甲型H1N1流感病毒样颗粒疫苗的初步研究

目的对甲型H1N1病毒样颗粒的免疫原性和保护效果进行初步研究,为研发不依赖鸡胚生产工艺的流感疫苗提供新的思路。方法使用昆虫-杆状病毒表达系统表达并纯化甲型H1N1流感病毒A/California/07/2009(H1N1)的病毒样颗粒;通过Western blot、血凝、单向免疫扩散,透射电镜等方法鉴定病毒样颗粒;使用A/California/07/2009(H1N1)裂解疫苗作为对照,腹腔注射免疫Balb/c小鼠,并使用A/Beijing/501/2009(H1N1)进行攻毒,评价病毒样颗粒疫苗的免疫原性及保护效果。结果经鉴定,纯化后的病毒样颗粒血凝滴度为1:512,HA含量为92.9μg/ml,颗粒大小在100 nm左右,二免10 d后IgG抗体效价7.9×103,血凝抑制实验测得血抑(Hemagglutination Inhibition,HI)效价384,对50LD50的A/Beijing/501/2009(H1N1)病毒的保护率达到100%。结论甲型H1N1流感病毒样颗粒的免疫原性显著高于裂解疫苗,为发展不依赖鸡胚生产的流感亚单位疫苗提供了技术保证。     

新型甲型H1N1流感病毒HA基因DNA疫苗诱导小鼠产生中和抗体

目的 探讨新型甲型流感病毒(2009H1N1)血凝素(HA)DNA疫苗诱导小鼠产生中和抗体特性.方法 构建2009H1N1或1918甲型流感病毒(1918H1N1)HA蛋白表达质粒2009HA和1918HA,采用25μg或200μg剂量2009HA质粒免疫小鼠,以2009HA或1918HA蛋白为包被抗原,测定小鼠血清中2009HA抗体总量或交叉反应抗体含量,分别用2009H1N1和1918H1N1两种假病毒(pp)测定抗体中和活性.结果 25 μg或200μg的2009HA质粒加强免疫小鼠后,4～16周内两组小鼠血清中2009HA总抗体水平以及对2009H1N1pp的中和抗体滴度相似(P>0.05),都含有与1918HA蛋白交叉反应抗体,对1918H1N1pp的交叉中和抗体滴度相似(P>0.05).结论 小剂量2009HA质粒DNA疫苗能够诱导小鼠产生持久的高水平中和抗体,对于预防新现流感病毒具有潜在应用价值.     

甲型H1N1流感病毒裂解疫苗的研制和临床观察初步结果

目的 研制甲型H1N1流感疫苗并进行人体安全性和有效性观察.方法 用WHO推荐的甲型H1N1流感疫苗毒种按照季节性流感裂解疫苗工艺研制甲型H1N1流感疫苗,成品参照流感病毒裂解疫苗质量标准进行各项指标检定,用2批血凝素含量不同的试制产品进行临床验证.结果 血凝素含量15μg/剂和30μg/剂各1批试制产品经检定并由中国药品生物制品检定所复检,符合暂定质量标准要求.临床观察显示,960名受试者接种15μg或30μg试验疫苗1针,21 d后血清抗体阳性率、保护率均大于70%.3～11岁、12～17岁、18～59岁及≥60岁,15μg组几何平均滴度(GMT)分别较免疫前增长15、39、37和25倍;30μg组GMT分别较免疫前增长26、72、68和36倍.安全性观察结果显示15μg和30μg组总的不良反应发生率为29.38%和43.75%,其中2级反应率为6.25%和15.42%,3级反应率为0.83%和1.46%,未观察到严重不良反应.结论 按照季节性流感生产工艺研制的甲型H1N1流感病毒裂解疫苗具有良好的安全性和有效性.     

