人可溶型破骨细胞分化因子的克隆原核表达及活性分析

目的克隆人可溶型破骨细胞分化因子（sODF）基因，构建融合表达载体，在大肠杆菌中表达，并制备抗体。方法采用反转录聚合酶链反应（RT-PCR）法得到人可溶型破骨细胞分化因子（ODF）编码区cDNA，克隆入原核表达载体pGEX-4T-3，转化大肠杆菌感受态细胞JM109，0．1mmol／L β—D一半乳糖苷（IPTG）诱导后收集菌体蛋白，行十二烷基硫酸钠-多丙烯酰胺冻胶电泳（SDS—PAGE）。纯化蛋白，体外诱导破骨细胞分化。结果获得人sODF基因cDNA，构建原核表达载体pGEX—ODF，转化菌株可表达人GST—ODF蛋白，蛋白相对分子质量约为43000，Western分析证实为GST—sODF融合蛋白。纯化后的表达产物体外培养人外周血淋巴细胞有破骨细胞形成，象牙片上有骨吸收陷窝形成。结论成功获得人sODF基因cDNA，并在大肠杆菌中表达了人GST-ODF融合蛋白。成功表达的人sODF蛋白有诱导破骨细胞分化的活性。为该蛋白的进一步应用研究提供了依据。     

人新的生长激素异形体基因的克隆及其在大肠杆菌中的表达

目的　构建含新的生长激素异形体 (GHv)基因全长编码区的pGEM TEasy质粒和GHv 谷胱甘肽转硫酶 (GST)融合蛋白的表达质粒并在大肠杆菌表达出其融合蛋白。方法　采用克隆和亚克隆技术构建GHv GST融合基因的表达质粒 ,转化大肠杆菌BL2 1并用异丙基硫代半乳糖苷 (IPTG)诱导其表达GHv GST融合蛋白 ,谷胱甘肽 Sepharose 4B纯化试剂盒纯化融合蛋白。 结果　含GHv GST融合基因表达质粒的大肠杆菌表达出相对分子质量约 43 0 0 0的GHv GST融合蛋白 ,SDS PAGE结果显示纯化的融合蛋白为单一条带。结论　成功构建成GHv GST融合基因的表达质粒并且GHv GST融合蛋白在大肠杆菌BL2 1中有高表达。上述工作为制备多抗 ,深入研究GHv基因的功能奠定了基础。     

甲状腺激素反应蛋白1的原核生物表达

目的 应用基因工程技术表达甲状腺激素反应蛋白1(TRP-1)。方法 应用RT-PCR方法，从新生大鼠脑组织RNA中扩增编码TRP-1的cDNA片段，构建表达型重组质粒，经DNA序列分析确证后，在大肠杆菌中表达，以Western印迹来初步鉴定是否表达了目的融合蛋白，用亲和层析纯化融合目的蛋白，并以SDS-PAGE电泳测定其相对分子质量。结果 所获特异PCR产物正确地重组入Pinpoint Xa-1表达载体中。Western印迹表明经异丙基硫代半乳糖苷诱导的原核细胞表达产生了目的融合蛋白，亲和层析得到了纯度较高、相对分子质量约23400的融合目的蛋白。结论 应用原核细胞表达体系成功地表达了TRP-1融合蛋白，为进一步研究其在脑发育中的所起的作用以及该蛋白的其它功能提供了可能。     

GST—FHL2融合蛋白的表达及纯化

目的高效表达和纯化可溶性GST—FHL2融合蛋白。方法（1）PCR法扩增FHL2（Four and a half LIM domains2）基因的编码片段，分别在5′端和3′端加上EcoR Ⅰ和Xho Ⅰ酶切位点，并克隆进入原核表达载体pGEX-4T-1；（2）利用异丙基硫代-β-D-半乳糖苷（IPTG）诱导重组质粒pGEX4T-1-FHL2在大肠杆菌B121（DE3）中表达同时带有谷胱甘肽-S-转移酶（GST）标签的融合蛋白；（3）超声法裂解大肠杆菌，应用谷胱苷肽琼脂糖树脂纯化可溶的GST—FHL2融合蛋白；（4）通过SDS—PAGE和Western blot验证GST—FHL2的表达。结果（1）成功构建pGEX-4T-1-FHL2重组质粒，测序结果证明FHL2与载体的GST在同一读框；（2）0．1mmol／L的IPTG在23℃的条件下能诱导可溶性GST—FHL2融合蛋白高效表达；（3）在Western blot分析中，GST—FHL2能被鼠抗GST单克隆抗体特异性识别，条带所在位置和GST-FHL2的分子量相符。结论正确构建pGEX-4T-1-FHL2重组质粒，在大肠杆菌BL21中高效表达GST—FHL2融合蛋白，经谷胱苷肽琼脂糖树脂纯化得到高纯度的可溶性GST-FHL2融合蛋白。     

