噬菌体抗体库中筛选特异结合新甲型H1N1病毒scFv分子的研究

目的从人源化噬菌体抗体库中筛选到能高亲和性、特异结合新甲型H1N1流感病毒的单链抗体。为分析H1N1病毒抗原中和表位的活性位点和人源化治疗单抗奠定基础。方法将细胞培养的新型H1N1病毒毒株，经超速离心浓缩纯化后，获得纯的新甲型H1N1病毒，以新甲型H1N1病毒为靶蛋白，以亲和结合为原理，筛选噬菌体scFv抗体文库，经过3轮筛选富集后，随机挑选了96个噬菌体克隆扩增培养，ELISA法挑选能特异性、高亲和性结合目的蛋白的噬菌体克隆。结果经过3轮富集性的亲和筛选．分别从96个噬菌体克隆中挑选到了4株能特异结合新甲型H1N1病毒，而不结合季节性H1N1病毒的单链抗体分子，且PCR扩增都得到了长为368、527和935bp的轻链、重链和轻链一连接片段一重链的基因片段。结论从噬菌体抗体库中筛选到的特异结合新甲型H1N1病毒的单链抗体片段．可为进一步研发新甲流的H1N1快速筛选试剂和人源性治疗抗体奠定基础，也可为鉴定新甲型H1N1病毒中的抗原决定簇提供结构信息。     

利用人源化噬菌体抗体库筛选骨唾液酸蛋白的单链抗体

目的:从人源化噬菌体抗体库中筛选高亲和、特异结合人骨唾液酸蛋白(BsP)的人源可变区单链抗体(scFv).方法:采用噬菌体表面展示系统,以重组的BSP蛋白为包被抗原,从噬菌体可变区scFv中筛选特异性结合BSP的小分子scFv,经过3轮亲和富集筛选后,再用ELISA方法进一步鉴定与抗原BSP有特异结合活性的scFv阳性克隆,PCR扩增鉴定阳性克隆的插入轻、重链基因片段,并对阳性scFv分子测序和序列分析.结果:筛选得到的scFv片段可以特异地结合BSP蛋白,PCR扩增都得到了长为368 bp、527 bp和935bp的轻链、重链和轻链-连接片段-重链的基因片段,测序结果分析发现上述scFv片段在轻链有11处的氨基酸组成不同,在重链区域氨基酸组成则有3处不同.基因序列分析结果表明符合人源单链可变区抗体基因序列的结构特征.结论:利用噬菌体抗体库技术成功获得BSP蛋白的特异性人源单链可变区抗体.     

抗人禽流感病毒H5N1人源化单链抗体噬菌体文库的构建和筛选

目的 利用抗人禽流感病毒H5N1 IgG抗体阳性的人禽流感康复患者外周血淋巴细胞,构建人源化Fv段单链抗体(seFv)噬菌体文库,并筛选与禽流感病毒相关蛋白有结合活性的scFv抗体文库.方法 提取人外周血淋巴细胞总RNA,逆转录成cDNA,以其为模板,利用家族特异性IgG基因的引物,扩增重链和轻链的可变区基因,并用合成的连接子将轻链和重链基因连接成单链抗体片段后,重组到噬菌粒载体pCANTAB5E中.将重组噬菌粒载体电转化大肠杆菌TG1,酶切和PCR鉴定抗体库的重组率,通过测定噬菌体抗体库的滴度计算抗体库的库容,用特异性禽流感病毒相关蛋白筛选表达的单链抗体.结果 构建了源于人禽流感康复患者血清的scFv抗体文库,库容为3.75×104;筛选出与禽流感病毒相关蛋白有结合活性的scFv抗体文库.结论 成功构建了抗人禽流感病毒H5N1的人源scFv噬菌体抗体库,并筛选出特异性结合人禽流感病毒相关蛋白的单链抗体,为进一步制备快速检测试剂和治疗研究提供了基础数据.     