禽流感病毒血凝素H5特异性单克隆抗体的制备及ELISA捕获法的建立

目的 制备和鉴定禽流感病毒(H5N1)血凝素(H5)特异性单克隆抗体(mAb),建立H5抗原的双抗体夹心ELISA捕获法.方法 以H5血凝素和携带H5全长基因的质粒免疫Balb/c小鼠制备mAb,利用血凝抑制(HI)实验筛选和鉴定,通过竞争抑制试验分析抗体识别表位,并采用抗体配对试验筛选捕获抗体和检测抗体,建立测定H5抗原的双抗体夹心ELISA捕获法.结果 获得16株特异性针对H5的单克隆抗体,与A型流感病毒H1、H3、H7、H9和B型流感病毒的血凝素无HI交叉反应,对H5血凝素的血凝抑制效价为1:100～1:51 200;通过配对实验,建立以单克隆抗体H5M9为捕获抗体,辣根过氧化物酶标记单克隆抗体H5M11为检测抗体的双抗体夹心ELISA;检测多株H5N1病毒和H5血凝素的最低检出值为1/32血凝单位,检测A型流感病毒H1N1、H3N2、B型流感病毒以及H7、H9血凝素均为阴性.结论 建立了一种灵敏度高、特异性强的测定H5抗原的ELISA捕获法,可应用于禽流感病毒H5N1感染的实验室早期诊断.     

甲型H7N9流感裂解疫苗制备及免疫保护效果的评价

目的构建、拯救重配甲型H7N9流感病毒疫苗候选株并制备甲型H7N9流感裂解疫苗,动物实验评价甲型H7N9流感裂解疫苗的免疫原性及免疫保护性效果。方法采用反向遗传学技术将A/Anhui/1/2013(H7N9)疫苗株的HA、NA基因和A/Puerto Rico/8/34(PR8)毒株的PB2、PB1、PA、NP、M、NS基因进行重配,转染细胞后筛选拯救甲型H7N9流感病毒疫苗候选株,制备rgPR8-H7N9流感裂解疫苗抗原。腹腔注射免疫Balb/c小鼠,检测血清IgG、IgG1、IgG2a、HI效价,进一步用野生株攻毒,评价rgPR8-H7N9流感裂解疫苗的免疫保护效果。结果成功拯救甲型H7N9流感病毒疫苗候选株rgPR8-H7N9。制备的重配甲型H7N9流感裂解疫苗对小鼠产生较高的HI抗体效价。IgG1/IgG2a亚型检测结果表明小鼠体内以诱导体液免疫为主。攻毒实验显示甲型H7N9流感裂解疫苗能够有效降低肺部的病毒载量,肺组织病变显著减轻、体质量下降后趋于稳定,疫苗剂量达到15μg即可全部存活。A/Anhui/1/2013(H7N9)野生株流感病毒攻毒,甲型H7N9流感裂解疫苗能够达到保护小鼠效果。结论成功拯救重配甲型H7N9流感病毒疫苗候选株rgPR8-H7N9,制备的甲型H7N9流感裂解疫苗具有较好的免疫原性及免疫保护性,为H7N9流感裂解疫苗的研发及进入临床研究提供了实验依据。     

江门市居民甲型H1N1流感病毒血清抗体的调查分析

目的了解江门市各年龄段人群甲型H1N1流感病毒抗体水平,为完善甲型H1N1流感疫情防控措施提供科学依据。方法采取多阶段分层随机抽样方法抽取研究对象480人,进行血清标本采集和问卷调查,应用红细胞血凝抑制方法检测甲型H1N1流感病毒抗体。结果人群甲型H1N1流感病毒抗体阳性率为19.38%,0～、6～、16～、25～和60岁以上组的甲型H1N1流感病毒抗体阳性率分别为22.09%、27.36%、24.47%、14.58%和8.16%。6～24岁的甲型H1N1流感病毒抗体阳性率较高,为51.83%,无症状抗体阳性率达12.61%。抗体阳性率在不同年龄、性别和职业上差异均有统计学意义（P〈0.05）。结论江门市甲型H1N1流感病毒感染状况各年龄段不同,青少年抗体阳性率较高,老年人抗体阳性率相对较低,男性抗体阳性率高于女性。     

