恶性疟原虫FCC—1／HN株环子孢子蛋白基因分枝杆菌——大肠杆穿梭表达重组质粒的构建及序列测定

本文对恶性疟原虫环子孢子蛋白(circumsporozoiteprotein,CSP)基因片段进行克隆和序列测定。根据恶性疟原虫837株基因编码序列设计合成一对引物,采用PCR技术从恶性疟原虫FCC-1/HN株基因组DNA中特异扩增CSP基因片段的Ⅰ区、中央重复区、重复区后可变区和Ⅱ区;经纯化的扩增产物用BamHⅠ和KpnⅠ双酶切后,定向克隆入大肠杆菌——分枝杆菌穿梭表达质粒,转化感受态大肠杆菌DH5α,重组克隆经抗性筛选和快速凝胶电泳鉴定,再经PCR和酶切鉴定,并对重组子进行序列测定。结果表明从恶性疟原虫FCC-1／HN株基因组DNA中可特异扩增出约1171bp的基因片段,阳性重组质粒经双酶切和PCR鉴定与预期的结果一致,序列测定表明所克隆的基因和编码环子孢子抗原的基因片段相符。     

恶性疟原虫GLURP基因的体外扩增，克隆及序列分析

通过体外扩增 ,克隆恶性疟原虫海南 (FCC1 HN)株GLURP基因 ,测定其基因序列 ,了解该基因的结构及在FCC1 HN株与其它分离株间的序列差异。根据GLURP基因已知序列设计合成 3对引物 ,用PCR技术从FCC1 HN株基因组DNA中扩增 3个部分序列重叠的GLURP基因片段 ;并分别克隆入测序用PMD 18T载体。用双脱氧链末端终止法测定GLURP基因序列 ,应用软件辅助分析基因结构及进行同源性比较。恶性疟原虫FCC1 HN株GLURP基因全长 3711bp ,编码 12 36个氨基酸。FCC1 HN株与F32株GLURP基因核苷酸序列同源性为 96 2 7% ;编码氨基酸序列同源性为 95 78%。本文为继续进行FCC1 HN株GLURP抗原的免疫原性和保护性研究奠定基础。     

寄生虫学

0500640 细胞因子表达质粒对恶性疟原虫顶端膜抗原1DNA免疫的调节作用，0500641 恶性疟原虫FCC1／HN株醛缩酶编码区基因的克隆及表达，0500642 伯氏疟原虫青蓠素抗性相关的消减cDNA文库构建，0500643 弓形虫SAG1基因的表达及其诱导的细胞免疫应答，0500644 弓形虫RH株致密颗粒蛋白GRA4基因的克隆与表达。     

巴尔通体(Bartonella tribocorum)P26蛋白原核重组表达研究

目的 构建巴尔通体表面蛋白p26基因的原核重组表达载体,表达和纯化重组表达蛋白P26,并鉴定其抗原性.方法 利用PCR方法从巴尔通体B.tribocorum厦门分离株的基因组DNA中扩增出p26蛋白基因,并将该基因的编码区克隆到pGEX-4T-1表达载体中,从而构建GST-p26融合蛋白原核重组表达载体.将表达载体转化...     

肾癌相关抗原G250的原核表达、纯化及抗原活性检测

目的 构建原核表达质粒pET-42a-hG250,表达并纯化肾癌相关抗原G250融合蛋白,并检测G250融合蛋白的抗原活性.方法 利用PCR从pGEM-T-G250质粒中扩增G250基因片段(112～1242 bp),测序正确后将其克隆至原核表达载体pET-42a中构建重组载体pET-42a-hG250.将其转化至大肠杆菌BL21(DE3)中,通过IPTG诱导G250蛋白的原核表达,随后将表达的融合蛋白进行纯化.经SDS-PAGE分析后,Western blot法检测纯化的蛋白,将纯化蛋白进一步包板后用ELISA对其抗原活性进行评价.结果 酶切和测序结果证实pET-42a-hG250原核表达载体构建成功;转化后可以成功诱导并纯化出大小与预期一致的G250融合蛋白;Western b1ot法和ELISA检测证实纯化的蛋白能与特异性的抗体发生反应,显示纯化后的G250融合蛋白具有良好的免疫原性.结论 成功构建了肾癌相关抗原G250基因的原核表达载体,纯化获得了G250融合蛋白,该蛋白具有良好的抗原活性.     

