【作者】 姜永玮；

【导师】 李明；

【作者基本信息】 南方医科大学 ， 免疫学， 2007， 硕士

【摘要】 人巨细胞病毒(Human Cytomegalovirus，HCMV)为疱疹病毒科β属的双螺旋DNA病毒，在世界范围内分布。HCMV不仅是人类先天性病毒感染的主要病原，而且是免疫功能低下人群(艾滋病患者、器官移植者、免疫抑制治疗者等)并发感染死亡的常见原因之一。由于HCMV感染者通常无症状或无特异性症状，建立HCMV快速、敏感、特异的实验室检测方法并采取早期治疗措施对优生、优育及阻止HCMV的传播具有重要意义。HCMV感染的检测方法包括病毒分离培养、核酸测定、抗原检测和血清学检测等，每种方法各有优缺点。HCMV原发感染的检测，主要采用血清学方法。HCMVIgM是机体受病毒抗原刺激后最早产生的抗体，是活动性HCMV感染的重要指标之一，因而是检测HCMV感染的常用指标。目前国内存在大量检测血清HCMV IgM的试剂盒，多使用从HCMV感染的人胚肺二倍体细胞中粗提取的完整病毒抗原，各种检测方法对相同血清的检测结果有很大差别，即是符合率较差。其主要原因为：1．病毒抗原的制备及纯化方法各不相同；2．提取物中含有多种抗原成分，很难进行标准化：3．制备抗原所需的HCMV要在人胚肺细胞中培养，此细胞的蛋白可能对制备的抗原产生污染；4．疱疹病毒有共同抗原，完整的HCMV抗原可以与其它非HCMV病毒感染产生的抗体发生交叉反应。由于病毒来源的抗原存在敏感性、特异性、标准化等问题，将通过基因工程方法获得的表达产物作为检测用抗原已引起各国学者重视。国内外研究工作者对人巨细胸病毒的一些抗原决定簇进行了免疫原性和特异性研究，期待能够应用于人巨细胞病毒感染的早期诊断。其中gp52(UL44)、pp150(ppUL32)、pp65(ppUL83)、ppUL80a均为强免疫原性蛋白且具有保守的DNA序列，能够特异性识别HCMV IgM抗体。因此，本研究选择HCMV gp52(UL44)、pp150(ppUL32)、pp65(ppUL83)、ppUL80a抗原性好、特异性强的部分基因片段作为目的基因，应用基因工程技术，得到高效表达的目的工程菌，并对表达产物进行了抗原性研究。此次研究从HCMV感染的细胞上清液中提取HCMV基因组，本试验用PCR方法从基因组中扩增获得pp150 aa595-614(A)、pp150 aa1006-1042(B)、gp52aa202-434(C)、pp65 aa297-510(D)、ppUL80a aa117-373(E)氨基酸之间的多肽片段的编码基因，用重组PCR的方法将前三个片段重组为新的DNA片段(F)，将D、E、F片段分别克隆至pMD-18T载体测序，核苷酸序列分析证明了PCR扩增目的序列的正确性。测序正确的片段克隆至原核表达载体pGEx-4T-1。构建重组质粒，转化大肠杆菌BL21，经IPTG诱导后成功表达了上述三种蛋白，分别命名为GST-ppUL83、GST-ppUL80a、GST-LINK。通过Western blotting检测其免疫特性，结果显示三种蛋白均能被羊抗人巨细胞病毒多克隆抗体及HOMV IgM阳性血清特异性识别，而载体蛋白与多克隆抗体及阳性血清并不能发生反应，证明这三种蛋白具有良好的抗原特异性，可期待应用于人巨细胞病毒感染的诊断。经蛋白电泳分析表达产物主要以包涵体的形式存在，根据这三种蛋白不同的理化特性，采用不同的方法进行蛋白纯化，获得了较好的纯化效果。时间分辨荧光免疫分析(Time-resolred fluoroimmunoassay，TRFIA)是上世纪八十年代兴起的一种新型非放射性标记技术，TRFIA巧妙利用镧系元素的荧光特点，克服了普通荧光检测中背景荧光强度大、干扰强的缺点，使强特异性荧光和背景荧光分辨开，几乎可以完全消除背景荧光的干扰，提高了检测的灵敏度。与现在广泛使用的放射免疫分析(RIA)、酶免疫分析(EIA)、化学发光免疫分析(CLIA)、电化学发光免疫分析(ECLIA)相比，具有灵敏度高、操作简便、示踪物稳定、标准曲线范围宽、不受样品自然荧光干扰、无放射性污染、对标记物分子生物活性影响小、多标记等优点，现已成为生物学研究和临床超微量生化检验中一项颇有发展前景的分析手段。为分析本实验所表达纯化的三种目的蛋白与HCMV IgM的反应能力，将目的蛋白及Microbix Biosystems公司的完整病毒裂解抗原CMV grade2 antigen，运用捕获法TRFIA检测已知HCMV IgM阳性及阴性的血清，两种反应结果进行直线相关分析。分析结果显示，三种目的蛋白单独应用检测血清的结果与完整裂解抗原的检测结果呈正相关，但效果不甚理想(r_s分别为0.947，0.879，0.957)。将三种目的蛋白联合应用检测血清的结果与完整裂解抗原的结果具有良好的相关性，r_s=0.989，P＜0.001，效果明显好于融合蛋白单独应用。其主要原因是虽然本实验所选择gp52、pp150、pp65、ppUL80a均为强免疫原性蛋白且具有保守的DNA序列，对HCMV IgM抗体具有一定的敏感性和特异性，但其可能只与HCMV感染的不同阶段机体产生的特异性IgM发生反应。将三者联合应用，检测效果与完整病毒裂解抗原的检测效果呈良好的正相关关系，说明目的蛋白联合应用与完整裂解抗原之间的相关关系更加密切，在检测血清HCMV IgM方面能更好地代表完整裂解抗原的作用。将三种目的蛋白按最佳比例应用检测100份随机血清，其结果与国内外有代表性的试剂盒检测结果进行比较，计算吻合度测量系数(κ)，吻合程度较高(国外κ=0.905，国内κ=0.701)。更多还原

【Abstract】 Human cytomegalovirus (HCMV) ,a beta herpesvirus,which has duplex DNA structure ,is ubiquitously distributed in human populations.Although rarely pathogenic in immunocompetent individuals,the virus poses a significant cause of morbidity and mortality in organ allograft ,AIDS patients, immunosuppression patients and so on.Pregnant women are also a risk group for this virus as HCMV is the most common cause of congenital infection.Sience the infections with HCMV either are asymptomatic or are accompanied by symptoms not specific for HCMV(such as fever and leucopenia),laboratory techniques are the sole means of diagnosing HCMV infection. So it has significant sense to build up a fast, sensitive and specific laboratory method for the diagnosis of HCMV, then doctors can take therapeutic measures in the early period of the infection which have positive meaning in aristogenesis and preventing the virus’s conveying.Diagnosis of HCMV infection can be obtained by direct demonstration of the virus or virus components in pathological materials or indirectly through serology. The most direct methods include culture of the virus or detection of viral components ,like viral DNA,RNA and antigens in body fluids or tissue biopsy specimens.Diagnosis of primary HCMV infection is exclusively accomplished by serological methods.Serologic assays are widely used for donor selection and to support the diagnosis of HCMV in the host and to determine whether it is an active or latent infection.Although it indirectly reflects viral activity ,serology provides a cheap alternative method that can readily be automated for routine use.HCMV-specific immunoglobulin M(IgM) is a sensitive and specific indicator of primary HCMV infection in immunocompetent subjects,while it is very often produced during viral reactivation in