甲型H1N1流行性感冒疫苗接种人群的血清抗体分析

目的:了解接种甲型H1N1流行性感冒(流感)疫苗后,人群中血清抗体的变化情况,为甲型H1N1流感疫苗的接种提供依据。方法:随机采集不同年龄已接种甲型H1N1流感疫苗人群的血清,采用血凝抑制实验检测血清中甲型H1N1流感抗体的血凝抑制滴度(HI滴度),HI滴度≥1∶40判定为阳性,同时调查采样对象的甲型H1N1流感疫苗与季节性流感疫苗的接种史。结果:甲型H1N1流感抗体阳性率为57.4%(402份/700份),抗体几何平均滴度(GMT)为1∶35.6;甲型H1N1流感抗体阳性率与GMT较高的是10～30岁组人群,较低的是60岁以上的人群;接种甲型H1N1流感疫苗后30～90天,GMT水平达到高峰(1∶56);随着季节性流感疫苗接种次数的增多,人群血清中甲型H1N1流感抗体的阳性率与GMT值反而降低。结论:青少年与成人接种甲型H1N1流感疫苗的免疫效果比儿童和老年人的好;甲型H1N1流感疫苗对人群的保护作用能持续90天左右;甲型H1N1流感抗体在0～10岁组,10～30岁组人群中持续的时间比30～60岁,>60岁组人群长;多次接种季节性流感疫苗可能会影响甲型H1N1流感抗体的产生。建议对儿童和老年人开展双倍剂量甲型H1N1流感疫苗接种;甲型H1N1流感疫苗的接种时间最好在流行期前1～3月内,并且应每年接种一次。     

甲型H1N1流感病毒疫苗株高适应性MDCK细胞的筛选及鉴定

目的筛选甲型H1N1流感病毒疫苗株高适应的MDCK单克隆细胞株,用于培养生产流感病毒疫苗,为细胞代替鸡胚生产制备流感病毒疫苗提供保证。方法通过有限稀释法将MDCK细胞进行单克隆化,扩大培养建立单克隆化细胞库,通过血凝和TCID50筛选甲型H1N1流感病毒疫苗株的高适应性单克隆化细胞株,并鉴定所获得的细胞株细胞表面NeuAcα2,6 Gal的丰度。结果共制备了97株单克隆化MDCK细胞,经过甲型H1N1流感病毒疫苗株的筛选,共筛选到2株高适应甲型H1N1流感病毒疫苗株的MDCK单克隆化细胞株,其TCID50分别为6.68 log10 TCID50/ml和6.77 log10 TCID50/ml,其表面NeuAcα2,6 Gal的丰度明显提高。结论成功培养了MDCK单克隆细胞株,经筛选获得的单克隆细胞株其血凝滴度,TCID50都比普通MDCK细胞有明显提高,为细胞培养生产流感病毒疫苗奠定了基础。     

2010年广州市越秀区居民新甲型H1N1流感病毒血清抗体调查

目的了解广州市越秀区居民各年龄段人群新甲型H1N1流感病毒抗体水平,为完善新甲型H1N1流感疫情防控措施提供科学依据。方法采取多阶段分层随机抽样方法抽取研究对象309人,进行血清标本采集和问卷调查,应用红细胞血凝抑制（HI）方法检测新甲型H1N1流感病毒抗体。结果人群新甲型H1N1流感病毒抗体阳性率为20.06%,0～岁组、6～岁组、10～岁组、16～岁组、25～岁组和60岁以上组的新甲型H1N1流感病毒抗体阳性率分别为18.84%、33.33%、46.67%、30.00%、6.67%和5.00%。6～24岁组的新甲型H1N1流感病毒抗体阳性率较高,为35.00%,无症状抗体阳性率达10.43%。抗体阳性率在性别上差异无统计学意义（P〉0.05）。结论广州市越秀区居民新甲型H1N1流感病毒感染状况各年龄段不同,青少年抗体阳性率较高,老年人抗体阳性率相对较低。     