牙龈卟啉菌外膜蛋白RagB的基因克隆、原核表达及其多克隆抗体的制备

目的:克隆牙龈卟啉菌外膜蛋白RagB基因、原核表达并制备牙龈卟啉菌外膜蛋白RagB多克隆抗体。方法:根据牙龈卟啉菌外膜蛋白RagB的DNA序列,设计特异性引物并经PCR扩增其基因。扩增产物经鉴定和测序分析后,与质粒pET-32a(+)重组形成pET-32a-ragB,继而转化大肠埃希菌。IPTG诱导重组蛋白的表达,再通过Ni-NTA亲和层析柱纯化后,用SDS-PAGE电泳和Western blot鉴定;获得的重组蛋白行常规家兔免疫,以制备多克隆抗体,并应用ELISA测定抗体效价。结果:PCR扩增获得编码区全长为1506bp可编码501个氨基酸的RagB基因,测序结果与GenBank公布的序列(AJ130872)完全一致;构建了pET-32a-ragB原核表达载体,获得了高表达的融合蛋白,SDS-PAGE电泳和Western blot表明具有较高的纯度;ELISA结果显示,特异性兔抗RagB的多克隆抗体效价为1×105。结论:在成功构建牙龈卟啉菌外膜蛋白RagB原核表达载体的基础上,高效表达及纯化了RagB融合蛋白,并制备了高效价的多克隆抗体。为进一步开展牙龈卟啉菌疫苗研究、建立牙龈卟啉菌感染的实验室诊断方法奠定了基础。     

人TLR2胞外区基因的原核表达及其抗体的制备

目的: 在原核系统中表达人Toll样受体2(TLR2)胞外区基因的融合蛋白并制备多克隆抗体.方法: 应用RT-PCR方法从人外周血单个核细胞中扩增TLR2胞外区基因, 将其克隆到表达载体pET-32a( )上构建重组原核表达质粒, 并在大肠杆菌中诱导表达, 目的融合蛋白经Ni-NTA亲和层析纯化, 用SDS-PAGE和 Western blot进行鉴定.用纯化的蛋白免疫新西兰白兔, 制备多克隆抗体, 采用Western blot对抗体进行鉴定, 并用ELISA分析抗体活性.结果: 成功构建了pET-32a( )-TLR2重组表达质粒并在大肠杆菌中进行表达, 获得了纯化重组蛋白, 重组蛋白免疫白兔后能够有效地刺激抗体产生, 并具有良好的免疫活性.结论: 成功地获得人TLR2胞外区基因的融合蛋白, 制备的多克隆抗体为进一步研究TLR2胞外区的功能和生物活性奠定基础.     

寄生虫学

0501961 用pfcrt点突变基因检测技术检测恶性疟原虫氯喹抗药性的初步研究；0501962 恶性疟原虫海南株FEN-1基因的克隆及序列分析；0501963 恶性疟原虫海南株（FCC1／HN）EBA-175Ⅱ区F2结构域基因的克隆与表达；0501964 抗恶性疟原虫谷螽酸脱氢酶单克隆抗体的研制与胶体金免疫层析方法的建立；0501965 伯氏疟原虫RC株和N株感染小鼠IFN-γ mRNA的表达及其对致病的影响。     

恶性疟原虫已糖转运体编码基因的体外扩增及克隆

目的：体外扩增恶性疟原虫海南分离株的已糖转运体基因(PfHT1)，并将该基因克隆至pEGFF真核表达载体内使其高效表达，为研究DNA疫苗创造条件。方法：特定PCR引物的设计；恶性疟原虫FCC1／HN株的体外培养；提取基因组DNA；PCR扩增和琼脂糖凝胶电泳分析；酶切、连接及PCR分析鉴定。结果：从恶性疟原虫簿南分离株基因组DNA中扩增出特异性的编码FfhTl的基因序列．片段太小为1516bp，克隆鉴定结果表明插人片段大小正确。结论：体外成功扩增出恶性疟原虫PfHTl编码序列，与预期长度相符，并成功构建pEGFF-Htl真核表达载体。为研究其结构、功能和免疫原性奠定基础，     