U251细胞血清饥饿特异抗原的ScFv噬菌体抗体库构建

目的：构建一个U251细胞血清饥饿特异抗原ScFv噬菌体抗体库。方法：将U251细胞进行48小时的血清饥饿培养,将其细胞裂解液免疫4周龄的BALB/c小鼠,提取被免疫小鼠脾脏细胞总RNA,反转录成cDNA,利用RT-PCR扩增免疫球蛋白IgG的重链可变区（VH）和轻链可变区（VL）基因,用一个柔性片段（Linker）连接VL和VH基因片段,将连接产物重组到pCANTAB-5E载体,转入TG1菌株中,随后加入辅助噬菌体（Help phage）M13K07超感染,构建单链抗体片段（Single chain fragment of variation,ScFv）噬菌体抗体库。结果：经过富集筛选后,库容量达到3×10^6cfu/L,随机挑取8个克隆进行ELISA检测,获得了1个阳性克隆。结论：成功构建了一个具有一定库容的U251细胞血清饥饿特异抗原的单链抗噬菌体抗体库,为筛选血清应答蛋白抗体、进一步克隆血清应答蛋白的基因奠定基础。     

抗人端粒酶蛋白亚基单域抗体制备和鉴定

目的　研制抗人端粒酶逆转录酶 (hTERT)蛋白亚基单域重组抗体。方法　以重组His taghTERT融合蛋白为抗原免疫小鼠 ,以逆转录 聚合酶链反应 (RT PCR)扩增小鼠脾细胞重链可变区 ;经克隆噬菌粒载体pCANTAB 5E及转染大肠杆菌建立噬菌体抗体展示文库 ,经过亲和性富集选择阳性克隆 ,并于大肠杆菌 (E .coli.HB2 15 1)中表达为可溶性单域抗体 ;Westernblot确定单域抗体结合特性。结果　以RT PCR扩增His taghTERT免疫小鼠脾细胞RNA ,得到 35 0bp小鼠重链可变区DNA片段 ;通过克隆及转染制备出噬菌体展示抗体文库 ,文库大小是 8× 10 4 ;经过抗原 抗体亲和性筛选 ,得到 4个克隆 ,并表达为可溶性单域抗体 (相对分子质量 16 0 0 0 ) ;WesternBlot分析显示 :其中 2个克隆的可溶性单域抗体可特异性结合His taghTERT融合蛋白 (相对分子质量 16 70 0 ) ,与His tag无关 ;与人癌细胞表达的天然hTERT蛋白 (相对分子质量 12 70 0 0 )分析亦显示其特异性结合能力。DNA序列分析证实可溶性单域抗体为小鼠抗体重链可变区VH基因编码。结论　利用噬菌体展示技术已初步制备出小鼠重链可变区编码的抗hTERT蛋白的特异单域抗体 ,为进一步探索其抑制端粒酶活性及抗肿瘤作用奠定了基础。     

HCV非结构蛋白NS5A人源单链可变区抗体基因的筛选与鉴定

目的 筛选、鉴定抗丙型肝炎病毒（HCV）非结构蛋白NS5A的人源单链可变区抗体（ScFv)的编码基因,为HCV NS5A蛋白质生物学功能的研究及抗HCV的基因治疗研究开辟新途径。方法 采用噬菌体表面展示技术,以重组纯化的HCV非结构蛋白NS5A为固相抗原,从噬菌体单链可变区抗体半合成库中经过5轮“吸附-洗脱-扩增”筛选过程,获得抗原结合活性和特异性较强的HCVNS5A人源单链可变区抗体基因片段的阳性克隆,并对其进行免疫检测及序列测定,结果 筛选得到的ScFv片段基因核苷酸序列为789nt,编码由262个氨基酸残基组成的多肽,具有典型的免疫球蛋白轻链和重链可变区的结构特点,基因编码产物具有HCV NS5A蛋白反应的免疫学活性和特异性。结论 利用噬菌体抗体库技术,成功获得了HCV NS5A人单链可变区抗本ScFv编码基因,并获得了可溶性单链抗体的表达。     

抗PSMA全人源单链抗体制备、鉴定及初步应用

目的 构建抗PSMA全人源单链抗体库,筛选抗PSMA人源单链抗体(ScFv)并鉴定,应用人源ScFv进行分子显像.方法 从健康人外周血淋巴细胞中提取总RNA,用RT-PCR方法扩增人轻链和重链可变区基因,重叠PCR拼接轻重链可变区基因,构建ScFv的表达载体pDF-ScFv,电转染大肠杆菌XL1 Blue,获得噬菌体单链抗体库,酶切鉴定及测序分析,用PSMA抗原进行富集筛选,阳性克隆用Western-Blot 和DNA指纹图谱鉴定.应用直接标记法将125I标记在ScFv上,通过尾静脉注入前列腺癌动物模型体内,2h后进行小动物SPECT/CT显像,观察人源ScFv对前列腺癌的定位性能.结果 成功构建抗PSMA全人源单链抗体库,库容约2.16×108,插入片段经酶切及DNA测序证实为人抗体轻链和重链可变区基因.筛选出6株ScFv阳性克隆,Western-Blot证实6株ScFv均能与PSMA抗原特异性结合,DNA指纹图谱鉴定6株ScFv可变区基因具有多样性.动物模型的分子显像:人源ScFv能与前列腺癌PSMA抗原特异性结合,并使肿瘤显像.结论 成功构建了一个全人源噬菌体单链抗体库,并筛选出了抗PSMA单链抗体,单链抗体在动物模型体内能靶向结合前列腺癌细胞,从而为前列腺癌的靶向诊断和治疗奠定了基础.     

