不同基因型戊型肝炎病毒的抗原表位分析

目的 研究不同基因型戊型肝炎病毒(HEV)的抗原表位特点。方法 以HEV基因工程重组蛋白p166Bur、p166Mex为免疫原，制备单克隆抗体(McAbs)。采用间接ELISA法及免疫印迹法(Western blot)，检测所制备的McAbs与7种不同基因型和亚型p166(p166Bur、p166Pak、p166Mor、p166Mex、p166Us、p166Nz和p166Chn)的交叉反应性。结果 获得4株稳定分泌基因型相关MeAb的杂交瘤细胞株，即1B5、1D10,1G10和D4A3。经检测，1B5、1D10,1G10分泌的McAbs只与p166Bur及p166Mor特异结合，D4A3分泌的McAb只与p166Mex特异结合。结论 不同基因型HEV存在不同的抗原表位。     

戊型肝炎病毒第Ⅳ基因型毒株中和抗原表位的鉴定

目的确定在我国新发现的戊型肝炎病毒（HEV）第Ⅳ基因型毒株的中和抗原表位特征及其与世界各地其他基因型HEV毒株中和抗原表位的异同。方法制备HEV第Ⅳ基因型ORF2重组衣壳蛋白p166Chn及其单克隆抗体（McAb），采用体外中和试验鉴定McAb的中和活性；通过间接ELISA和免疫印迹法测定中和性McAb与不同基因型HEVORF2编码蛋白p166的免疫反应性，并结合相加ELISA确定p166Chn中和抗原表位的分布和性质。结果获得6株稳定分泌抗-p166Chn McAb的杂交瘤细胞株。所得McAb能在体外中和HEV中国毒株（第Ⅳ基因型）对PLC/PRF／5细胞的感染性，并与来源于HEV4种不同基因型代表株的7种p166重组蛋白（p166Chn、p166Bur、p166Mor、p166Pak、p166Mex、p166Us和p166Nz）均能发生阳性反应。抗体间的相加ELISA结果为阴性。表明6株McAb识别p166上的同一个中和抗原表位。结论在我国新发现的HEV第Ⅳ基因型毒株与分布在世界各地的其他基因型毒株具有共同的中和抗原表位。     

GST与戊型肝炎病毒ORF2编码蛋白融合表达形成的新抗原表位的鉴定

目的:以戊型肝炎病毒(HEV)ORF2编码的重组蛋白p166为例,研究蛋白标签GST对融合表达的重组蛋白抗原结构的影响.方法:以HEV中国株重组蛋白p166Chn-GST为免疫原,制备单克隆抗体(mAb),与代表HEV 4个基因型的摩洛哥株、墨西哥株、美国株和中国株p166的GST或His融合蛋白、中国株非融合重组蛋白p179Chn以及GST融合的HEV无关蛋白进行ELISA检测,鉴定mAb所识别的抗原表位. 结果:获得3株稳定分泌抗p166Chn-GST的杂交瘤细胞株,分泌的mAb 1A8、9B4和8H10与p166Chn-GST反应,与GST不反应.其中1A8和9B4可与带GST标签的4种p166-GST蛋白以及N和C端截短的p146Chn-GST、p137Chn-GST反应,而不与4种p166-His蛋白反应,也不与p179Chn反应,与HEV病毒颗粒竞争试验阴性,与GST融合的HEV无关蛋白无交叉反应性,表明1A8和9B4识别的抗原表位不是HEV病毒颗粒表面天然存在的抗原表位,而是GST与HEV ORF2编码蛋白的465-601aa区段序列共同形成的新的抗原表位.结论:GST能够赋予基因工程重组蛋白以新的抗原特性,它与融合表达的重组蛋白可以共同形成新的抗原表位.     

不同基因型戊型肝炎病毒重组抗原p166用于抗体检测的价值

目的探讨不同基因型和亚型戊型肝炎病毒（HEV）ORF2重组蛋白p166用于抗体检测的价值,为研发准确可靠的戊型肝炎诊断试剂提供新的途径.方法用等浓度的不同基因型和亚型HEV p166作为包被抗原,对血清标本进行酶联免疫吸附试验测定.用HEV多基因型通用性引物逆转录套式PCR（RT-nPCR）扩增标本中HEV RNA,并测序、分型.结果8种不同p166抗原对30份健康献血者血清无抗原性,对182份来自世界不同国家和地区的已知HEV抗体阳性血清和7份HEV实验感染动物血清标本均呈阳性反应,但所得血清抗体滴度的高低与所用抗原的基因型有明显关系.RT-nPCR检测的50份中国血清标本中,19份阳性,基因分型均为Ⅳ型,与Ⅳ型中国株p166抗原反应最好.而以同属于第Ⅲ基因型的猪HEV新西兰株和人HEV美国株重组p166检测血清标本,两者结果差异无统计学意义.以多基因型p166混合抗原建立的ELISA抗体检测法与两种市售试剂盒比较,前者敏感性高,特异性好.结论不同基因型和亚型的HEV重组蛋白p166对不同血清标本HEV抗体检测的敏感性高低不同,因此多基因型和亚型p166的混合抗原是HEV抗体检测的最佳抗原.     

