胰腺癌潜在肿瘤标志物的探索及在胰腺癌中的应用

目的:探索胰腺癌新的潜在标志物,建立夹心法ELISA体系,并初步应用于胰腺癌患者的血清检测。方法:应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术对胰腺癌患者术前后血清进行分析,提纯分析差异蛋白并命名为DAP44,通过杂交瘤技术制备出抗DAP44单克隆抗体,用HRP标记法标记抗体,间接ELISA法检测抗体滴度,用制备出的抗体对胰腺癌组织和癌旁组织进行组化染色,采用夹心ELISA(DAS-ELISA)法制备抗DAP44检测试剂盒,检测胰腺癌病人和正常人血清DAP44值,比较两者差异。结果:对差异蛋白进行肽段测序和生物信息分析,并融合了3株能稳定分泌抗DAP44单克隆抗体的杂交瘤细胞(2D6H5,1E4D6,5B8H12),3株杂交瘤细胞分泌的抗体效价均在107以上,通过抗体配对筛选确定以2D6H5为包被抗体,1E4D6为酶标抗体时,DAS-ELISA法敏感性最高。两株抗体组化染色结果显示:癌组织DAP44表达量远高于癌旁。DAS-ELISA法标准曲线线性范围在0.78-25 ng/mL,检测下线为0.78 ng/mL,此方法检测到的胰腺癌病人和正常人血清DAP44平均含量分别为19.707±1.464和10.653±2.221,两者之间有统计学差异(P0.001)。结论:DAP44可能作为潜在的胰腺癌肿瘤标志物,建立的抗DAP44 DAS-ELISA法体系能够初步用于胰腺癌的临床诊断和疗效评估指标。     

抗HLJ1单克隆抗体的制备及抗原检测方法的建立

为制备抗人肝脏DnaJ-like蛋白(Human liver DnaJ-like protein, HLJ1)的单克隆抗体, 并建立免疫组化和双抗体夹心ELISA检测HLJ1的方法。采用淋巴细胞杂交瘤技术, 获得两株能稳定分泌抗HLJ1单克隆抗体的杂交瘤细胞株A4C7和 C4C8。经鉴定, 两株单抗的亚类均为IgG1, 并且效价高、特异性好。以单抗A4C7和C4C8作为一抗, 对人胎肝组织石蜡切片进行免疫组化染色, 结果表明, 两株单抗均为阳性染色, 且HLJ1主要定位于胎肝细胞的胞浆。选取A4C7进行HRP酶标记, 并以HRP- A4C7作为酶标抗体, 以C4C8作为包被抗体, 建立双抗体夹心ELISA方法, 并进行棋盘滴定确定抗体的最佳工作浓度。该检测方法的线性范围是15~750 ng/mL, 灵敏度下限达15 ng/mL, 特异性良好。所建立的免疫组化和双抗体夹心ELISA 法可用于快速、灵敏地检测组织及血清中的HLJ1蛋白, 为HLJ1的肿瘤相关性研究提供了有力的工具。     

抗猪鼻支原体单克隆抗体的研制及双抗体夹心ELISA检测法的建立

目的 制备多种抗猪鼻支原体的单克隆抗体,建立双抗体夹心ELISA方法用于该病原体的检测。方法用猪鼻支原体CVCC361免疫BALB／c小鼠,采用杂交瘤技术和酶联免疫吸附实验筛选出抗该病原体的单克隆抗体;运用免疫双向扩散试验、Western blotting确定I异G亚类及针对抗原的相对分子质量;筛选出配对抗体,建立双抗体夹心ELISA的检测方法,并评价其灵敏度和特异性。结果共筛选出17株单克隆抗体,抗体亚类分别为IgG1、IgG2a、IgG2b、IgG3,免疫印迹结果表明单抗ZB1、ZB2及ZB16与相对分子质量为35×103的抗原有特异性结合,而ZB3和ZBIO与相对分子质量为70×10^3的抗原有特异性结合。确定了2个配对抗体（ZB1-ZB1-HRP和ZB1-ZB2-HRP）,可检出最小抗原量为30ns／mL,检出猪鼻支原体活菌8．34×10^2CFU／mL,与人呼吸道常见的致病菌及支原体均无非特异性反应。结论筛选的单克隆抗体具有较高的特异性和敏感性,应用双抗体夹心ELISA方法可用于猪鼻支原体的检测。     

