幽门螺杆菌hpaA核酸疫苗的构建及免疫原性检测

目的 构建含幽门螺杆菌(Hp)hpaA基因的核酸疫苗。方法 抽提Hp标准菌株CCUG17874基因组DNA，应用聚合酶链式反应(PCR)技术从基因组DNA扩增hpaA基因，克隆入pUCmT载体，检测hpaA基因序列，经过一系列酶切、连接反应将其克隆入真核表达载体plRES，转入大肠杆菌，筛选阳性克隆，通过PCR和酶切反应鉴定。通过脂质体法将构建好的重组载体pIRES-hpaA转染COS-7细胞，SDS-PAGE及Western印迹法检测pIRES-hpaA表达HpaA蛋白的免疫原性。结果 成功扩增出长约750bp的hpaA基因．测序结果表明扩增出的hpaA基因与Hp hpaA序列一致，PCR和酶切鉴定结果证实hpaA基因克隆入真核表达载体pIRES，成功构建了含hpaA基因的Hp核酸疫苗pIRES-hpaA，并经Western印迹法检测到特异性蛋白条带。结论 构建了hpaA基因的Hp核酸疫苗，为进一步探索其免疫作用奠定了基础。     

幽门螺杆菌hpaA基因在乳酸菌中的表达及免疫原性分析

摘要 目的 在乳酸菌中表达幽门螺杆菌(HP)hpaA基因,口服免疫小鼠后检测其免疫原性。方法 PCR方法从基因组中扩增基因hpaA,将其克隆至表达载体pNICE:sec,将重组质粒转化乳酸菌NZ9000株,筛选鉴定阳性菌落,诱导表达的hpaA蛋白用SDS-PAGE和Western blot进行鉴定。将雌性ICR(CV级)小鼠随机分为4组,分别用PBS、携带空质粒的乳酸菌、携带pNICE:sec-hpaA的乳酸菌、灭活的HP灌胃。免疫7次后检测其特异性IgG和IgA的产生。结果 扩增hpaA基因并构建了重组原核表达质粒pNICE:sec-hpaA,转化乳酸菌NZ9000后经nisin诱导可表达Mr分子量约29KD的重组蛋白,表达量约占菌体总蛋白量的9.3%,Western blot分析其能与幽门螺杆菌抗血清特异性反应,具有良好的免疫原性。携带pNICE:sec-hpaA质粒的乳酸菌刺激产生的IgG水平明显高于携带空质粒组,与灭活HP组没有明显的差异,但其刺激产生的IgA水平明显高于其它组（P<0.05）。结论 表达hpaA蛋白的乳酸菌口服免疫小鼠后,能够刺激小鼠产生特异的IgG和IgA,可作为幽门螺杆菌口服疫苗候选抗原。为乳酸菌作为抗原递送载体的研究和HP口服疫苗的开发提供一定的实验基础。     

幽门螺杆菌金属蛋白酶原核表达载体的构建与重组表达及纯化北大核心CSCD

目的构建幽门螺杆菌(Helicobacter pylori, Hp)金属蛋白酶(metalloprotease, Mtp)基因mtp与麦芽糖结合蛋白基因mbp融合表达载体,诱导Mtp重组表达并纯化表达产物,为Hp致病机制和疫苗研究奠定基础。方法应用PCR从Hp郑州分离株MEL-Hp27的DNA扩增mtp基因,经纯化回收后与克隆载体pMD19-T连接,并进行测序验证。对重组质粒pMD19-T-mtp双酶切,酶切目的片段克隆至表达载体pMAL-c2X,然后用重组质粒pMAL-mtp转化大肠埃希菌(E.coli TB1)。从大肠埃希菌重组子中提取重组质粒,进行酶切和测序鉴定。用分光光度计测定重组菌的生长曲线。用IPTG诱导mtp表达,用SDS-PAGE法分析表达产物,并用Amylose树脂预装柱纯化Mtp蛋白。结果 PCR扩增的mtp基因片段长度为621 bp,重组菌株的酶切和测序鉴定正确;重组菌生长曲线显示重组质粒的转入对受体菌的生长无显著影响;Mtp诱导表达和纯化产物的SDS-PAGE显示,Mtp表达产物为相对分子质量为66.4×10^3的水溶性融合蛋白(rMtp),纯化产物的纯度约为85.6%。结论成功构建mtp基因与麦芽糖结合蛋白mbp基合表达载体,并纯化制备了重组蛋白,为探索Mtp在Hp感染发病机制和抗Hp感染疫苗研究奠定了基础。     