人、猪、禽戊型肝炎病毒血清学关系的研究

目的研究人、猪、禽戊型肝炎病毒（HEV）的血清学关系。方法应用ELISA分别以人、猪、禽HEVORF2重组蛋白p166human、p166swine、p268avian检测人、猪、鸡血清及其他标本中抗．HEVIs,G，用SAS软件进行统计学分析，同时进行序列同源性比较。结果p166human和p166swine对HEV实验感染动物血清、HEVORF2重组蛋白免疫血清和单克隆抗体均呈阳性反应，而p268avian均呈阴性反应。以p166human、p166swine、p268avian检测人、猪、鸡血清抗．HEVIgG，戊型肝炎患者血清检出率分别为98．5％、97．7％和1．5％，正常人血清为10．0％、10．0％和4．0％，猪血清为26．9％、25．6％和1．3％，鸡血清为4．3％、2．2％和33．3％。p268avian与p166human或p166swine的检出率差异有统计学意义（P〈0．001）。相关性分析表明，p166human和p166swine对不同样本的检测均呈直线正相关，而p268avitm与p166human或p166swine无直线相关性。人和猪HEVpORt2的序列同源性在88．2％。99．2％，而禽HEV与人、猪HEVpORt2的同源性仅为45．5％～46．1％，其中含有多个插入和缺失突变。结论人和猪HEV血清学关系密切，而禽HEV与人、猪HEV抗原性差异有统计学意义，无血清学相关性。因此禽HEV与人、猪HEV的关系应予进一步考证。     

酵母表达的重组戊型肝炎病毒结构区ORF2蛋白的抗原性鉴定

目的 对应用巴氏毕赤酵母表达系统制备的重组戊型肝炎病毒(HEV)结构区ORF2蛋白的抗原性进行鉴定。方法 以原核细胞表达的重组HEV ORF2抗原作为对照，应用间接ELISA方法鉴定重组HEV ORF2蛋白在HEV IgM和IgG抗体检测中的特异性和敏感性；并测定不同保存时间对抗原稳定性的影响。同时，比较了两种重组抗原与国外5株HEV ORF2单克隆抗体的反应特性。结果 应用酵母抗原可检测到HEV抗体的最低包被量为12．5ng／ml，可检测到HEV IgM、IgG抗体的血清最大稀释度皆为l：51200重组蛋白与其他类型肝炎患者血清无交叉反应，37℃加速试验证实重组蛋白4℃保存12个月可保持良好的抗原性。各株单克隆抗体与酵母表达的HEV ORF2蛋白有更好的反应性，其中4B2、2E2与酵母表达的HEV ORF2蛋白的反应性比与原核表达抗原的反应性分别高出125倍和25倍。结论 应用巴氏毕赤酵母表达系统制备的重组HEV ORF2蛋白可能包含了更为广泛的构象依赖性抗原表位，具有良好抗原性、特异性和稳定性。提示其可作为开发戊型肝炎诊断试剂的一个独具优势的候选抗原。     

抗HIV p24和人A型红细胞双特异性单克隆抗体的研制

目的：制备抗HIV p24和人A型红细胞双特异性单抗(mAb)，并建立检测HIV p24的间接血凝试验。方法：将分泌抗HIV p2d mAb的杂交瘤株2-E4和分泌抗人A型红细胞mAb的杂交瘤株S2，分别用8-Ag和5-BrdU驯化，使成为HAT敏感株。将两者常规融合，筛选分泌双特异性mAb的杂交-杂交瘤株。然后制备并纯化双特异性mAb，用其建立的间接血凝法检测p24。结果：共获得6株杂交-杂交瘤细胞株，以其分泌的双特异性mAb建立了检测HIV p24的间接血凝法，敏感性可达400ng/L。结论：获得可稳定分泌双特异性mAb的杂交-杂交瘤株，并用纯化的双特异性mAb建立了快速检测HIV p24的间接血凝法。     