重组流感血凝素三聚体蛋白疫苗的制备及免疫原性评价

目的制备重组流感病毒血凝素(hemagglutinin, HA)三聚体蛋白疫苗并进行相关表征分析, 研究重组HA三聚体在小鼠模型中的免疫原性。方法构建稳定表达HA三聚体蛋白的CHO细胞株, 通过Western blot、单向免疫扩散试验、蛋白质粒径检测和N-糖基化位点分析对重组蛋白进行定性及定量分析。根据剂量、佐剂等处理条件的不同将BALB/c小鼠分为11个组, 并进行一致的免疫程序。初次免疫后56 d检测血清中和抗体效价, 以评价免疫原性。结果构建的CHO细胞株能够分泌表达HA三聚体。HA三聚体具备生物学活性能够在单向琼脂免疫扩散试验中形成沉淀环, 蛋白质粒径大小约为18.79 nm, 具有10个N-糖基化位点, 包括高甘露糖型、复合糖型和杂合糖型;HA三聚体重组蛋白疫苗在2次免疫小鼠后, 3.75 μg剂量配伍RFH01佐剂与对照组单价疫苗原液15 μg引起的中和抗体效价差异无统计学意义(P=0.431 2, U=36), 配伍佐剂组免疫后血清抗体效价均高于未加佐剂组, 其中15 μg HA三聚体+RFH01组效价最高, 为1 280。结论成功制备了流感病毒重组HA三聚体蛋白, 其...     

HLA-DRB1, -DQA1, -DQB1, -DPA1 and -DPB1 genes in Japanese multiple sclerosis patients

Japanese MS patients and controls were examined for the distribution of HLA-DRB1, -DQA1, -DQB1, -DPA1 and -DPB1 alleles using in vitro amplification of genomic DNA and probing with sequence-specific oligonucleotides. No significant difference in frequency of the examined alleles was observed among the two groups. This is in contrast to Norwegian MS patients, where an association to a combination of certain DQA1 and DQB1 alleles has previously been demonstrated.     

NDE1 and NDEL1: Multimerisation,alternate splicing and DISC1 interaction

Nuclear Distribution Factor E Homolog 1 (NDE1) and NDE-Like 1 (NDEL1) are highly homologous mammalian proteins. However, whereas NDEL1 is well studied, there is remarkably little known about NDE1. We demonstrate the presence of multiple isoforms of both NDE1 and NDEL1 in the brain, showing that NDE1 binds directly to multiple isoforms of Disrupted in Schizophrenia 1 (DISC1), and to itself. We also show that NDE1 can complex with NDEL1. Together these results predict a high degree of complexity of DISC1-mediated regulation of neuronal activity.     

韩国人CYP450 1A1、CYP450 2E1和GSTM1、GSTT1、GSTP1基因座遗传多态性分析

目的 调查代谢相关的CYP4501A1、CYP4502E1和GSTM1、GSIT1、GSTP1基因座在韩国人群中的遗传多态性分布状况。方法 采用多重聚合酶链式反应、聚合酶链式反应-限制性片段长度多态性技术，分析300名韩国健康大学生的CYP1A1基因3′端限制性内切酶Msp Ⅰ位点、CYP2E1基因5′端转录调节区Pst Ⅰ位点和GSTM1、GSTT1缺失与存在、GSTP1基因第5外显子BsmA Ⅰ位点的基因型，计算基因型和基因频率。结果 CYP1A1基因型频率为ml／ml型39．7％、ml／m2型49．7％、m2／m2型10．7％，基因频率为ml 0．645、m2 0．355。CYP2E1基因型频率为cl／cl型66．7％、cl／c2型30％、c2／c2型3．3％，基因频率为C1 0．818、C2 0．182。GSTM1基因缺失型频率为53．3％。GSTT1基因缺失型频率为54．7％。GSTP1基因型频率为Ile／Ile型62％、Ile／Val型34．3％、VaL／Val型3．7％，基因频率为Ile 0．792、Val 0．208。基因分布符合Hardy-Weirtberg平衡定律。结论 韩国人CYP1A1、CYP2E1、GSTM1、GSTT1基因分布与我国人群较为相近，半数以上人缺乏GSTM1和GSTT1基因，纯合缺失型频率超过印度人的3倍。     

Maternal genetic polymorphisms in CYP1A1, GSTM1 and GSTT1 and the risk of hypospadias