人源Fab抗体库的构建和抗hPRLR抗体的筛选鉴定

目的构建人源Fab噬菌体抗体库,筛选抗hPRLR抗体片段并进行初步鉴定。方法从乳腺癌患者外周血提取总RNA,通过RT-PCR扩增人抗体轻链和重链基因,构建抗hPRLR人源Fab抗体库。分别以His-hPRLR融合蛋白、BSA-hPRLR表位多肽融合蛋白和GST-hPRLR融合蛋白作为抗原包板,经过3轮循环的吸附-洗脱-扩增的筛选及1轮交叉筛选,挑单克隆用Phage-ELISA、DNA测序筛选阳性克隆,将筛选到的阳性克隆FabG2转化至Top10’受体菌,诱导表达可溶性Fab抗体,通过Western blot和ELISA进行特异性的鉴定。结果构建的人源Fab库容为1.0×109,4轮的筛选,获得6株能与hPRLR结合的人源抗体克隆。选取的FabG2能够进行可溶性Fab抗体蛋白的表达,ELISA初步鉴定,能够与hPRLR进行特异性地结合。结论成功构建了人源Fab噬菌体抗体库,筛选并鉴定了1株抗hPRLR Fab抗体的克隆。hPRLR特异性Fab抗体的获得将为高表达hPRLR乳腺癌的生物免疫治疗奠定了基础。     

人源性抗HBc单链抗体的筛选、鉴定及基因序列测定

目的:筛选人源性抗乙型肝炎病毒核心蛋白(HBc)单链抗体(ScFv)并在体外原核表达、鉴定和基因序列测定, 为抗HBcScFv的进一步研究奠定基础。方法: 采用噬菌体表面展示技术, 以乙肝病毒核心抗原为包被抗原, 从人源性单链噬菌体抗体库中经过3轮"吸附-感染-扩增"的筛选过程, 获得抗原结合活性较强的人源性抗HBc单链抗体阳性克隆, 并在大肠杆菌HB2151中进行ScFv可溶性表达, 对其进行免疫学鉴定和序列测定。结果:筛选出的ScFv具有抗HBc的特异性, 并可在大肠杆菌中进行可溶性表达;基因序列测定表明, 该ScFv的重链段基因与人类免疫球蛋白重链可变区相符, 轻链段基因为人类免疫球蛋白κ轻链可变区。结论:利用噬菌体抗体库技术, 成功筛选出人源性抗HBcScFv并获得其基因序列。     

抗肝癌细胞噬菌体单链抗体库的构建及筛选

构建抗人肝癌细胞单链抗体库 ,从中筛选与肝癌细胞特异结合的高亲和力单链抗体。从HepG2细胞免疫的BALB/c小鼠脾脏提取总RNA ,RT PCR扩增小鼠抗体重、轻链可变区基因 ,用 (Gly4Ser) 3 连接肽基因 ,经重叠延伸反应 ,在体外将VH 和VL 连接成单链抗体 (scFv)基因 ,并克隆入噬菌粒载体pCANTAB5E中 ,构建噬菌体单链抗体库。以HepG2细胞为抗原对抗体库进行淘选 ,ELISA法鉴定各单克隆与肝癌细胞的结合活性 ,并对阳性克隆进行表达。成功构建了库容为 1 1× 10 6抗肝癌细胞的噬菌体单链抗体库 ,经筛选得到了与HepG2细胞具有较强结合能力的单链抗体 ,实现了scFv在大肠杆菌中的可溶性表达。序列测定结果表明 ,VH 和VL 基因符合小鼠抗体可变区特征 ,scFv基因拼接正确     

Modes of reinforcement and gender and culture of experimenter and subject: effects of "alien" and external reinforcement for Japanese and American men and women

In this study, we examined external and "alien" reinforcement (ER and AR. respectively) as a factor in social learning, and studied the combined effects of culture and reinforcement mode. A female (Experiment 1) and a male (Experiment 2) experimenters conducted experimental sessions. Both men and women, who grew up in the same culture as the experimenter, participated and performed the experimental task. A three-way interaction effect of experimenter gender, culture, and reinforcement mode was found on task performance. And the effect was more pronounced for a Japanese experimenter. A female and a male experimenters conducted Experiments 3 and 4, respectively; however participants this time were men and women who grew up in different cultures than the experimenter. Results indicated that the pattern of the subject gender and reinforcement mode interaction effect, when the experimenter was Japanese with American subjects, was exactly opposite to that when the experimenter was American. These experiments showed that AR was as effective for social learning as ER, and that the cultural backgrounds of experimenter and subject influenced AR and ER effectiveness.     

