乙型肝炎病毒全长基因的扩增及克隆

目的 建立血清标本经聚合酶链反应(PCR)扩增乙型肝炎病毒(HBV)全长基因的新方法,并初步进行克隆分析,从而为HBV分子生物学及致病机理研究奠定基础.方法 针对位于整个HBV基因序列中的两个缺口区设计含酶切位点的引物,扩增3.2 kb HBV全长DNA,经酶切及连接后克隆人PUC18载体.结果 用PCR方法成功获得了3.2 kb HBV DNA,经Sal Ⅰ酶切克隆入PUC18载体,获得重组质粒PUC18/HBV3.2,酶切鉴定和PCR扩增证实重组质粒中含有3.2 kb HBV全长DNA,成功构建了HBV全基因克隆.结论 成功建立了从患者血清中克隆HBV全基因组的方法,为从全基因水平深入研究乙型肝炎病毒变异与致病机理的关系奠定了基础.     

乙型肝炎病毒拉米夫定耐药株的重组逆转录病毒载体及其包装细胞系的建立

目的构建1.3拷贝乙型肝炎病毒(HBV)拉米夫定耐药株的重组逆转录病毒载体及其包装细胞系。方法在野毒株1.3拷贝HBV载体的基础上采用定点突变的方法将rtL180M,rtM204V位点突变,构建拉米夫定耐药病毒株,并通过PCR、限制性内切酶酶切,分别以正向和反向将野毒株和耐药株分别连接于逆转录病毒载体pLNCX2的相应酶切位点,构建成重组逆转录病毒载体,经双酶切及PCR扩增鉴定。重组载体转染包装细胞,筛选培养细胞克隆并进行病毒滴定。结果通过双酶切、PCR扩增、测序鉴定证实成功构建了1.3拷贝HBV拉米夫定耐药株的逆转录病毒载体。成功建立包装细胞系并测得病毒滴度均为1×105cuf/ml。结论含正向及反向1.3拷贝HBV拉米夫定耐药的重组逆转录病毒载体及其包装细胞系构建成功,可以用于拉米夫定耐药细胞模型研究。     

340例慢性乙型肝炎患者乙型肝炎病毒多位点耐药相关突变分析

目的 分析慢性乙型肝炎患者HBV逆转录酶基因与核苷(酸)类似物耐药相关的12个位点上的突变情况及其临床意义.方法 提取血清HBV DNA,扩增HBV逆转录酶基因,对PCR产物进行DNA双向测序,对测序成功的样本进行基因型分析.检测逆转录酶基因12个位点上的碱基突变情况,分析不同核苷(酸).类似物使用情况、患者的耐药相关突变情况及不同核苛(酸)类似物耐药的突变形式. 结果 检出拉米夫定耐药突变63例,阿德福韦耐药突变10例,恩替卡韦耐药突变8例,替比夫定耐药突变1例.拉米夫定耐药突变中以M204V和M204I最常见,前者通常伴随L180M突变,后者常单独出现,阿德福韦耐药中以N236T±A181位碱基替换为主;恩替卡韦耐药突变发生在拉米夫定耐药基础上,以T184位碱基替换为主;替比夫定的耐药突变为M204I.少数未接受过核苷(酸)类似物治疗的患者也可检出耐药相关突变.结论 检测HBV逆转录酶基因多位点耐药相关突变,有助于临床及时发现和确认乙型肝炎患者是否存在HBV耐药,合理进行抗病毒治疗.     