Hypospadias is one of the most common congenital anomalies. Increased exposure to environmental factors (endocrine-disrupting chemicals and smoking) or maternal endogenous estrogen may cause hypospadias because male sexual differentiation is dependent on normal androgen homeostasis. Moreover, interactions between genetic factors and cigarette smoking and other chemicals have been suggested. It has been demonstrated that the CYP1A1 metabolizes not only environmental chemicals but also estrogens, and glutathione-S-transferases (GSTs) are detoxification enzymes that protect cells from toxicants by conjugation with glutathione. In this study, to investigate the association of CYP1A1 (MspI), GSTM1 and GSTT1 polymorphisms with hypospadias, a case-control study of 31 case mothers who had boys with hypospadias and 64 control mothers was performed in Japan. These polymorphisms were investigated by PCR-based methods using DNA from peripheral lymphocytes. We found that the heterozygous CYP1A1 and heterozygous and homozygous CYP1A1 were less frequent in the case mothers than in the control mothers [adjusted odds ratio (OR)=0.17, 95% confidence interval (CI)=0.04-0.74, OR = 0.28, 95% CI = 0.08-0.97, respectively]. We found no effect of maternal smoking on the hypospadias risks among the gene polymorphisms. The results suggest that mothers with the CYP1A1 MspI variant allele may have a decreased risk for hypospadias.     

Rb1cc1 is critical for myoblast differentiation through Rb1 regulation

Rb1-inducible coiled-coil 1 (Rb1cc1) expressed at high levels is associated with the maturation of human embryonic musculoskeletal cells. To clarify the molecular role of Rb1cc1 in muscular differentiation, we investigated the expression of Rb1cc1 and other genes that regulate differentiation in murine embryonic tissues and in C2C12 myoblasts. We also evaluated the effects of RNA interference (RNAi)-mediated Rb1cc1 knockdown on C2C12 myoblast differentiation. After Rb1cc1, Rb1 and myosin heavy chain (Myhc) were expressed in mouse embryonic muscles. The synchronous expression of Rb1cc1 and Rb1 predicted Myhc expression during C2C12 myoblast differentiation. RNAi-mediated knockdown of Rb1cc1 led to Rb1 suppression, and C2C12 myoblasts failed to differentiate. These results indicated that Rb1cc1 is a potent regulator of the Rb1 pathway and a novel mediator that plays a crucial role in muscular differentiation. Rb1cc1 expression is, thus, a prerequisite for myogenic differentiation.     

Circulatinglong non-coding RNA FEZF1-AS1 and AFAP1-AS1 serve as potential diagnostic biomarkers for gastric cancer

BackgroundGrowing evidence indicates that two long non-coding RNAs (lncRNAs), FEZ family zinc finger 1 antisense RNA 1 (FEZF1-AS1) and Actin filament associated protein 1 antisenseRNA1 (AFAP1-AS1), are highly expressed in different cancers, including gastric cancer (GC). However, the expression pattern and clinical utility of these two lncRNAs are still unknown.MethodsSerum expression levels of FEZF1-AS1 andAFAP1-AS1 were measured by quantitative real-time polymerase chain reaction (qRT-PCR). CEA and CA19-9 were detected by ARCHITET I2000 SR. Analyses were all performed using SPSS software version 20.0 (SPSS Inc., Chicago, USA). P < 0.05 was considered statistically significant.ResultsDetection of serum FEZF1-AS1 and AFAP1-AS1 showed both of them were up-regulated in GC patients compared with the normal controls (p < 0.0001), and high serum expression levels were correlated with tumor size, tumor-node-metastasis (TNM) stage and lymph node metastasis. Besides, the area under the ROC curve (AUC) demonstrated the two lncRNAs had higher diagnostic utility than CEA and CA19-9. Furthermore, when combined the two lncRNAs as a model, it yielded an AUC of 0.866, and the combination of the model, CEA and CA19-9 could observably improve diagnostic sensitivity to 95.5 %. What’s more, circulating FEZF1-AS1 and AFAP1-AS1 were significantly decreased after the GC patients underwent the operation (both p < 0.001).ConclusionOur study indicated that serum FEZF1-AS1 and AFAP1-AS1 had better sensitivity and efficiency for the diagnosis of GC and the combination of the two lncRNAs might be used as a potential prognostic indicator in GC.     