Sj28GST基因克隆和高效表达产物的纯化

目的　从日本血吸虫中国大陆株成虫cDNA文库中克隆 2 8kDa谷胱甘肽 -S -转移酶 (Sj2 8GST)基因 ,获得用于疫苗和T细胞表位研究的高纯度重组融合蛋白 2 8GST -TRX。方法　应用引物特异性PCR技术 ,从日本血吸虫中国大陆株成虫cDNA文库中扩增Sj2 8GST基因 ,经琼脂糖凝胶电泳结合碱基序列分析鉴定后 ,采用定向克隆技术构建Sj2 8GST -TRX融合蛋白重组表达载体并诱导其在大肠杆菌BL2 1(DE3)的表达 ,用Western -blotting测定以融合蛋白方式表达的Sj2 8GST的免疫原性 ,通过包涵体纯化结合Ni+柱亲和层析获得高纯度重组融合蛋白。结果　Sj2 8GSTPCR产物为约 6 4 0bp ,其碱基序列 (6 33bp)和推导的氨基酸序列 (2 11aa)与GenBank报道一致 ;获得了Sj2 8GST -TRX重组质粒 ;2 8GST -TRX融合蛋白在大肠杆菌以包涵体形式高效表达 ;在分子量为约 4 3kDa处有一能与羊抗Sj2 8GST抗体产生特异性反应的含 2 8GST融合蛋白条带 ;包涵体纯化法结合Ni+ 亲和层析可获得高纯度的重组融合蛋白。结论　获得到了日本血吸虫中国大陆株 2 8GST分子的全长基因和高纯度重组 2 8GST -TRX融合蛋白 ,该融合蛋白具有天然Sj2 8GST免疫原性。     

广州管圆线虫ASP基因的克隆及原核表达

目的对广州管圆线虫ASP基因的完整开放读码框进行克隆、表达及重组蛋白的免疫性分析。方法以广州管圆线虫幼虫cDNA文库中含有ASP基因的质粒为模板,扩增目的基因,进一步将其克隆到原核表达质粒pET-30a(+)中,重组质粒转化大肠杆菌BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,Ni-IDA亲和层析纯化表达产物。免疫印迹(Western blotting)分析重组蛋白的免疫性。结果广州管圆线虫ASP基因的编码区含有366个碱基,编码121个氨基酸,相对分子量(Mt)为13398.26Da。重组质粒pET-30a(+)-ASP构建成功,IPTG诱导获得可溶性表达的重组蛋白,经亲和层析获得的纯化蛋白可被广州管圆线虫病人血清识别。结论广州管圆线虫ASP基因可在原核表达系统中获得具有免疫性的高效表达。     

重组人神经肽Y融合蛋白的原核表达、纯化及其多克隆抗体制备与鉴定

目的：构建人神经肽Y（NPY）基因的原核表达载体,诱导表达重组蛋白,制备兔抗人NPY抗血清。方法：取已构建好且经测序确认无误的再组质粒pET28a—NPY转化大肠杆菌BL21（DE3）,IPTG诱导表达融合蛋白,并经SDS—PAGE检测和Western印迹鉴定,表达产物包涵体经Ni^2＋-NTA亲和层析纯化,以纯化后的融合蛋白pET28a—NPY为抗原免疫家兔,获得抗血清,Western blotting、ELISA法鉴定获得的抗血清。结果经IPTG诱导含有pET28a—NPY重组质粒的DE3菌,表达出重组人NPY融合蛋白。重组蛋白经Ni^2＋-NTA亲和层析进行纯化后,得到了较高纯度的融合蛋白,用纯化的融合蛋白免疫家兔制备了多克隆抗体,Western blotting、ELISA法证实多克隆抗体制备成功。结论：成功表达了人NPY蛋白并获得了其多克隆抗体,为进一步研究人NPY蛋白的功能奠定了基础。     

华支睾吸虫F0-ATP合酶b亚基基因的克隆表达和重组蛋白的免疫原性分析

目的 对华支睾吸虫F₀-ATP合酶b亚基（CsF₀-ATP-synt_B）基因进行克隆、表达及重组蛋白的免疫原性分析。 方法 以华支睾吸虫成虫cDNA文库中含有F₀-ATP合酶b亚基基因的质粒为模板，扩增该基因（去除线粒体靶向序列的成熟肽基因），将其克隆到原核表达质粒pET-28a（+）中，转化大肠埃希菌BL21（DE3），异丙基硫代-β-D-半乳糖苷（IPTG）诱导表达，经亲和层析获得的纯化表达产物免疫SD大鼠，制备抗血清。蛋白质印迹（Western blotting）分析重组蛋白的免疫原性。 结果 华支睾吸虫F₀-ATP合酶b亚基成熟肽基因的编码区含813个碱基，编码271个氨基酸，相对分子质量（Mr）为31 171.9。经亲和层析获得的重组蛋白可被其免疫的大鼠血清识别。 结论 华支睾吸虫F₀-ATP合酶b亚基基因可在原核表达系统中获得具有免疫原性的高效表达。     

