幽门螺杆菌重组VacA-HpaA IgY的制备

目的:采用基因工程技术大量表达、提纯幽门螺杆菌(H pylori)细胞空泡毒素毒性片段,和黏附素片段的融合蛋白,以此为抗原,制备高效价的抗VacA-HpaA蛋黄抗体(IgY)．方法:大量培养重组菌pQE30-VacA-HpaA- DH5α,诱导表达获得融合蛋白VacA—HpaA, Ni2 -NTA树脂纯化．将纯化的重组蛋白免疫鸡,水稀释法提取IgY,硫酸铵沉淀法纯化浓缩VacA—HpaA IgY．ELISA法测定抗体产生的时间-效价变化,SDS—PAGE分析纯度, Bradford法测定蛋白含量,Western blot检测其分别对VacA和HpaA抗原的特异性,ELISA法检测效价．结果:重组蛋白主要以包涵体形式表达,免疫鸡后提取的IgY可与该蛋白发生免疫反应, IgY效价总体上随免疫时间增加而升高．经纯化、浓缩后,获得纯度为60％左右,效价为1: 12 800的VacA-HpaA IgY,蛋白浓度为22 g／L, Westem blot显示在Mr27 000和Mr30 000处分别有相应条带,ELISA检测与VacA和HpaA反应的效价分别为1:3200和1:6400(P<0．01)．结论:成功制备了高浓度、高效价的抗重组VacA-HpaA的特异性IgY．     

抗幽门螺杆菌粘附素HpaA卵黄抗体的制备

目的研制高效价特异性的抗幽门螺杆菌黏附素鸡卵黄抗体免疫球蛋白抗体。方法用纯化重组幽门螺杆菌黏附素(Helicobacter pyloriadhesin A,rHpaA)抗原免疫22周龄罗曼鸡,母鸡免疫应答后,在卵黄中产生抗幽门螺杆菌黏附素抗体;经硫酸铵法初步纯化浓缩后;以间接ELISA鉴定抗体效价及免疫学活性;采用Bradford法测定蛋白含量;并用SDS-PAGE法测定分子量及鉴定抗体纯度。结果母鸡初次免疫第10d即有抗体产生,初次免疫后75d左右抗体效价超过1∶10000;最高可达为1∶12800。卵黄抗体经硫酸铵法纯化后,用SDS-PAGE分析,出现两条蛋白带,纯度达95%以上。其含量为10.56mg/ml蛋黄。结论本研究首次研制并鉴定抗黏附素卵黄抗体,开拓了我国预防幽门螺杆菌感染的新领域,分析了抗体在产蛋期卵黄液中表达的进度,为今后该抗体的持续、高质量诱导奠定基础。有望获得高效价、高产量的抗黏附素的IgY抗体(IgY-HpaA),为制备口服制剂开辟了广阔前景。     

抗弓形虫特异性鸡卵黄抗体的制备与鉴定

目的　制备高效价特异性鸡卵黄免疫球蛋白 ( yolkimmunoglobulin ,IgY)抗体 ,为弓形虫病防治的研究探索新的方法和途径 ;方法　用提纯的弓形虫RH株抗原免疫育龄种鸡 ,获取大量含高效价IgY的卵黄 ,经水稀释法初步纯化浓缩后 ,用ELISA检定其免疫学活性 ,应用SDS -PAGE分析、鉴定IgY的分子基础 ;结果　获得ELISA效价为 1× 10 7的抗弓形虫IgY抗体 ,经SDS -PAGE分析 ,出现 1条蛋白带 ,其分子量大小根据电泳迁移率计算 ,初步鉴定为 4 6kDa用弓形虫抗原免疫Lyh ern种鸡制备的IgY抗体效价高、特异性强 ;水稀释法初步提纯获得的IgY抗体分子量为 4 6kDa,为在弓形虫病的特异性免疫学诊断、预防和治疗中的应用奠定基础。     