幽门螺杆菌金属蛋白酶原核表达载体的构建与重组表达及纯化

目的构建幽门螺杆菌(Helicobacter pylori, Hp)金属蛋白酶(metalloprotease, Mtp)基因mtp与麦芽糖结合蛋白基因mbp融合表达载体,诱导Mtp重组表达并纯化表达产物,为Hp致病机制和疫苗研究奠定基础。方法应用PCR从Hp郑州分离株MEL-Hp27的DNA扩增mtp基因,经纯化回收后与克隆载体pMD19-T连接,并进行测序验证。对重组质粒pMD19-T-mtp双酶切,酶切目的片段克隆至表达载体pMAL-c2X,然后用重组质粒pMAL-mtp转化大肠埃希菌(E.coli TB1)。从大肠埃希菌重组子中提取重组质粒,进行酶切和测序鉴定。用分光光度计测定重组菌的生长曲线。用IPTG诱导mtp表达,用SDS-PAGE法分析表达产物,并用Amylose树脂预装柱纯化Mtp蛋白。结果 PCR扩增的mtp基因片段长度为621 bp,重组菌株的酶切和测序鉴定正确;重组菌生长曲线显示重组质粒的转入对受体菌的生长无显著影响;Mtp诱导表达和纯化产物的SDS-PAGE显示,Mtp表达产物为相对分子质量为66.4×10~3的水溶性融合蛋白(rMtp),纯化产物的纯度约为85.6%。结论成功构建mtp基因与麦芽糖结合蛋白mbp基合表达载体,并纯化制备了重组蛋白,为探索Mtp在Hp感染发病机制和抗Hp感染疫苗研究奠定了基础。     

幽门螺杆菌lpp20基因的克隆与表达

目的克隆及表达幽门螺杆菌(Helicobacter pylori)lpp20基因,为探索高效的防治幽门螺杆菌感染的疫苗抗原和诊断抗原奠定基础.方法用PCR方法从幽门螺杆菌的染色体DNA上扩增出lpp20基因片段,将目的基因插入的表达载体pMAL-C2X中,用重组质粒转化大肠杆菌(E.coli TB1),并在E.coli TB1中进行表达.用SDS-PAGE方法对表达产物进行分析.结果用PCR方法扩增的lpp20基因长度约为540 bp;经酶切、PCR和测序分析,插入到载体的基因片段与文献报道的基因序列一致;SDS-PAGE的结果显示,目的基因表达产物的相对分子质量为18k1Da,融合蛋白的表达量占全菌总蛋白的34%.结论本研究中构建的pMAL-C2X与lpp20基因重组质粒在E coli TB1中能够高效表达目的基因.该重组子的构建为幽门螺杆菌lpp20基因的研究建立了重要的基础.     

幽门螺杆菌1pp20基因的克隆与表达

目的 克隆及表达幽门螺杆菌（Helicobacter pylori)lpp20基因，为探索高效的防治幽门螺杆菌感染的疫苗抗原和诊断抗原奠定基础。方法 用PCR方法从幽门螺杆菌的染色体DNA上扩增出lpp20基因片段，将目的基因插入的表达载体pMAL-C2X中，用重组质粒转化大肠杆菌（E.coli TB1），并在E.coli TB1中进行表达。用SDS-PAGE方法对表达产物进行分析。结果 用PCR方法扩增的lpp20基因长度约为540bp；经酶切、PCR和测序分析，插入到载体的基因片段与文献报道的基因序列一致；SDS-PAGE的结果显示，目的基因表达产物的相对分子质量为18kDa，融合蛋白的表达量占全菌总蛋白的34%。结论 本研究中构建的pAML-C2X与lpp20基因重组质粒在E.coli TB1中能够高效表达目的基因。该重组子的构建为幽门螺杆菌lpp20基因的研究建立了重要的基础。     

