幽门螺杆菌尿素酶B亚单位编码基因的克隆与序列分析

目的 幽门螺杆菌(Helicobacter pylori)尿素酶B亚单位的编码基因(ureB)的克隆和序列分析，为H．pylori基因工程疫苗的研究奠定基础。方法应用PCR方法获得国内分离H．pylori菌株MEI，HP27和国际标准参考株H．pylori的ureB基因，通过定向克隆的方法分别插入克隆载体pNEB193中，用质粒酶切电泳和特异PCR方法鉴定重组质粒。克隆基因经测序后进行核苷酸和氨基酸的同源性比较。结果 重组质粒经双酶切后得到1．71kb的ureB基因片段，特异PCR可扩增出ureB基因片段，证实H．pylori ureB基因的重组克隆质粒构建成功。经测序，国内分离H．pylori MEL-HP27的ureB基因全长1710bp( Genbank收录号：AY295085)，编码由569个氨基酸残基组成的肽链，ureB基因序列与GenBank公布的H．pylori相应基因同源性高达96．08％～98．30％，氨基酸序列同源性在98．77％．99．82％之间。结论 成功克隆了MEL-HP27菌株的ureB基因，其核苷酸序列与国际参考株NCTC11637的同源性为97．67％。     

幽门螺杆菌热休克蛋白A亚单位编码基因的克隆与序列分析

目的 幽门螺杆菌(Helicobacter pylori)热休克蛋白A亚单位的编码基因(hspA)克隆和序列分析，为H.pyiori基因工程疫苗的研究奠定基础。方法 应用PCR方法获得国内分离H.pylori菌株MEI，HP27和国际标准参考株H.pylori的hspA基因，通过定向克隆的方法分别插入克隆载体pNEB193中，用质粒酶切电泳和特异PCR方法鉴定重组质粒。克隆基因经测序后进行核苷酸和氨基酸的同源性比较。结果 重组质粒经双酶切后得到0．35kb的hspA基因片段，特异PCR可扩增出hspA基因片段，证实H.pylori hspA基因的重组克隆质粒构建成功。经测序，国内分离HpMEL-HP27的hspA基因全长357bp(Genbank收录号：AY295084)，编码由118个氨基酸残基组成的肽链，hspA基因序列与GenBank公布的H.pylorl相应基因同源性高达95．20％～97．48％，氨基酸序列同源性在95．76％～97．46％之间。结论 克隆了H．pylori菌株MEL-HP27的hspA基因，其核酸序列与国际参考株NCTC11637同源性为97．48％。     

幽门螺杆菌NCTC 11637 cagT基因的克隆及序列分析

目的:克隆幽门螺杆菌(H pylori)NCTC 11637 cag T(HP0532)的编码基因,并分析其核苷酸序列.方法:应用PCR技术从H pylori基因组DNA中扩增cag T编码基因片段,克隆至pGEM-T载体后,再将其定向插入pQE30载体中,双酶切鉴定筛选阳性克隆,并进行序列分析.结果:NCTC 11637 cag T基因全长843 bp(GenBank登录号为EF114758),编码280个氨基酸,与GenBank公布的其他H pylori菌株基因序列的核苷酸同源性为97%-99%.结论:成功克隆了cag T基因,为进一步研究其生物学功能奠定了基础.     

幽门螺杆菌热休克蛋白60核酸疫苗的构建和免疫原性鉴定

背景:核酸疫苗是新兴的第三代疫苗。目前幽门螺杆菌(H.pylori)疫苗的研究主要集中在减毒、灭活和亚单位疫苗方面,有关H.pylori核酸疫苗的研究罕见报道。目的:构建含H.pylori热休克蛋白60(hsp60)基因的核酸疫苗,并鉴定其表达蛋白的免疫原性。方法:抽提H.pylori标准菌株CCUG17874基因组DNA为模板,应用聚合酶链反应(PCR)扩增hsp60基因并插入测序载体pUCmT,测定插入的hsp60基因的核苷酸序列。通过一系列酶切、连接反应将hsp60基因克隆入真核表达载体pIRES,然后转化入感受态大肠杆菌DH5α,筛选阳性克隆,行酶切和PCR鉴定。采用脂质体法将所构建的重组载体pIRES鄄hsp60转染真核细胞COS鄄7,蛋白质印迹法(Western blotting)检测pIRES鄄hsp60表达蛋白的免疫原性。结果:以H.pylori基因组DNA为模板,PCR扩增出约1 640 bp的hsp60基因片断,测序结果表明其与GenBank中H.pylorihsp60原序列的同源性达98%。酶切和PCR鉴定结果证实hsp60基因已克隆入真核表达载体pIRES,成功构建了含hsp60基因的H.pylori核酸疫苗pIRES鄄hsp60。蛋白质印迹法检测显示经pIRES鄄hsp60转染的COS鄄7细胞在约60 kDa(1 Da=0.992 1 u)处出现特异性蛋白条带。结论:成功构建了具有免疫原性的含hsp60基因的H.pylori核酸疫苗,为进一步探索其免疫作用奠定了     