载体和基因片段的选择对戊型肝炎病毒基因免疫抗原表达的影响

目的比较不同载体和不同大小的目的基因片段对戊型肝炎病毒(HEV)基因免疫抗原表达的影响,为基于HEV中和抗原表位的HEV基因免疫提供一定的依据。方法将含Ⅳ型HEV中国株中和抗原表位的p166和p179片段编码基因分别克隆入pTR421和pCDNA3.1两种真核表达载体,用脂质体介导基因转染HepG2人肝癌细胞系,经间接免疫荧光和Western blot分析以及将质粒注射小鼠局部肌肉组织后经免疫组织化学染色检测,分析目的基因在体内外的表达水平。结果成功构建了pTR421-166、pTR421-179、pCDNA3.1-166和pCDNA3.1-179四种重组质粒,经限制性内切酶双酶切和核苷酸测序鉴定,编码基因正确无误;pTR421-179转染的细胞以及注射小鼠的局部肌肉组织可以检测到p179的表达,而pCDNA3.1-179、pCDNA3.1-166以及pTR421-166均检测不到目的基因在体内、外的抗原表达。结论载体和目的基因片段的选择显著影响HEV抗原在体内、外的表达,直接关系到基因免疫的成功与否。     

烟台沿海地区人源和猪源戊型肝炎病毒基因型别的研究

目的 调查烟台市沿海地区人源与猪源戊型肝炎病毒(HEV)基因型别的相关性.方法 应用逆转录一巢式聚合酶联反应( RT-nPCR)方法对当地急性散发戊型肝炎患者、正常人群中抗HEV-IgM阳性者和当地养猪场的猪进行HEV RNA检测,并对HEV RNA阳性标本进行克隆测序和序列分析.结果 16例急性散发戊型肝炎患者中有7例粪便标本HEV RNA阳性;51份lgM阳性正常人群血清标本中有1份HEV RNA阳性;34份猪胆汁标本中有1份HEV RNA阳性.序列分析发现该地区HEV人株与猪株在ORF2部分区域的核苷酸序列同源性为87％～ 98.1％.7株患者的戊肝病毒基因型和1株猪的戊肝病毒基因型均为Ⅳ型,基因序列同源性在87％ ～ 98.1％之间;其中有6例患者和猪的基因序列同源性在93.9％ ～98.1％之间,为Ⅳ型a亚型;1例患者和猪的基因序列同源性为87％,为Ⅳ型d亚型.正常人群的1例戊肝病毒基因型为Ⅰ型d亚型.该地区人与猪HEV的ORF2的部分基因片段与HEV Ⅰ～Ⅳ型的代表株进行比较,核苷酸序列同源性分别是82.5％～100％,81.7％ ～92.9％,81.4％ ～93.9％,84.9％ ～ 100％.结论 该地区人群中流行的HEV存在2个基因型3个亚型,主要以基因Ⅳa型为主,与猪群中流行的HEV基因Ⅳa型同源性较高;HEV Ⅰ型在人群中散在存在.     

沙眼衣原体质粒蛋白pgp3单克隆抗体制备及鉴定

目的:原核表达沙眼衣原体(Ct)质粒分泌性蛋白pgp3,制备其单克隆抗体(mAbs)并鉴定其基本生物学特征。方法:构建pGEX-6p2-pgp3原核表达载体,在大肠杆菌中表达GST-pgp3融合蛋白作为抗原免疫BALB/c小鼠,取免疫后小鼠脾细胞与骨髓瘤细胞SP2/0融合,ELISA法筛选分泌抗pgp3蛋白mAbs的细胞株,对mAbs特异性、型别、类及亚类和效价进行鉴定。结果:GST-pgp3融合蛋白表达成功,且成功筛选出5株稳定分泌mAbs的杂交瘤细胞株,其中4株为分泌抗pgp3mAbs的杂交瘤细胞株(P1B3、P2A1、P2B6、P2C2),mAbs类型为IgG1/κ型,剩余1株为分泌抗GST蛋白的杂交瘤细胞株(P3B4),mAbs类型为IgG2b/κ型。交瘤细胞株P1B3、P2A1、P2B6、P2C2、P3B4培养上清中的mAbs效价分别为1∶6400、1∶3200、1∶12800、1∶6400和1∶6400。结论:成功制备出抗pgp3mAbs,为进一步研究Ct质粒蛋白pgp3功能及为Ct检测方法的建立奠定了基础。     

Efficient computation of lod scores: genotype elimination, genotype redefinition, and hybrid maximum likelihood algorithms

Calculation of multilocus lod scores presents challenging problems in numerical analysis, combinatories, programming, and genetics. It is possible to accelerate these computations by exploiting the simple pedigree structure of a CEPH-type pedigree consisting of a nuclear family plus all four grandparents. Lathrop et al. (1986) have done' this by introducing likelihood factorization and transformation rules and Lander & Green (1987) by the method of 'hidden Markov chains'. The present paper explores an alternative approach based on genotype redefinition in the grandparents and systematic phase elimination in all pedigree members. All three approaches accelerate the computation of a single likelihood. Equally relevant to multilocus mapping are search strategies for finding the maximum likelihood estimates of recombination fractions. Hybrid algorithms that start with the EM algorithm and switch midway to quasi-Newton algorithms show promise. These issues are investigated in the context of a simulated 10 locus example. This same example allows us to illustrate a simple strategy for determining locus order.     