幽门螺杆菌重组Bb-hpaA-vacA疫苗的构建

目的 构建幽门螺杆菌( Hp) 重组双歧杆菌( Bb) Bb-hpaA-vacA疫苗。方法 通过 PCR 分别扩增hpaA和vacA抗原编码基因, 然后采用gene SOEing 剪接hpaA和vacA, 得到hpaA-vacA融合基因;将该融合基因定向克隆到大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT, 构建重组质粒 pGEX-hpaA-vacA,电穿孔法将该质粒导入Bb, 构建幽门螺杆菌重组hpaA-vacA 疫苗。结果 PCR成功扩增出分子量约为1500 bp的 hpaA-vacA融合基因,双酶切证实hpaA-vacA融合基因成功插入pGEX-1λT中,并成功转化入双歧杆菌,构建rBb-hpaA-vacA疫苗。结论 成功构建螺杆菌rBb-hpaA-vacA疫苗,为该疫苗的进一步研究奠定了基础。     

幽门螺杆菌lpp20基因与麦芽糖结合蛋白基因的融合表达与纯化

目的 研究幽门螺杆菌(Helicobacter pylori，H．pylori)lpp20基因与麦芽糖结合蛋白基因的融合表达产物的纯化方法，为幽门螺杆菌基因工程疫苗的制备建立基础。方法 采用本研究室分离的HpMEL-HP27菌株提取染色体DNA，用PCR方法从HP染色体DNA上扩增出lpp20基因片段，将目的基因插人到表达载体pMAL-c2X中，用重组质粒转化大肠杆菌(E．coil TB1)。采用IPTG进行诱导表达。用多糖树脂(amylose resin)作为填充料，制备层析柱，将可溶性菌体蛋白进行亲和层析。应用SDS-PAGE方法对纯化产物进行分析。结果 融合蛋白的分子量约为60kDa，融合蛋白的表达量约占全菌总蛋白的34％；纯化后的融合蛋白纯度达90％以上。结论 与麦芽糖结合蛋白基因融合的lpp20基因在E．coli TB1中能够高效表达；用多糖树脂作为填充料，进行亲和层析的方法，具有良好的纯化效果。     

幽门螺杆菌融合蛋白HCTV的表达及免疫原性研究

目的构建表达幽门螺杆菌融合蛋白粘附素-细胞空泡毒素A-霍乱毒素B亚单位(HpaA-CtxB-VacA,HCTV)的原核表达载体,并诱导表达,为制备具有治疗与预防作用的多联疫苗奠定基础。方法用PCR技术从pQE-hctB扩增hpaA和ctxB目的基因片段,从pQE30-V质粒扩增出vacA基因,同时插入表达载体pQE-30,构建成pQE-hctv。再将pQE-hctv转化大肠杆菌DH5α,经IPTG诱导表达。SDS-PAGE分析表达结果。Western blotting鉴定其抗原性。结果融合蛋白的相对分子量约为68000,可溶性表达占全菌的15%以上,经亲和层析后可获得纯度为90%以上的重组蛋白。Western blotting分析其能分别与HpaA抗血清、VacA抗血清和CT抗血清特异性反应。结论重组表达质粒pQE30-hctv表达成功,而且具有各自蛋白的抗原性,为进一步研究融和蛋白的功能和制备集预防和治疗为一体的Hp候选口服疫苗提供基础。     

幽门螺杆菌融合蛋白HspA-UreB在大肠杆菌中的表达与鉴定

目的在大肠杆菌中表达幽门螺杆菌(简称Hp)HspA-UreB融合蛋白,并探索其免疫反应性,为Hp基因工程疫苗的研制奠定基础。方法用PCR方法扩增郑州分离Hp菌株MEL-HP27的hspA和ureB基因,分别克隆入pNEB193中。测序后,回收两种基因片段,并以hspA-ureB的顺序连接插入原核表达载体pMAL-C2x进行融合表达。采用蛋白印迹法对表达产物进行鉴定。结果特异PCR法和酶切鉴定证实融合基因hspA-ureB克隆入表达载体中;重组质粒转化大肠杆菌TB1后,经IPTG诱导3h,SDS-PAGE电泳显示在119kDa处出现一条特异蛋白带,即麦芽糖结合蛋白(MBP)与HspA-UreB的融合表达形式,约占细菌总体蛋白含量的31%;该融合蛋白与Hp免疫小鼠血清和Hp阳性病人血清的Westernblot分析结果显示,在119kDa处出现特异杂交带。结论成功地在大肠杆菌中实现了Hp融合蛋白HspA-UreB的高效表达,并证实其具有良好的免疫反应性。     

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.     

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.     

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.