transplant recipients, ,AIDS patients and so on.In current serologic assays complex viral lysates are commonly used.However, detection of HCMV-specific IgMvaries widely,and poor agreement has been found among the results obtained with different commercially available kits.The use of these viral lysates has disadvantages because they consist of many viral antigens whose exact compositions are difficult to standardize.Preparation of lysates requires culture of HCMV in fibroblasts,resulting in potential contamination with cellular proteins. Since many transplant recipients may temporarily develop autoantibody responses ,a false-positive reactivity may result.Another problem can arise,since herpesviruses share multiple protein homologues,which can give rise to cross-reactivity in assays based on complex viral lysates.In order to overcome these problems the viral lysate should be replaced by a defined antigen preparation,preferably consisting of a combination of HCMV-specific and immunodominant antigens,in order to achieve the highest sensitivity and specificity.Recombinant proteins from genetic engineering can be used to detect antibodies to the parent protein.Combinations of such defined immunodominant proteins or peptides may ideally be suited as replacements for complex protein-antigen mixtures. Some antigenic determinants were investigated to seek immunogenic and protective for potential diagnosis.gp52(UL44)、pp150 (ppUL32)、pp65 (ppUL83)、ppUL80a are all immunogenic antigen determinants with most conservative DNA sequences,and can recognize the HCMV-IgM in the serum specifically.In this study,these five antigenic determinant genes(ppl50 aa595-6149 (A) ,ppl50 aa 1006～1042 (B),gp52 aa202～434 (C), pp65 aa297～510 (D) ,ppUL80a aa117～373 (E)) have been amplified successfully from HCMV genome and then the first three fractions were recombinated into a new fraction(F) by Overlap PCR.Then the three fractions(D、E、F)were cloned into a cloning vector,and the DNA sequencing proved that the fractions amplified by PC R are totally correct.The correct gene fragments were cloned into a prokaryotic expression vector to generate fusion proteins named GST- ppUL83, GST- ppUL80a, GST-LINK respectively, which were induced by IPTG and well expressed in E coli.The HCMV immuno-specificity of these three fusion protein weredetected by western-blotting.All three recombination proteins could be specially recognized by HCMV IgM sera,however the vector protein GST could not react with the infected sera,indicating the ideal antigenicity of the three recombination protein and the application of the HCMV diagnosis.According to the different bio-characteristic,different purification methods were used to acquire purified antigen,and the three antigens had been purified successfully. Time- resolved fluoroimmunoassay(TRF1A) is a kind of new non- radio marked technique rised from 80’s last century. It makes use of the special fluorescence characteristic of the lanthanum series and their chelate to mark antigens,antibodies,probes of nucleic acid and so on.After the reaction system(for example:antigen-antibody reaction,hybridization of nucleic acid probes) occures ,determinant the fluorescence intensity of the reaction product by the TRFIA tester. According to the specific value of the outcome fluorescence intensity and the opposing fluorescence intensity, we can decide the concentration of the analyte in the reaction system. The TRF1A dexterously utilize the special fluorescence characteristics of the lanthanum series :first,the decay time of the fluorescence of the lanthanum series’ chelate is very long, which is 10~3-10~6 times of traditional fluorescences ;second:The Stokes bias between the excitation light and the emission light can amount to 290 nm, while the common fluorescences’ Stokes bias is only 28 nm. Thus it overcomes the weakness of the common fluorescence examination :strong intensity of the background fluorescence and interference, thusdistinguish the strong particularity fluorescence and the background fluorescences .It can almostly remove the interference of the background fluorescence, raising the sensitivity of the examination. Compared with the common