金黄葡萄球菌蛋白A与SUMO标签的融合表达及应用研究

目的 从金黄葡萄球菌菌株(ATCC6538)中克隆得到蛋白A的全长基因并对其结构及应用进行研究.方法 从该菌株的基因组DNA中克隆得到全长的葡萄球菌蛋白A基因,对其基因结构分析之后,将该基因的成熟区全长片段和抗体结合功能区片段重组到pHisSUMO原核分泌表达载体上与SUMO标签融合表达,通过ELISA的方法来鉴定融合蛋白的抗体结合活性及其稳定性,并将抗体结合功能区片段耦联到CNBr活化的琼脂糖凝胶上进行抗体的纯化.结果 首次获得了此株菌的全长蛋白A基因并发布于GenBank中,登录号为EU695225.蛋白A全长蛋白和功能区蛋白在pHisSUMO载体上得到正确高效表达,通过ELISA鉴定SUMO标签的存在,没有影响全长蛋白和功能区蛋白的抗体结合活性并有效提高了功能区蛋白的稳定性.在抗体纯化实验中,应用表达的蛋白A能够得到浓度和纯度较高的抗体并具有较好的效价.结论 克隆得到一个新的葡萄球菌蛋白A全长基因.蛋白A功能区蛋白与SUMO标签融合表达,在保持抗体结合活性的基础上提高了稳定性,并成功应用于抗体的纯化.     

Opportunities and challenges in the prevention and control of cancer and other chronic diseases: children's diet and nutrition and weight and physical activity

OBJECTIVE: The purpose of this article is to review the role of behavioral research in disease prevention and control, with a particular emphasis on lifestyle- and behavior-related cancer and chronic disease risk factors--specifically, relationships among diet and nutrition and weight and physical activity with adult cancer, and tracking developmental origins of these health-promoting and health-compromising behaviors from childhood into adulthood. METHOD: After reviewing the background of the field of cancer prevention and control and establishing plausibility for the role of child health behavior in adult cancer risk, studies selected from the pediatric published literature are reviewed. Articles were retrieved, selected, and summarized to illustrate that results from separate but related fields of study are combinable to yield insights into the prevention and control of cancer and other chronic diseases in adulthood through the conduct of nonintervention and intervention research with children in clinical, public health, and other contexts. RESULTS: As illustrated by the evidence presented in this review, there are numerous reasons (biological, psychological, and social), opportunities (school and community, health care, and family settings), and approaches (nonintervention and intervention) to understand and impact behavior change in children's diet and nutrition and weight and physical activity. CONCLUSIONS: Further development and evaluation of behavioral science intervention protocols conducted with children are necessary to understand the efficacy of these approaches and their public health impact on proximal and distal cancer, cancer-related, and chronic disease outcomes before diffusion. It is clear that more attention should be paid to early life and early developmental phases in cancer prevention.     

Pain and Effusion and Quadriceps Activation and Strength

Context:

Quadriceps dysfunction is a common consequence of knee joint injury and disease, yet its causes remain elusive.

Objective:

To determine the effects of pain on quadriceps strength and activation and to learn if simultaneous pain and knee joint effusion affect the magnitude of quadriceps dysfunction.

Design:

Crossover study.

Setting:

University research laboratory.

Patients or Other Participants:

Fourteen (8 men, 6 women; age = 23.6 ± 4.8 years, height = 170.3 ± 9.16 cm, mass = 72.9 ± 11.84 kg) healthy volunteers.

Intervention(s):

All participants were tested under 4 randomized conditions: normal knee, effused knee, painful knee, and effused and painful knee.

Main Outcome Measure(s):

Quadriceps strength (Nm/kg) and activation (central activation ratio) were assessed after each condition was induced.

Results:

Quadriceps strength and activation were highest under the normal knee condition and differed from the 3 experimental knee conditions (P < .05). No differences were noted among the 3 experimental knee conditions for either variable (P > .05).