幽门螺杆菌27kDa外膜蛋白的基因克隆和特性鉴定

背景：幽门螺杆菌（H.pylori)是全球人群中感染范围最广的致病菌，现已被公认为慢性胃炎和消化性溃疡的重要致病因子，并与胃腺癌和胃淋巴瘤的形成密切相关。而H.pylori疫苗可能成为控制这一全球范围感染的有效措施。其研制和开发已成为目前研究热点。目的：探索研制H.pylori疫苗的新途径，对H.pylori27kDa外膜蛋白（OMP27）进行基因克隆和特性鉴定。方法：培养和收集H.pylori菌株NCTC11637，采用酚；氯仿抽提和纯化基因组DNA。分别设计引物P1和P2，并以该基因组DNA为模板，以聚合酶链反应（PCR）方法扩增OMP27基因片段。构建pQE30-OMP27重组表达载体时，pQE30质粒载体和纯化的PCR产物均用限制性内切酶KpnⅠ和HindⅢ双酶切，再用T4DNA连接酶将双酶切后的目的基因片段OMP27重组于pQE30的相应酶切位点之间，连接产物转化大肠杆菌XL1-Blue。挑选转化克隆，提取质粒，并进行KpnⅠ和HindⅢ双酶切和PCR方法鉴定，1%琼脂糖凝胶电泳观察酶切和PCR扩增结果，经测序分析确认后，筛选出插入目的基因的阳性克隆，命名为pQE30-OMP27，挑取单个含重组质粒pQE30-OMP27的工程菌（XL1-Blue)阳性克隆，进行培养和IPTG诱导表达后，经SDS-聚丙烯酰胺凝胶电泳（PAGE）和Western blot进行蛋白表达和抗原性鉴定。结果：该目的基因的PCR产物全长723bp。编码241个氨基酸残基组成的多肽（约27kDa)。SDS-PAGE和Western blot检测表明，电泳图谱上显示出1条相对分子量约27kDa的新生蛋白带，占细菌总蛋白的5%，并能与H.pylori感染小鼠血清发生特异性反应，表明重组H.pyloriOMP27具有良好的抗原性。结论：H.pyloriOMP27有望成为新的H.pylori疫苗候选分子，并可作为抗原用于H.pylori感染患者血清膜蛋白抗体的检测。     

幽门螺杆菌尿素通道蛋白基因ureI的检测、克隆及序列分析

目的:检测镇江地区幽门螺杆菌(Helicobacterpylori,H pylori)尿素通道蛋白基因ureI,并进行克隆和序列分析.方法:从胃十二指肠疾病患者胃黏膜组织中分离培养获得60例H pylori,PCR扩增检测ureI基因,部分菌株的ureI基因克隆至pMD18-T载体上,进行测序和序列分析.结果:60例H pylori菌株的ureI检出率为100%,成功克隆了8株来源于慢性胃炎、消化性溃疡和胃癌的H pylori菌株ureI并进行了序列分析.结果表明不同来源H pylori菌株之间ureI核苷酸和氨基酸序列同源性均高达95.6%以上.结论:ureI基因在H pylori中高度保守,可以作为鉴定H pylori的分子诊断标志.     