抗登革病毒2型E蛋白单克隆抗体的制备和生物学特性的鉴定

为了制备可用于胶体金快速检测试纸条的抗登革病毒2型（DEN2）E蛋白单克隆抗体（mAb）,通过基因克隆获得E基因与质粒pET32a（＋）的重组质粒,将重组质粒转化入大肠杆菌BL21,IPGT诱导表达重组蛋白。以DEN2重组E蛋白免疫BALB／c小鼠,采用杂交瘤技术制备抗DEN2E蛋白的mAb,以间接ELISA法和Western blot进行mAb特异性鉴定;同时采用间接ELISA法鉴定mAb的Ig亚类。结果表明获得1株可分泌特异性mAb的杂交瘤细胞（7C7）,其抗体亚类为IgG1。Western blot显示该株mAb能特异识别重组pET32a-DEN2E蛋白。因此,成功制备出抗DEN2E的1株mAb,为建立快速特异橙测登革病毒感染的实验方法提供了有力的工具。     

hGH单克隆抗体的筛选及双抗体夹心ELISA检测方法的建立

采用杂交瘤法制备单克隆抗体,并用辛酸-硫酸铵法纯化单抗,通过ELISA方法和Western blotting测定抗体的效价与特异性,并进行抗体类型、相对亲和力测定;应用纯化的单抗建立hGH双抗体夹心ELISA检测方法。筛选出两株可以稳定分泌抗hGH单抗的杂交瘤细胞株,分别命名为3E11、2G9,抗体类型均为IgG1,抗体滴度均可达10-10,特异性好,相对亲和力高,以筛选到的两株单抗建立的双抗夹心ELISA法线性范围为0.09～1.5625ng/mL,R2>0.9,灵敏度为0.09ng/mL。筛选出高效抗hGH的单抗,并建立了hGH双抗体夹心ELISA检测方法。     

抗人甲状腺球蛋白单克隆抗体的制备

目的:制备分泌特异性抗人甲状腺球蛋白(thyroglobulin,Tg)单克隆抗体的杂交瘤细胞株,为建立高灵敏度的Tg检测方法做准备。方法:以天然人源甲状腺球蛋白为抗原经皮下免疫BALB/c小鼠,通过细胞融合制备分泌抗人甲状腺球蛋白单克隆抗体,并对其进行特异性鉴定,建立检测Tg的双抗体ELISA(enzyme-linked immunosorbent assay)夹心法。结果:获得7株可稳定分泌抗人甲状腺球蛋白单克隆抗体的杂交瘤细胞株,经ELISA鉴定,筛选抗体可与Tg抗原有良好的特异性反应。建立的双抗体夹心ELISA方法敏感性可达1 ng/mL。结论:成功制备了抗人Tg单克隆抗体并建立了检测人Tg双抗体夹心ELISA方法,为进一步研发Tg快速诊断试剂盒提供了原料。     

抗嗜肺军团菌血清8型单克隆抗体的制备及双抗体夹心酶联免疫吸附试验检测法的建立

目的：制备针对嗜肺军团茵血清8型的单克隆抗体,并建立双抗体夹心酶联免疫吸附试验（ELISA）检测方法。方法：用甲醛灭活的嗜肺军团菌血清8型菌免疫BALB／c小鼠,采用杂交瘤技术制备抗嗜肺军团菌血清8型单克隆抗体,建立双抗夹心ELISA检测方法。结果：研制出8株能特异性分泌抗嗜肺军团菌血清8型单克隆抗体的杂交瘤细胞株,Ig类型分别为IgM（2株）、IgG,（1株）和IgG,（5株）;利用IgG1型单抗6G10与6C7配对,建立了双抗夹心ELISA检测方法,该方法的最低检出浓度为2．6×10^5cfu／mL,除与金黄色葡萄球菌有微弱的交叉反应外,与14株其他血清型嗜肺军团菌、17株非嗜肺军团菌及11株非军团菌均无交叉反应,具有较高的特异性。结论：制备了具有高特异性和亲和力的抗嗜肺军团菌血清8型单克隆抗体,并建立了双抗夹心EUSA检测方法。     

玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的建立

玉米赤霉烯酮具有较强的生物毒性,检测谷物中的玉米赤霉烯酮在食品和饲料安全中具有重要的作用.将玉米赤霉烯酮与牛血清白蛋白的偶联物免疫BALB/c小鼠制备单克隆抗体,并建立基于单克隆抗体的酶联免疫法作为检测玉米赤霉烯酮的方法.结果共筛选到4株抗玉米赤霉烯酮单克隆抗体,3株抗体亚类为IgG1,1株为IgG2b.选择其中的一株杂交瘤细胞2C9制备小鼠腹水,纯化后测定了抗体效价为1/40 000.以此单抗建立的间接竞争ELISA方法,其半数抑制率(IC50)为1.90 ng/mL,检测限(IC10)为0.051 ng/mL,检测区间(IC20-IC80)为0.115-13.900 ng/mL;且对玉米赤霉烯酮有很好的特异性.回收率检测在样品含1.46-93.80 μg/kg时回收率为96.5％-113.0％.本实验建立的检测方法可用于多种谷物及饲料样本中玉米赤霉烯酮的检测.     

抗人脂蛋白相关磷脂酶A2单抗制备及应用

本研究旨在制备抗人脂蛋白相关磷脂酶A2 (Lp-PLA2)单克隆抗体,对单抗进行初步评价,采用免疫层析方法学,建立一种可在社区医疗机构应用的Lp-PLA2快速检测方法。首先从NCBI获得人Lp-PLA2全长基因序列构建表达载体,在CHO-K1细胞中表达Lp-PLA2重组蛋白。以获得的重组蛋白免疫BALB/c小鼠,利用细胞融合技术筛选杂交瘤细胞,小鼠腹水诱生单克隆抗体。通过SDS-PAGE、ELISA等方法评价抗体性能。利用双抗夹心法制备Lp-PLA2量子点荧光免疫层析检测试剂,并使用便携式检测仪器评估试剂性能。制备并筛选得到亲和力达到1×10~(–8)的配对单克隆抗体PLA1与PLA5,抗体亚类为IgG1,能特异性识别血液中Lp-PLA2蛋白。自制Lp-PLA2量子点荧光免疫检测试剂可检测线性范围为20–2000ng/mL,与进口试剂的检测相关系数R达到0.99。综上,本研究制备得到一对高亲和力、高特异性的抗人Lp-PLA2单克隆抗体,并建立了Lp-PLA2免疫层析检测方法,该方法检测准确、操作简便,适合Lp-PLA2检测应用于社区医疗机构,为居民心血管健康管理提供了新途径。     

抗埃博拉病毒核蛋白抗体的制备与双抗夹心ELISA检测方法的建立

目的:制备抗埃博拉病毒核蛋白(EBOV NP)单克隆抗体和多克隆抗体,建立针对EBOV NP的ELISA检测方法。方法:以重组EBOV NP免疫动物并制备多克隆抗体和单克隆抗体。在此基础上,通过优化抗体浓度、包被液等条件建立检测EBOV NP的双抗夹心ELISA方法。结果:制备出了兔多克隆抗体,筛选出2株可分泌单克隆抗体的鼠源杂交瘤细胞株。Western blot实验结果表明兔多抗与鼠单抗的结合区域均为N端1~35氨基酸。通过优化,建立了针对EBOV NP的双抗夹心ELISA检测方法。其线性范围是31.2~1 000 ng/ml,最低检测限为2.6 ng/ml。结论:制备出了抗EBOV核蛋白的高特异性多克隆抗体和单克隆抗体,建立了定量检测EBOV核蛋白的方法。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.