Rates of oxygen consumption and of anaerobic glycolysis in renal cortex and medulla of adult and new-born rats and guinea-pigs

1. Rates of oxygen uptake and of anaerobic glycolysis were estimated in slices from the renal cortex and medulla (a) of adult rats and guinea-pigs, (b) of new-born (1-, 5- and 21-day-old) rats and of guinea-pigs of 1, 12, 21, 24 and 120 hr age.2. In the adult rat, Q(O2) values for the cortex were 12.55 +/- 0.20 (22) and for the medulla: 8.56 +/- 0.17 (22) mul./hr.mg dry weight, while in the new-born rat (24 hr old) they were 10.99 +/- 0.46 (12) and 9.33 +/- 0.18 (9) mul./hr.mg dry weight respectively.3. Values for Q(CO2) (N2) (anaerobic glycolysis) in the 14 hr old new-born rat were in the renal cortex 9.65 +/- 0.35 (5) and in the medulla 7.39 +/- 0.43 (5) mul./hr.mg dry weight; while in the adult they were 2.25 +/- 0.08 (16) and 5.76 +/- 0.14 (16) mul./hr.mg dry weight, respectively.4. In the adult guinea-pig values for Q(CO2) (N2) were of the same order as in the adult rat, though the rate of O(2) uptake was for the cortex 8.12 +/- 0.22 (12) and for the medulla 5.02 +/- 0.23 (11) mul./hr.mg dry weight.5. Though the Q(O2) values in the renal cortex and medulla were smaller in the 1 hr old new-born guinea-pig, they were already increasing in the 12 hr old neonate.6. The results are discussed in the light of enzyme changes occurring during the process of maturation of the nephron as indicated by histochemical observations.     

Timing and intensity of early fevers and the development of allergies and asthma

BACKGROUND: Early childhood fevers appear to protect against later allergies and asthma. What is not known is the time in which fevers exert this effect and whether the degree of temperature increase is important. OBJECTIVE: We sought to examine the relationship between the time and degree of early fevers and later allergies and asthma. METHODS: Eight hundred thirty-five children from southeast Michigan were enrolled at birth. Clinic records from their first 2 years were abstracted for episodes of fever. At age 6 to 7 years, children underwent allergy testing. We examined fevers occurring within 6-month intervals in the first 2 years of life and outcomes at age 6 to 7 years. The primary outcome measures were allergic sensitization, asthma, asthma with allergic sensitization, and asthma without allergic sensitization. RESULTS: In the unadjusted analysis each episode of fever between 7 and 12 months of age was associated with a lower odds of allergic sensitization (odds ratio [OR], 0.71; 95% CI, 0.54-0.93) and asthma with allergic sensitization (OR, 0.43; 95% CI, 0.21-0.90) at age 6 to 7 years. Likewise, every 1 degrees C increase in the maximum temperature between 7 and 12 months was associated with a lower odds of allergic sensitization (OR, 0.77; 95% CI, 0.61-0.96) and asthma with allergic sensitization (OR, 0.62; 95% CI, 0.40-0.94). After adjusting for potential confounders, each episode of fever between 7 and 12 months was associated with a lower likelihood of asthma with allergic sensitization (adjusted OR, 0.33; 95% CI, 0.11-0.94) at age 6 to 7 years. CONCLUSIONS: Both the timing and intensity of childhood fevers appear to be important factors in the development of allergies and asthma.     

纳米水平分子成像与诊疗一体化研究进展与挑战

分子成像能以非侵入性的方式重现活体细胞的生理功能和生物学过程,提高疾病的早期和特异性诊断水平。纳米颗粒/材料具有物理性质可控性高、易于表面修饰、血液循环时间长和可功能化等优点,在疾病诊断与治疗中显示出巨大潜力。但如何阐明纳米材料多功能间的内在联系、解决其代谢及安全性等关键机制难题、实现纳米颗粒/材料多功能性到临床多功能...