Conclusions:

Both pain and effusion led to quadriceps dysfunction, but the interaction of the 2 stimuli did not increase the magnitude of the strength or activation deficits. Therefore, pain and effusion can be considered equally potent in eliciting quadriceps inhibition. Given that pain and effusion accompany numerous knee conditions, the prevalence of quadriceps dysfunction is likely high.Key Words: arthrogenic muscle inhibition, central activation failure, voluntary activation, muscles

Key Points

• Knee pain and effusion resulted in arthrogenic muscle inhibition and weakness of the quadriceps.
• The simultaneous presence of pain and effusion did not increase the magnitude of quadriceps dysfunction.
• To reduce arthrogenic muscle inhibition and improve muscle strength, clinicians should employ interventions that target removing both pain and effusion.

Quadriceps weakness is a common consequence of traumatic knee joint injury^1,2 and chronic degenerative knee joint conditions.^3,4 Arthrogenic muscle inhibition (AMI), a neurologic decline in muscle activation, results in quadriceps weakness and hinders rehabilitation by preventing gains in strength.⁵ The inability to reverse AMI and restore muscle function can lead to decreased physical abilities,⁶ biomechanical deficits,⁷ and possibly reinjury.⁵ Furthermore, researchers^8,9 have suggested that quadriceps weakness resulting from AMI may place patients at risk for developing osteoarthritis in the knee. In light of the substantial influence of quadriceps AMI on these clinically relevant outcomes, we need to improve our understanding of the factors that contribute to this neurologic decline in muscle activity so efforts to target and reverse it can be implemented and gains in strength can be achieved more easily.Joint injury and disease are accompanied by numerous sequelae (ie, pain, swelling, tissue damage, inflammation), so ascertaining which one ultimately leads to neurologic muscle dysfunction is difficult. Whereas a joint effusion can result in AMI,^10–12 the effects of pain are less understood despite many clinicians attributing AMI to pain. Using techniques that introduce knee pain without accompanying injury may provide insights into the role of pain in eliciting AMI.The degree of knee joint damage may play a role in the quantity of AMI that manifests. Hurley et al^13,14 demonstrated that quadriceps AMI, measured using an interpolated-twitch technique, was greater in patients with extensive traumatic knee injury (eg, fractured tibial plateau, ruptured medial collateral ligament, and medial meniscectomy) than patients with isolated joint trauma (ie, isolated anterior cruciate ligament [ACL] rupture). Similarly, patients with more knee joint symptoms (ie, greater number of symptoms and increased severity of symptoms) may present with greater magnitudes of quadriceps inhibition. Recently, investigators¹⁵ have suggested that patients with more pain display less quadriceps strength, supporting this tenet. Given that effusion and pain often present simultaneously with joint injuries and diseases, such as ACL injury and osteoarthritis, examining both the isolated and cumulative effects of these sequelae appears warranted to determine if they influence the magnitude of muscle inhibition.Experimental joint-effusion and pain models are safe and effective experimental methods that allow for the isolated examination of their effects on muscle function. The effusion model, whereby sterile saline is injected directly into the knee joint capsule,⁷ produces a clinically relevant magnitude of the joint effusion that may be present with traumatic injury. Effusion is thought to activate group II afferents responding to stretch or pressure,^16–18 which in turn may facilitate group Ib interneurons and result in quadriceps AMI.⁵ The pain model involves injecting hypertonic saline into the infrapatellar fat pad to produce anteromedial knee pain similar to that described in patients with patellofemoral pain syndrome.¹⁹ Pain is considered to initiate AMI through activation of group III and IV afferents that act as nocioceptors to signal damage or potential damage to joint structures.^16–18 The firing of these afferents then may lead to facilitation of group Ib interneurons, the flexion reflex, or the gamma loop, ultimately resulting in quadriceps inhibition.²⁰ Thus, these models allow us to create symptoms that are associated with knee injury and have the added benefit of providing a way to examine their effects in isolation.Therefore, the purpose of our study was to determine the effects of pain on quadriceps strength and activation and to learn if simultaneous pain and knee joint effusion would affect the magnitude of quadriceps dysfunction. We hypothesized that pain alone would result in quadriceps inhibition and that the magnitude of inhibition would be greater when effusion and pain were present simultaneously.     