埃及伊蚊黄蛋白c基因的克隆、表达及其体外抗凝血作用

目的 克隆表达埃及伊蚊黄蛋白c (Aael-yellow-c)基因，并探讨r Aael-yellow-c蛋白体外抗凝血作用。方法 RT-PCR扩增Aael-yellow-c成熟肽基因，纯化后的片段与pEASY-E1载体连接，转化至大肠埃希菌DH5α感受态细胞，取菌液进行PCR、双酶切和测序鉴定。0.1 mol/L异丙基-β-D-硫代半乳糖苷诱导蛋白表达后，镍柱亲和层析纯化重组蛋白，采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹（Western blotting）分析重组蛋白表达情况。比浊法观察不同浓度r Aael-yellow-c蛋白对二磷酸腺苷诱导的血小板聚集活性影响。手工法检测不同浓度r Aael-yellow-c蛋白对人血浆凝血酶原时间（PT）、部分凝血活酶时间（APTT）及凝血酶时间（TT）的影响。采用SPSS 18.0统计软件进行统计学分析，组间差异比较采用t检验。结果Aael-yellow-c基因CDS区为1 287 bp，含27个氨基酸组成的信号肽，成熟肽序列长1 200 bp，编码399个氨基酸。RT-PCR扩增获得Aael-yellow-c...     

重组人神经肽Y融合蛋白的表达、纯化及生物信息学分析研究

目的 在大肠杆菌中表达人神经肽Y,并对之进行纯化、鉴定及生物信息学分析.方法 取已构建好且经测序确认无误的重组质粒pET28a-NPY转化大肠杆菌BL21(DE3),IPTG诱导表达融合蛋白,并经SDS-PAGE检测和Western印迹鉴定,表达产物包涵体经Ni2+-NTA亲和层析纯化.然后利用相关在线软件进行生物信息学分析NPY蛋白.结果 经IPTG诱导含有pET28a-NPY重组质粒的DE3菌,表达出重组人NPY融合蛋白.重组蛋白经Ni2+-NTA亲和层析进行纯化后,得到了较高纯度的融合蛋白.经相关在线软件分析后获得了NPY的相关生物学特性.结论 重组质粒pET28a-NPY在大肠杆菌DE3中成功表达,亲和层析纯化后获得较高纯度融合蛋白,并对NPY蛋白的生物学特征进行了预测,为进一步研究其生物学功能及其抗体的研制奠定了基础.     

Three layer functional model and energy exchange concept of aging process

Relying on a certain degree of abstraction, we can propose that no particular distinction exists between animate or living matter and inanimate matter. While focusing attention on some specifics, the dividing line between the two can be drawn. The most apparent distinction is in the level of structural and functional organization with the dissimilar streams of ‘energy flow’ between the observed entity and the surrounding environment. In essence, living matter is created from inanimate matter which is organized to contain internal intense energy processes and maintain lower intensity energy exchange processes with the environment. Taking internal and external energy processes into account, we contend in this paper that living matter can be referred to as matter of dissipative structure, with this structure assumed to be a common quality of all living creatures and living matter in general. Interruption of internal energy conversion processes and terminating the controlled energy exchange with the environment leads to degeneration of dissipative structure and reduction of the same to inanimate matter, (gas, liquid and/or solid inanimate substances), and ultimately what can be called ‘death.’ This concept of what we call dissipative nature can be extended from living organisms to social groups of animals, to mankind. An analogy based on the organization of matter provides a basis for a functional model of living entities. The models relies on the parallels among the three central structures of any cell (nucleus, cytoplasm and outer membrane) and the human body (central organs, body fluids along with the connective tissues, and external skin integument). This three-part structural organization may be observed almost universally in nature. It can be observed from the atomic structure to the planetary and intergalactic organizations. This similarity is corroborated by the membrane theory applied to living organisms. According to the energy nature of living matter and the proposed functional model, the decreased integrity of a human body's external envelope membrane is a first cause of the structural degradation and aging of the entire organism. The aging process than progresses externally to internally, as in single cell organisms, suggesting that much of the efforts towards the restoration and maintenance of the mechanisms responsible for structural development should be focused accordingly, on the membrane, i.e., the skin. Numerous reports indicate that all parts of the human body, like: bones, blood with blood vessels, muscles, skin, and so on, have some ability for restoration. Therefore, actual revival of not only aging tissue of the human body's membrane, but the entire human body enclosed within, with all internal organs, might be expected. We assess several aging theories within the context of our model and provide suggestions on how to activate the body's own anti-aging mechanisms and increase longevity. This paper presents some analogies and some distinctions that exist between the living dissipative structure matter and inanimate matter, discusses the aging process and proposes certain aging reversal solutions.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status—the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.