BCG_3174基因敲除卡介苗菌株的构建及鉴定

目的为了证实BCG_3174是BCG基因组中存在的潜在抑制凋亡功能基因,拟构建BCG_3174敲除菌株并进行鉴定。方法以BCG中国株的基因组DNA为模板,PCR扩增BCG_3174基因,扩增产物用Van91Ⅰ酶切并回收产物。载体p0004S转化E.coli DH5α培养,用Van91I酶切载体p0004S。将酶切BCG_3174基因片段的左、右臂以及酶切载体p0004S的3.6 kb和1.6 kb两个片段连接,转化入DH5α中培养,筛选阳性克隆并测序鉴定,构建重组质粒p0004S-ΔBCG_3174。将载体phAE159转化E.coli HB101培养,提取phAE159后以PacI酶切,同时以PacI酶切重组质粒p0004S-ΔBCG_3174,以T4连接酶连接。加入E.coli HB101感受态细胞并培养,提取质粒后用PacI酶切鉴定,阳性克隆命名为phAE159-ΔBCG_3174,电击转化耻垢分枝杆菌mc2155感受态细胞。取BCG菌液与噬菌体裂解液混合共培养,提取敲除菌株的基因组DNA,鉴定正确的菌株命名为ΔBCG_3174。结果凝胶成像显示扩增产物与预期目标大小一致,左臂大小为887 bp,右臂大小为931 bp。阳性克隆测序确证左、右臂正确连入p0004S且序列无突变,p0004S-ΔBCG_3174序列与预期一致。PacⅠ酶切phAE159载体产生约42 kb和4 kb两条片段,酶切p0004S-ΔBCG_3174质粒产生7.1 kb片段,该片段亚克隆入PacⅠ酶切的phAE159载体后产生重组质粒phAE159-ΔBCG_3174,酶切鉴定证实phAE159-ΔBCG_3174构建正确。BCG_3174基因敲除的BCG基因组PCR扩增产物大小约为5.6 kb,与预期大小相符,BCG_3174基因敲除BCG菌株ΔBCG_3174构建成功。结论成功制备BCG_3174基因敲除的BCG菌株ΔBCG_3174,为BCG_3174基因的功能研究奠定了基础。     

乙肝病毒核苷类似物耐药位点分析

目的:建立巢式PCR结合测序检测乙型肝炎病毒(HBV)耐药位点的方法,确定患者的HBV基因型和耐药突变情况。方法:采用特异性引物进行巢式PCR扩增HBV DNA,产物测序后,利用MEGA 3.1对测序结果进行分析判断有无耐药突变的产生。结果:100例乙肝病毒载量阳性患者共检出48例有耐药突变,其中概率最高的是rt204位点的YVDD 15例、YIDD 13例;拉米夫定组的耐药概率明显高于对照组;耐药组的ALT和HBV DNA水平明显高于非耐药组。结论:传统的核苷类似物如拉米夫定会产生耐药,耐药一旦发生肝功能和HBV DNA会明显升高,巢式PCR结合测序法可对患者体内的HBV耐药突变位点进行有效检测,有利于指导临床用药。     

弓形虫ROP2基因的克隆与鉴定

目的 体外扩增弓形虫棒状体分泌抗原2（ROP2）靶基因，构建真核表达载体pc-DNA3-ROP2。 方法 收集、纯化RH株弓形虫速殖子，提取基因组DNA；根据基因库ROP2基因序列设计合成1对引物，应用PCR扩增ROP2基因片段，回收纯化后克隆入TA载体质粒pUCm-T；用限制性内切酶EcoRⅠ、HindⅢ双酶切该重组子，将切下的ROP2基因在T4DNA连接酶作用下插入真核细胞表达载体质粒pc-DNA3，并进一步作双酶切、PCR及测序鉴定。 结果 以弓形虫基因组DNA为模板，PCR扩增出1.7 kb ROP2基因片段，克隆于pUCm-T载体中，再将ROP2基因亚克隆于真核表达载体质粒pc-DNA3，经筛选鉴定，构建pc-DNA3-ROP2重组质粒；测序结果显示，重组质粒包含了ROP2蛋白基因读码框内的完整序列，能完整表达ROP2的抗原蛋白。 结论 弓形虫ROP2基因片段，经TA克隆及亚克隆，构建弓形虫pc-DNA3-ROP2重组质粒。     