Beta-endorphin and drug-induced reward and reinforcement

Although drugs of abuse have different acute mechanisms of action, their brain pathways of reward exhibit common functional effects upon both acute and chronic administration. Long known for its analgesic effect, the opioid beta-endorphin is now shown to induce euphoria, and to have rewarding and reinforcing properties. In this review, we will summarize the present neurobiological and behavioral evidences that support involvement of beta-endorphin in drug-induced reward and reinforcement. Currently, evidence supports a prominent role for beta-endorphin in the reward pathways of cocaine and alcohol. The existing information indicating the importance of beta-endorphin neurotransmission in mediating the reward pathways of nicotine and THC, is thus far circumstantial. The studies described herein employed diverse techniques, such as biochemical measurements of beta-endorphin in various brain sites and plasma, and behavioral measurements, conducted following elimination (via administration of anti-beta-endorphin antibodies or using mutant mice) or augmentation (by intracerebral administration) of beta-endorphin. We suggest that the reward pathways for different addictive drugs converge to a common pathway in which beta-endorphin is a modulating element. beta-Endorphin is involved also with distress. However, reviewing the data collected so far implies a discrete role, beyond that of a stress response, for beta-endorphin in mediating the substance of abuse reward pathway. This may occur via interacting with the mesolimbic dopaminergic system and also by its interesting effects on learning and memory. The functional meaning of beta-endorphin in the process of drug-seeking behavior is discussed.     

PTEN与信号转导及肿瘤

ＴＥＮ[1] (ｐｈｏｓｐｈａｔａｓｅａｎｄｔｅｎｓｉｎｈｏｍｏｌｏｇｙｄｅｌｅｔｅｄｏｎｃｈｒｏｍｏｓｏｍｅｔｅｎ)又名ＭＭＡＣ1 [2 ] (ｍｕｔａｔｅｄｉｎｍｕｔｉｐｌｙａｄａｎｃｅｄｃａｎｃｅｒ 1 )和ＴＥＰ1 [3 ] (ＴＧＦ -βｒｅｇｕｌａｔｅｄａｎｄｅｐｉｔｈｅｌｉａｌｃｅｌｌ -ｒｉｃｈｅｄｐｈｏｓｐｈａｔａｓｅ 1 ) (以下均称为ＰＴＥＮ) ,是 1 997年由 3个研究小组先后发现的一个具有双特异磷酸酶活性的抑癌基因。ＰＴＥＮ基因异常广泛存在于人类多种恶性肿瘤 ,如恶性神经胶质瘤、前列腺癌、子宫内膜癌、黑色素瘤等…     

Tobacco and alcohol and the risk of head and neck cancer

Summary We carried out two case-control studies on the relative risk of head and neck cancer in association with tobacco and alcohol consumption. The first study carried out at the ENT Department of the University hospitals of Heidelberg and Giessen (FRG) comprised 200 male patients with squamous cell cancer of the head and neck and 800 control subjects matched for sex, age, and residential area (1:4 matching design). Of the tumour patients, 4.5% had never smoked, in contrast to 29.5% of the control group. The average tobacco and alcohol consumption of the patients was approximately twice as high as in the control subjects. The highest alcohol and tobacco consumption was observed in patients suffering from oropharyngeal cancer. Tobacco and alcohol increased the risk of head and neck cancer in a dose-dependent fashion and acted as independent risk factors. In heavy smokers (> 60 pack-years) a relative risk of 23.4 (alcohol adjusted) was calculated. Combined alcohol and tobacco consumption showed a synergistic effect. The risk ratio increased more in a multiplicative than in an additive manner. Oral and laryngeal cancer were associated with the highest tobacco-associated risk values. The highest ethanol-associated risk values were associated with oropharyngeal and laryngeal cancer. The second study was carried out at the ENT Department of the University of Heidelberg on 164 males with squamous cell carcinoma of the larynx and 656 control subjects matched for sex, age and residential area (1:4 matching design). Of the cases, 4.2% had never smoked, compared with 28.5% of the control subjects. The risk of laryngeal cancer by tobacco consumption was dose dependent, reaching a maximum value of 9.1 (adjusted for alcohol) for a consumption of more than 50 tobacco-years (TY). The relative risk of laryngeal cancer associated with alcohol intake was also dose dependent, reaching a value of 9.0 (adjusted for tobacco) for a mean daily consumption of more than 75 g alcohol. An analysis of subsite specific risks showed that heavy smokers (> 50 TY) carried a nearly ten times higher risk of supraglottic cancer than of glottic cancer. The risk of supraglottic cancer from alcohol consumption was also higher than that of glottic cancer.     