抗旋毛虫肌幼虫ES抗原IgY的制备及鉴定

目的制备抗旋毛虫肌幼虫排泄-分泌(excretory-secretory,ES)抗原的鸡卵黄免疫球蛋白(IgY),测定其效价及用于检测抗原的敏感性。方法 4只24w龄罗曼母鸡用旋毛虫肌幼虫ES抗原经大腿外侧与胸部肌肉免疫4次(首次剂量为500μg/只,加强剂量为250μg/只),每次间隔10d。取免疫前和首次免疫后42d的鸡蛋卵黄,用水稀释法提取IgY,考马斯亮蓝法测定蛋白含量,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹(Western blot)及间接荧光抗体试验(IFAT)对IgY进行分析,ELISA检测纯化后IgY的效价及检测抗原的敏感性。结果罗曼鸡经ES抗原免疫后,每枚鸡蛋经提纯后均可得到约70mg抗体,SDS-PAGE表明纯化的IgY有2条主要蛋白带,分子量为67kDa、23kDa,Western blot与IFAT发现提纯的IgY可识别肌幼虫虫体与ES抗原。IgY的抗体效价为1∶107,IgY-夹心ELISA检测旋毛虫抗原的敏感性为1.17ng/mL。结论制备的抗旋毛虫ES抗原的IgY具有较高的效价与敏感性。     

重组刚地弓形虫Peroxiredoxin蛋白的纯化及抗血清制备

目的对原核系统表达的重组刚地弓形虫Peroxiredoxin(rTgPrx)蛋白进行镍亲和层析纯化,免疫大鼠,制备抗血清。方法优化rTgPrx表达条件,获得大量的可溶性蛋白,经镍亲和层析法纯化后皮下注射免疫Wistar大鼠,间接ELISA法测定血清抗体效价。结果在表达菌液A600为0.5,IPTG浓度为0.2 mmol/L,37℃诱导10 h时,可溶性rTgPrx表达量较高;用镍亲和层析纯化获得纯的目的蛋白,免疫大鼠后诱导产生高滴度抗体血清,效价达1∶12 800以上。结论镍亲和层析法纯化的rTgPrx具有免疫原性,用该蛋白免疫大鼠可获得高效价的抗rTgPrx血清。     

幽门螺杆菌重组VacA蛋黄抗体的体外活性

目的:制备幽门螺杆菌(Helicobacter pylon)重组VacA的鸡蛋黄抗体IgY(VacA-IgY),了解其理化特性和生物学活性,评价其抗H pylori感染的作用．方法:以纯化的重组VacA免疫产蛋鸡,水稀释法提取蛋黄抗体(VacA-IgY),硫酸铵沉淀法纯化IgY．将一定浓度的VacA-IgY在不同温度的水浴中维持一定时间,评价其耐热性;在不同 pH的Tris-HCI中孵育一定时间,评价其耐酸性;加入胃蛋白酶并作用一定时间后,评价其对酶消化的耐受作用．以上评价均采用ELISA 法测定抗体效价变化．采用Hela细胞体外培养 MTT法分析VacA-IgY对H pylori细胞毒活性的中和作用．结果:本实验制备的VacA-IgY在70℃水浴 15 min后活性保持约50％;在pH≥5时,抗体活性几乎无改变,pH<5时,抗体活性下降较快,pH2．0左右,抗体活性几乎丧失;pH4．0时, 60 kU／L胃蛋白酶作用1 h,抗体活性几乎无变化,2 h后活性仍保持50％以上．VacA-IgY能浓度依赖性地中和H pylori菌体蛋白的Hela细胞毒活性．20 mg／L超声提取物即可降低1／2的 Hela细胞增殖能力,80-320 mg／L的VacA-IgY 能完全中和H pylori菌体蛋白的Hela细胞毒活性(P<0．01)．结论:成功制备了重组VacA的IgY,且具有较好的耐热性、耐胃酶消化和一定的耐酸性; 在其体外具有中和H pylori细胞毒活性的作用．     