emanation such as radioimmunoassay(RIA),enzyme-immunoassay(EIA),chemiluminescence immune assay(CLIA) ,the TRFIA has some advantages ,for example,the high sensitivity,the simple operation process,the stabilization of the tracer, the wide extent of the standard curve,no radioaction contaminate,the little interference in the bioactivity of the marked mass,and so on.Now,TRFIA is one of the excellent analysis methods in biological investigation and clinical ultramicro-examination of biochemistry.To check whether the three pured purpose proteins contain the antigenic determinant that can react specially with the HCMV lgM in the sera,we make use of the purpose proteins and the complete virus antigen of schizolysis(CMV grade2 antigen of Microbix Biosystems company), utilizing the TRFIA method to detect the serums which we have already known the positive or negative of HCMV IgM, then the results were analyzed by Linear Correlation. The analysis result demonstrat that the result of the three purpose proteins used prospectively has positive correlation with the result of CMV grade2 antigen, but the result is not very well.While the result of the three purpose proteins used together has fine positive correlation with the result of CMV grade2 antigen, r_s=0.989. The main reason may be although the proteins we chosed in this experiment:gp52, pp150, pp65, ppUL80a are all proteins with strong immunogenicity, conservative sequence of DNA and have the ability to react with HCMV IgM sensitivity and specificity, they can only react with HCMV IgM of particular stage of HCMV infection. The analysis of the humoral immune response elicited during natural infection has repeatedly shown that the basic phosphoprotein of 150 KDa encoded by UL32(ppUL32) and localized in the viral tegument is highly immunlgenic and is recognized by sera from nearly 100% of the HCMV-seropositive subjects tested.In this molecule at least two epitopes have been identified and shown to react efficiently with human immunolobulins. In particular, the analysis of several ppUL32 fusion proteins showed that a region localized in the last 43 amino acids at the carboxy terminus of the molecule(amino acids 1006 to 1048)reacts with more than 80% IgM-positive serum samples. When chemically synthesized, antother region localized between anino acids 595 and 614 gave a positive reaction with almost 100%IgG-positive serum samples. When it is expressed as recombinant protein,this region also reacted efficiently with HCMV=specific IgM.The two coding regions were recently fused together and were shown to produce a double-epitope fusion protein which can replace the entire p150 molecule in its lgM-binding ability. Another HCMV protein which reacts very well with IgM is ppUL44, the nonstructural DNA-binding protein of 52 kDa. The carboxy-terminal part of th molecule was chosen because it does not contain relevant amino acid sequences cross-reacting with the homologous protein of other members of the Herpesviridae family. The carboxy-terminal half of this protein was expressed and tested with human sera alone and in combination with the double-ppUL32 epitope fusion protein. Besides ppUL32 and ppUL44, two other HCMV proteins were chosen:the major matrix protein ppUL83 and the assembly protein(ppUL80a). The first is known to induce a strong IgM response, and antibodies reacting exclusively to this protein were described during primary infection. The second protein was included in the study because in HCMV-seropositive transplant recipients undergoing viral reactivation, IgM to this protein is very often the first marker to be detected. Use the three proteins together to detect the serums which we have already known the positive or negative of HCMV IgM, the result has fine positive correlation with the result of CMV grade2 antigen,which demonstrate that the chosen proteins contains the antigenic determinants that can react sensitively and specifically with the HCMV IgM. Use the three proteins together according to the optimum to check 100 random sera, this result compared with the results of commercial kits from internal and abroad having excellent efficiency, didn’t show significant difference.更多还原