Postinfectious immunodeficiency and autoimmunity: pathogenic and clinical values and implications

Autoimmunity is still a mystery of clinical immunology and medicine as a whole. The etiology and pathogenesis of autoimmune disorders remain unclear and, thus, are assessed as a balance between hereditary predisposition, triggering factors and the appearance of autoantibodies and/or self-reactive T cells. Among the immunological armamentarium, molecular mimicry, based on self-reactive T- and B-cell activation by cross-reactive epitopes of infectious agents, is of special value. Hypotheses regarding the possible involvement of molecular mimicry in the development of postinfectious autoimmunity are currently very intriguing. They provide new approaches for identifying etiological agents that are associated with postinfectious autoimmunity, paired microbial- and tissue-linked epitopes targeted for autoimmune reaction determination, postinfectious autoimmunity pathogenesis recognition and specific prevention, and therapy for autoimmune disorder development.     

类赖氨酰氧化酶2与肿瘤转移和发生

类赖氨酰氧化酶2(lysyl oxidase-like 2,LOXL2)是赖氨酰氧化酶(lysyl oxidase,LOX)基因家族的成员之一,其表达产物能促进胶原沉积.LOXL2的过表达能促进纤维化,并与肿瘤侵袭、转移及不良预后有关.目前大部分学者认为LOXL2是一种转移促进基因,也有实验支持其是一种肿瘤抑制基因.研究发现LOXL2可以通过激活Snail/Ecadherin通路或Src/FAK通路促进转移.LOXL2有望作为肿瘤生物标志物,用于预后判断,成为一个新的治疗靶点.     

Muscle strength and serum testosterone,cortisol and SHBG concentrations in middle-aged and elderly men and women

Forty healthy males (M) and females (F) divided into two different age groups i.e. M50 years (range 44–57; n= 9), F50 years (range 43–54; n= 9), M70 years (range 64–73; n= 11) and F70 years (range 63–73; n= 11) volunteered as subjects for examination of muscle cross-sectional area (CSA) and maximal voluntary isometric force production characteristics of the leg extensor muscles and serum androgen and sex hormone binding globulin (SHBG) concentrations. The CSA in the male groups was greatly larger (P < 0.01) than in the female groups and both elderly groups demonstrated slightly (n.s.) smaller values in the CSA than the two middle-aged groups. Maximal force of 2854 ± 452 N in M50 was greater (P < 0.05) than that of 2627 ± 752 N recorded for F50 as well as the force of 2787 ± 843 in M70 was greater (P < 0.001) than that of 1849 ± 295 recorded for F70. The force between F50 and F70 differed significantly (P < 0.05) from each other. The maximal rate of force production in M50 was greater (P < 0.01) than in F50 as well as in M70 greater (P < 0.001) than in F70. Both middle-aged groups demonstrated greater (P < 0.05) values than the respective elderly groups of the same sex. The individual values in the CSA correlated with the values in maximal force both in the middle-aged subjects (r= 0.66; P < 0.01) and in the elderly subjects (r= 0.69; P < 0.01). The mean concentration of serum testosterone in M50 was slightly (n.s.) greater than in M70 and in F50 significantly (P < 0.05) greater than in F70. Serum SHBG levels were lower in the males (P < 0.01) than in the females and serum testosterone/SHBG ratio in M70 and in F70 were lower (P < 0.05) than in M50 and in F50, respectively. In the females significant positive correlations were observed between the individual values in serum testosterone concentration and the values both in the CSA (r= 0.46; P < 0.05) and in maximal force (r= 0.62; P < 0.01) as well as between serum testosterone/SHBG ratio and both the CSA (r= 0.55; P < 0.05) and maximal force (r= 0.68; P < 0.01). The present results imply that the decreasing basal level of blood testosterone over the years in aging people, especially in females, may lead to decreasing anabolic effects on muscles thus having an association with age-related declines in the maximal voluntary neuromuscular performance capacity in aging people.