抗日本血吸虫SEA鸡卵黄抗体的制备与鉴定

目的 制备特异性抗日本血吸虫可溶性虫卵抗原（SEA）的鸡卵黄免疫球蛋白（IgY）并检测其特异性、敏感性。 方法 用日本血吸虫SEA经翅膀下静脉和皮下免疫25周龄海兰母鸡4次（首次剂量为60 μg/只，加强剂量为30 μg/只），每次间隔10 d。取免疫前和首次免疫后35 d的鸡蛋卵黄，分别用水稀释法提取IgY，BCA法测定蛋白含量，并进行十二烷基磺酸钠?鄄聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹（Western blotting）分析，ELISA检测纯化后IgY特异性和敏感性。分别提取免疫后不同时间的卵黄抗体，观察抗体效价的变化。 结果 海兰母鸡经SEA首次免疫后35 d，每枚蛋经提纯后可得到约61 mg抗体，经SDS-PAGE和Western blotting分析，纯化的IgY有1条主要蛋白带，相对分子质量（Mr）为130 000，并可被SEA识别。母鸡初次免疫后第10天，经SDS-PAGE和ELISA分析，鸡蛋卵黄内即有抗体产生，初次免疫后31 d效价可达1∶1 600。双抗体夹心ELISA结果显示纯化后的IgY有较高的敏感性，可检测到的SEA达2.4 ng/ml。 结论 制备的抗SEA鸡卵黄抗体的特异性和敏感性均较高。     

抗旋毛虫鸡卵黄抗体的活性检测

目的制备特异性抗旋毛虫的鸡卵黄免疫球蛋白,研究其免疫应答规律及其稳定性。方法用纯化的旋毛虫幼虫免疫育龄种鸡,经水稀释法初步纯化浓缩后获得卵黄抗体。应用酶联免疫吸附试验检测卵黄抗体的效价及其敏感性和稳定性。结果通过免疫可成功诱导出高效价的抗旋毛虫抗体,效价可达1∶106以上,免疫应答持续时间可达34周以上。免疫后卵黄抗体敏感性和血清抗体敏感性无显著性差异(P>0.05)。稳定性试验表明IgY敏感性高,在pH4～10及温度不超过60℃条件下活性稳定。结论成功制备出高效价的特异性抗旋毛虫卵黄抗体,其敏感性高,有一定的耐热、耐酸碱性,可进一步用于旋毛虫感染的研究。     

兔抗阴道毛滴虫重组蛋白AP65多克隆抗体的制备、纯化及鉴定

目的制备、纯化并鉴定兔抗阴道毛滴虫重组蛋白AP65的多克隆抗体。方法日本大耳兔多次免疫重组蛋白AP65．间接ELISA法检测抗体效价，用饱和硫酸铵法和nprotein Asepharose4FF柱纯化多克隆抗体，Western blot检测免疫反应性。结果第3次加强免疫后，间接ELISA法检测血清效价达到1：25600，用硫酸铵法纯化多克隆抗体纯度为56％，而后采用亲合层析柱nprotein Asepharose 4FF再次纯化。纯度达到80％。Western blot鉴定兔抗重组蛋白AP65多克隆抗体能识别重组蛋白AP65。结论成功制备了高效价、高纯度的抗阴道毛滴虫重组蛋白AP65多克隆抗体。     

酶联免疫吸附技术测定鸡蛋黄中抗幽门螺杆菌IgY活性

目的:探索测定抗Hpylori-IgY的方法. 方法:用间接ELISA法测定幽门螺旋杆菌(Hpylori)超声破碎物免疫鸡所产鸡蛋中抗H pylori-IgY的效价.用间接ELISA法检测抗Hpylori-IgY的酸碱及酶稳定性. 结果:用幽门螺旋杆菌的超声破碎物免疫产蛋母鸡,可得到高效价的抗幽门螺旋杆菌IgY.IgY的酸稳定性好,耐胃蛋白酶. 结论:间接ELISA法简单、准确,可作为测定IgY的标准方法.     

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.     

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.     

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.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.