幽门螺杆菌OipA、HopX、HopW基因的检测及序列分析

目的:检测幽门螺杆菌(H pylori)标准菌株NCTC11637及临床菌株外膜蛋白的三种基因(OipA、HopX、HopW及比较NCTC11637中三种基因与国际标准菌株(26695和J99)的同源性,为Hpylori疫苗的筛选提供实验依据.方法:运用PCR方法检测标准菌株NCTC 11637及27例临床菌株中的三种基因,并对其进行测序.运用GenBank比较与国际标准菌株(26695和J99)的同源性.结果:在标准菌株NCTC11637中检测到三种基因.在27例临床菌株中,有10例检测到OipA基因.在23例中检测到HopX基因,在27例中均检测到HopW基因.NCTC11637中OipA基因与26695和J99的核苷酸序列同源性分别为95.38%、94.60%,均低于两株国际标准菌株的同源性95.50%.HopX基因的同源性分别为94.90%和95.15%,同样低于两株国际标准菌株的同源性96.49%.HopW基因与26695和J99的同源性分别为97.38%、94.90%,两株标准菌株的同源性为95.20%.结论:在标准菌株NCTC11637和临床菌株中检测到三种基因.H pylori HopW基因核苷酸序列具有一定的保守性.     

幽门螺杆菌Mr26000外膜蛋白真核载体的构建及表达蛋白的鉴定

目的　构建含人幽门螺杆菌 (Helicobacterpylori,H pylori) 2 6 0 0 0外膜蛋白编码基因的真核重组载体 ,并在COS- 7细胞中表达 ,为核酸疫苗的开发奠定基础。方法　从原核表达质粒 pET32a(+) / 5 94中 ,酶切H pylori 2 6 0 0 0外膜蛋白编码基因片段 ,将目的基因与同样进行酶切、纯化的载体pcDNA3 1进行连接 ,而后转化并筛选含有目的基因的重组载体pcDNA3 1/ 5 94 ,并在COS - 7细胞中表达 ,以RT -PCR ,Dotblot法检测其表达产物。 结果　经酶切证实插入的基因片段为H pylori 2 6 0 0 0外膜蛋白编码基因 ;采用RT -PCR方法 ,能够从转染COS - 7细胞中扩增出一条与目的基因大小一致的DNA片段 ;同时Dotblot法等检测显示 ,该重组质粒能够在COS - 7细胞中表达目的蛋白。结论　成功地构建了真核重组载体 pcDNA3 1/ 5 94 ,并在COS - 7细胞中表达 ,为H pylori核酸疫苗的研制奠定了良好的基础。     

幽门螺杆菌黏附素HpaA蛋白在大肠杆菌中表达及其免疫保护作用

目的表达和纯化幽门螺杆菌(Helicobacter pylori,H.pylori)黏附素HpaA蛋白,研究其免疫活性和对小鼠的免疫保护作用。方法诱导TB1(pMAL-c2X-hpaA)表达H.pylori黏附素HpaA蛋白,Amyloss树脂预装柱进行分离、纯化,Western blot鉴定其免疫学活性;纯化蛋白免疫小鼠后,H.pylori国际标准菌株NCTC11637攻击感染,观察H.pylori定植情况及免疫保护效果。结果获得了高纯度且免疫活性良好的目的蛋白;HpaA蛋白免疫小鼠的保护率为53.33%(8/15),与对照组统计学差异显著(P=0.002)。结论纯化HpaA蛋白对小鼠有免疫保护作用,可作为H.pylori基因工程疫苗的候选组分。     

携带幽门螺杆菌热休克蛋白B亚单位基因重组活减毒鼠伤寒沙门疫苗菌的构建和鉴定

背景：幽门螺杆菌(h.pylori)是慢性活动性胃炎和消化性溃疡的重要致病菌，以减毒鼠伤寒沙门菌为载体构建活疫苗己成为探索新型H．pylori疫苗的重要途径。目的：构建携带H．pylori热休克蛋白B亚单位(hspB)基因的重组活减毒鼠伤寒沙门疫苗菌。方法：应用基因工程技术将1640bp的hspB基因克隆入原核表达质粒pTrc99A。对重组质粒进行序列测定，并将测序结果与基因文库中H.pylori-hspB的基因和蛋白序列进行BLAST分析，再将重组质粒导入活减毒鼠伤寒沙门菌SL3261。结果：重组质粒经聚合酶链反应(PCR)和双酶切，证实构建了携带hspB基因的重组原核表达质粒pTrc99A—hspB，后者成功转化活减毒鼠伤寒沙门菌SL3261。所构建的重组质粒pTrc99A—hspB中所含的H.pylori-hspB与基因文库中量H.pylori-hspB基因和蛋白的同源性均为97％。结论：成功构建并鉴定了携带量H.pylori-hspB基因的重组活减毒鼠伤寒沙门疫苗菌，为研制H.pylori口服疫苗奠定了基础。     

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.     

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.     

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.