Costello syndrome: Clinical phenotype,genotype, and management guidelines

Costello syndrome (CS) is a RASopathy caused by activating germline mutations in HRAS. Due to ubiquitous HRAS gene expression, CS affects multiple organ systems and individuals are predisposed to cancer. Individuals with CS may have distinctive craniofacial features, cardiac anomalies, growth and developmental delays, as well as dermatological, orthopedic, ocular, and neurological issues; however, considerable overlap with other RASopathies exists. Medical evaluation requires an understanding of the multifaceted phenotype. Subspecialists may have limited experience in caring for these individuals because of the rarity of CS. Furthermore, the phenotypic presentation may vary with the underlying genotype. These guidelines were developed by an interdisciplinary team of experts in order to encourage timely health care practices and provide medical management guidelines for the primary and specialty care provider, as well as for the families and affected individuals across their lifespan. These guidelines are based on expert opinion and do not represent evidence‐based guidelines due to the lack of data for this rare condition.     

Ethnicity,gender, genotype,and anger as related to nocturnal dipping

Bishop, Pek, and Ngau (2005) found a significant interaction in Singapore between anger and nocturnal dipping among Indians but not Chinese and Malays. The current study examines the role of 5‐HTTLPR genotype in this relationship. Two hundred thirty‐one undergraduates participated in up to 4 days of 24‐h ambulatory monitoring, completed the State‐Trait Anger Expression Inventory, and provided blood samples for genotyping of 5‐HTTLPR. Results indicated individuals with two copies of the short allele (SS) showed reduced dipping when they were high in Outward Anger (OA) but increased dipping when they were low in OA. Further, for Indian men only, dipping was reduced for individuals having the SS genotype when they were low on Anger In and increased when they were high on Anger In. These data provide further evidence for the role of 5‐HTTLPR in cardiovascular risk as well as ethnic differences in the 5‐HTTLPR–phenotype relationship. They also provide further evidence for 5‐HTTLPR as a “plasticity gene.”     

Hepatitis C virus genotype diversity in Shanghai, China

We studied 67 hepatitis C virus (HCV) isolates from 64 hospitalized patients in Shanghai, China. Genotype 1 was prevalent, and genotypes 2, 3, 6 were found for the first time in Shanghai. A rare mixed infection with three subtypes (1a, 1b, 2a) was found. The complete genome sequence of a subtype 3b isolate was determined and analyzed.     

A new genotype of lymphocystivirus, LCDV-RF, from lymphocystis diseased rockfish

Summary. Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease. In this study, nucleotide sequences of the major capsid protein (MCP) gene were analyzed among LCDV isolates from Japanese flounder and rockfish. A phylogenetic tree revealed three clusters for lymphocystiviruses. The first cluster included Japanese flounder isolates; the second cluster consisted of rockfish isolates; and the remaining one consisted of LCDV-1. Nucleotide sequence identities were ≥99.6% among Japanese flounder isolates and 100% among rockfish isolates, while between each cluster they were ≤85.2%. Experimental infections with Japanese flounder and rockfish isolates revealed that Japanese flounder and rockfish were infected by the respective homologous isolate but not by the heterologous isolate. These findings suggest that at least three genotypes exist in the genus Lymphocystivirus.     

Phenotype-directed analysis of genotype in early-onset, familial breast cancers

Considerable heterogeneity of morphology and disease outcome exists within breast cancers (BC), which likely reflects variable molecular pathogeneses within this broad clinical group. AIM: To evaluate the underlying genomic alterations associated with familial, early-onset BC (EOBC) phenotypes, in order to improve the management of this disease. METHODS: Using hierarchical clustering of morphological and immunophenotypical parameters, 116 EOBC were stratified into six groups. Conventional and array-based comparative genomic hybridisation was used to analyse the genomic alterations. RESULTS: Specific areas of genomic imbalance were associated with individual phenotypes. The largest phenotypical group was high grade, oestrogen receptor and HER-2 negative. This group contained the majority of BRCA1 germline mutation-associated tumours and commonly showed loss of chromosomal regions 5cent-5q13, 5q14-22 and 4q28-32. High mitotic rate, an important indicator of tumour cell proliferation and poor prognosis, was associated with gain of 19p, mapped within 7 Mb of the telomere. This region contains the candidate oncogene CDC34, the protein product of which is involved in ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor, p27Kip1. CONCLUSION: Phenotype-based analysis can be used to determine the genetic changes important in subtypes of BC. Further, the different morphological phenotypes could act as a cost-effective surrogate for genotypical stratification to facilitate optimal management of this disease.