神经系统RNA结合蛋白Musashi1 RRM1的初步结晶

目的 对Musashi1发挥功能的 RRM1结构域进行结晶,得到可用来衍射的蛋白晶体,为之后的结构解析打基础。方法 通过构建Musashi1RRM1的原核表达载体,并在BL21中表达、纯化高纯度的蛋白质,通过筛选结晶体条件得到蛋白晶体。结果 通过系统筛选和优化晶体生长条件得到了蛋白晶体。结论 Musashi1 RRM1的蛋白晶体质量较好,满足蛋白晶体衍射和数据收集的要求。     

HIV-1 Nef stabilizes AP-1 on membranes without inducing ARF1-independent de novo attachment

HIV-1 Nef affects the trafficking of numerous cellular proteins to optimize viral replication and evade host defenses. The adaptor protein (AP) complexes, which form part of the cytoplasmic coat of endosomal vesicles, are key cellular co-factors for Nef. Nef binds these complexes and alters their physiologic cycle of attachment and release from membranes. Specifically, while AP-1 normally becomes cytosolic when attachment events are blocked by inhibition of the GTPase cycle of ADP-ribosylation factor-1 (ARF1), the complex remains membrane-associated in Nef-expressing cells. To investigate the mechanism of this effect, we used a permeabilized cell system to detect the de novo attachment of exogenous AP-1 to endosomal membranes. Nef did not mediate de novo attachment independently of ARF1, despite its ability to maintain the association of AP-1 with endosomal membranes when the activity of ARF1 was blocked. We conclude that Nef stabilizes AP complexes on endosomal membranes after ARF1-dependent attachment. This stabilization may facilitate coat formation and stimulate the trafficking of multiple cellular proteins.     

IL—1受体相关激酶—1和—2协同调控IL—1诱导的AP—1的活化

目的研究白介素 - 1受体相关激酶 - 1(IRAK- 1)和 IRAK- 2在白介素 - 1(IL - 1)诱导 AP- 1活化中的作用。方法L ipofectin介导反义 IRAK- 1寡核苷酸和反义 IRAK- 2寡核苷酸转染 Hep G2细胞。用逆转录 PCR法检测 IRAK - 1和 IRAK- 2m RNA表达水平 ;Western blot分析 IRAK- 1和 IRAK - 2蛋白表达水平。以 Sandwich EL ISA法检测 AP- 1的活化。结果反义IRAK- 1寡核苷酸和反义 IRAK- 2寡核苷酸通过抑制各自靶基因 m RNA和蛋白表达抑制 IL- 1诱导的 AP- 1活化 ;反义 IRAK-1寡核苷酸与反义 IRAK- 2寡核苷酸共转染 Hep G2细胞对 AP- 1的抑制作用较两者单独转染明显增强。结论 IRAK- 1和 I-RAK- 2在调控白介素 - 1诱导的 AP- 1活化时协同作用。     

Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not involved in the risk of recurrent pregnancy loss

The etiology of recurrent pregnancy loss (RPL) remains unclear, but it may be related to a possible genetic predisposition together with involvement of environmental factors. We examined the relation between RPL and polymorphisms in four genes, human aryl hydrocarbon (Ah) receptor, cytochrome P450 (CYP) 1A1, CYP1A2 and CYP1B1, which are involved in the metabolism of a wide range of environmental toxins and carcinogens. All cases and controls were women resident in Sapporo, Japan and the surrounding area. The Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotypes were assessed in 113 Japanese women with recurrent pregnancy loss (RPL) and 203 ethnically matched women experiencing at least one live birth and no spontaneous abortion (control). No significant differences in Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotype frequencies were found between the women with RPL and the controls [Ah receptor: Arg/Arg (reference); Arg/Lys and Lys/Lys, odds ratio (OR)=0.67; 95% confidence interval (CI)=0.40-1.11, CYP1A1: m1m1 (reference); m1m2 and m2m2, OR = 0.86; 95% CI = 0.53-1.40, CYP1A2: C/C and C/A (reference); A/A, OR = 1.16; 95% CI = 0.71-1.88, CYP1B1: Leu/Leu (reference); Leu/Val and Val/Val, OR = 1.18; 95% CI = 0.68-2.02]. The present study suggests that the Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not major genetic regulators in RPL.