and express fusion protein included gG-2 unique domain by using prokaryotic expression vector, then detect expression protein can specifically bind with GST polyclonal antibody by Western blot. Purified protein is our needed interest protein. Indirect ELLISA proves expressed protein can take reaction with serum of HSV-2 infection patiens, not r-Globulin and HSV-1 patiens, which manifest prokaryotic expression protein has immunologic competence. Above study provides information for inventing type specific diagnosis kit of HSV-2 infection, genital herpes seroepidemiological survey and type specific vaccine study.更多还原