幽门螺杆菌glmM DNA重组质粒的构建及体外表达

目的构建幽门螺杆菌(Hp)pVAX1-glmMDNA重组质粒,体外转染SGC-7901细胞并鉴定其表达蛋白的抗原性。方法RT-PCR方法从国际标准株NCTC11637中获取glmM全长基因,克隆插入T载体。测序后经酶切、连接反应将glmM的开放读码框架定向克隆入真核表达载体pVAX1,酶切初步鉴定后测序确认。通过脂质体法将pVAX1-glmMDNA重组质粒转染SGC-7901细胞,检测其转染效率,确定转染成功后RT-PCR法检测glmM在转录水平的表达,ELISA法鉴定表达蛋白的抗原性。结果DNA测序结果显示扩增出的glmM全长基因与GenBank公布的HpglmM序列有96%的同源性。PCR、酶切和测序结果证实glmM目的基因成功克隆入真核表达载体pVAX1。重组质粒转染的细胞经RT-PCR扩增获得1.4kb片段,与目的基因大小相符。ELISA结果显示重组质粒转染细胞的超声破碎物及培养上清液中抗原的滴度比对照组高2倍以上。结论成功构建了pVAX1-glmMDNA重组质粒,其表达蛋白具有良好的抗原性,为进一步研究其抗Hp感染的效果及制备相应有效DNA疫苗奠定基础。     

CARP基因重组腺病毒载体的构建

目的：利用Adeasy-1系统，构建并鉴定CARP基因重组腺病毒载体。方法：PCR扩增含有CARP全长cDNA的片段，经测序验证无误后，亚克隆至pAdTrack-CMV穿梭质粒，再与pAdEasy．1质粒在大肠杆菌BJ5 183中进行同源重组产生腺病毒载体质粒。经过抗性筛选以及酶切鉴定得到阳性的重组质粒，再在293细胞中进行包装扩增，利用Adeasy系统上的绿色荧光蛋白标签鉴定病毒表达。结果：测序证实PCR产物为CARP全长cDNA；抗性筛选及酶切鉴定均表明重组腺病毒载体构建成功；转染293细胞3天后可见绿色荧光，回收病毒可以重复感染293细胞，证明病毒包装成功。     

乙型肝炎病毒C型重组杆状病毒的构建及鉴定

目的构建乙型肝炎病毒（HBV）中国株及其拉米夫定耐药相关变异株的重组杆状病毒.方法将1.2倍HBV基因组（血清adr型、基因C型）连接至杆状病毒的转移载体pFastBacI上,然后转化入DH10Bac菌株中,在细菌内的辅助质粒辅助作用下与杆状病毒基因组Bacmid发生位点特异性转座,形成含有HBV基因组的重组Bacmid,转染昆虫sf9细胞后,包装产生HBV重组杆状病毒vAcHBVc.采用连续聚合酶链反应（PCR）引入方法进行定点突变,构建YVDD变异株质粒,并按照上述方法构建HBV YVDD变异株重组杆状病毒vAcHBVYVDD,扩增并滴定.为便于观察重组杆状病毒的转染和扩增效率及病毒滴度测定,同时构建了含绿色荧光蛋白（GFP）的HBV重组杆状病毒vAcGFPHBVc.结果 HBV野毒株和变异株重组杆状病毒均构建成功,通过免疫空斑及TCID50定量测定病毒滴度为106～108pfu/ml左右.结论应用Bac to Bac杆状病毒表达系统可有效快速地构建HBV C型重组杆状病毒,以用于HBV体外复制系统的建立,为HBV野毒株及变异株生物学特性的研究及抗病毒药物的筛选提供技术平台.     

乙型肝炎病毒表达载体pHY106在筛选抗病毒药物中的意义

目的应用新的HBV表达载体,建立体外筛选临床抗HBV药物的方法。方法克隆对拉米夫定耐药的CHB患者体内HBV全基因组,然后将其亚克隆到HBV真核表达载体pHY106,体外转染Huh7细胞,检测转染后不同时间HBsAg、HBeAg、HBV DNA及HBV复制中间体水平。分析拉米夫定和阿德福韦对HBV基因表达和复制的抑制作用,指导临床用药。结果成功构建全部8个HBV临床分离株全基因组真核表达质粒,HBV聚合酶基因YMDD有5个产生YVDD突变,3个产生YIDD突变。该质粒上HBV基因能在Huh7细胞中进行复制和表达,拉米夫定不能体外抑制HBV复制,阿德福韦抑制了HBV在Huh7细胞中的复制,抑制程度与药物浓度相关。阿德福韦在患者体内也抑制了HBV的复制。结论新建立的体外筛选抗HBV药物的方法能用于临床快速筛选抗HBV药物,对CHB的治疗用药具有指导意义。     

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.     

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.     

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.