鸡传染性支气管炎病毒核蛋白基因在大肠杆菌中的表达及抗原性初步研究

为了研究重组鸡传染性支气管炎病毒核蛋白能否作为群特异性诊断抗原加以应用,将鸡传染性支气管炎病毒国内分离株IBV—LX4核蛋白基因亚克隆于大肠杆菌原核表达载体pPROEX^TM HT中。构建拟表达重组鸡传染性支气管炎病毒核蛋白的重组质粒pPROEX^TM HT—N。经核苷酸序列测定后,阳性质粒转化大肠杆菌DH5α,并进行诱导表达。表达产物经SDS-PAGE、Western blot鉴定,表明核蛋白在大肠杆菌中获得了表达,表达产物是分子量分别为约56kD和45kD的蛋白,其中分子量为56kD的蛋白表达量约为菌体总蛋白的13％。包涵体被6mol盐酸胍裂解后。通过镍离子亲和树脂进行了纯化,并用纯化的分子量为56kD的重组蛋白免疫新西兰白兔,所获得的兔抗鸡传染性支气管炎病毒核蛋白多克隆抗体,分别与不同致病型的鸡传染性支气管炎病毒进行琼脂扩散反应,结果表明。该多克隆抗体可与各不同致病型毒株发生反应。这初步表明重组鸡传染性支气管炎病毒核蛋白。可作为群特异性诊断抗原用于该病毒的诊断中。     

慢性支气管炎患者下呼吸道感染病原菌分布及耐药性分析

目的:研究老年慢性支气管炎患者合并下呼吸道感染病原菌分布以及耐药性。方法:选取2009年1月到2013年12月我院收治的老年慢性支气管炎患者合并下呼吸道感染患者261例,采集所有患者的痰液,然后进行病原菌鉴定和药敏试验。结果:261例患者中,144例革兰阴性杆菌感染(55.2%),51例革兰阳性杆菌感染(19.5%),66例真菌感染(25.3%),其中混合感染者36例(13.8%)。革兰阴性杆菌以肺炎克雷伯菌最多(18.4%),革兰阳性杆菌以金黄色葡萄球菌最多(9.2%)。革兰阴性杆菌对亚胺培南的耐药性最低,其次是头孢哌酮和阿米卡星,对氨苄西林耐药率最高。金黄色葡萄球菌和表皮葡萄球菌对青霉素的耐药率均为100.0%,均对万古霉素敏感,其次是对环丙沙星敏感。结论:老年慢性支气管炎患者合并下呼吸道感染以革兰阴性杆菌感染为主,真菌和混合感染也占一定的比例,应该引起注意。     

α-Galactosidase from Alfalfa Seeds (Medicago sativa L.)

An α-galactosidase from alfalfa seeds was purified 140-fold by ammonium sulfate fractionation, and column chromatography on Sephadex G-100, DEAE- and CM-Sephadex. Polyacrylamide-gel electrophoresis of the purified enzyme showed a single protein band. The molecular weight was estimated to be approximately 57,000 by gel-filtration. The purified enzyme hydrolyzed p-nitrophenyl α-d-galactoside more rapidly than raffinose. The maximal enzyme activities were obtained at pH 4.0 and 5.5 for p-nitrophenyl α-d-galactoside and at 4.5 for raffinose. The enzyme was shown to be inhibited by Hg²⁺ and Ag⁺ ions, and d-galactose.     

Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166–247 aa, S1 gene; 501–515 aa, S1 gene; 8–30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 μg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD₄₅₀ value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.     

Visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus

Autophagy is a highly conserved cellular response to starvation that leads to the degradation of organelles and long-lived proteins in lysosomes and is important for cellular homeostasis, tissue development and as a defense against aggregated proteins, damaged organelles and infectious agents. Although autophagy has been studied in many animal species, reagents to study autophagy in avian systems are lacking. Microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) is an important marker for autophagy and is used to follow autophagosome formation. Here we report the cloning of avian LC3 paralogs A, B and C from the domestic chicken, Gallus gallus domesticus, and the production of replication-deficient, recombinant adenovirus vectors expressing these avian LC3s tagged with EGFP and FLAG-mCherry. An additional recombinant adenovirus expressing EGFP-tagged LC3B containing a G120A mutation was also generated. These vectors can be used as tools to visualize autophagosome formation and fusion with endosomes/lysosomes in avian cells and provide a valuable resource for studying autophagy in avian cells. We have used them to study autophagy during replication of infectious bronchitis virus (IBV). IBV induced autophagic signaling in mammalian Vero cells but not primary avian chick kidney cells or the avian DF1 cell line. Furthermore, induction or inhibition of autophagy did not affect IBV replication, suggesting that classical autophagy may not be important for virus replication. However, expression of IBV nonstructural protein 6 alone did induce autophagic signaling in avian cells, as seen previously in mammalian cells. This may suggest that IBV can inhibit or control autophagy in avian cells, although IBV did not appear to inhibit autophagy induced by starvation or rapamycin treatment.     

Genetic susceptibility for chronic bronchitis in chronic obstructive pulmonary disease

Background

Chronic bronchitis (CB) is one of the classic phenotypes of COPD. The aims of our study were to investigate genetic variants associated with COPD subjects with CB relative to smokers with normal spirometry, and to assess for genetic differences between subjects with CB and without CB within the COPD population.

Methods

We analyzed data from current and former smokers from three cohorts: the COPDGene Study; GenKOLS (Bergen, Norway); and the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE). CB was defined as having a cough productive of phlegm on most days for at least 3 consecutive months per year for at least 2 consecutive years. CB COPD cases were defined as having both CB and at least moderate COPD based on spirometry. Our primary analysis used smokers with normal spirometry as controls; secondary analysis was performed using COPD subjects without CB as controls. Genotyping was performed on Illumina platforms; results were summarized using fixed-effect meta-analysis.

Results

For CB COPD relative to smoking controls, we identified a new genome-wide significant locus on chromosome 11p15.5 (rs34391416, OR = 1.93, P = 4.99 × 10^-8) as well as significant associations of known COPD SNPs within FAM13A. In addition, a GWAS of CB relative to those without CB within COPD subjects showed suggestive evidence for association on 1q23.3 (rs114931935, OR = 1.88, P = 4.99 × 10^-7).

Conclusions

We found genome-wide significant associations with CB COPD on 4q22.1 (FAM13A) and 11p15.5 (EFCAB4A, CHID1 and AP2A2), and a locus associated with CB within COPD subjects on 1q23.3 (RPL31P11 and ATF6). This study provides further evidence that genetic variants may contribute to phenotypic heterogeneity of COPD.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00608764","term_id":"NCT00608764"}}NCT00608764, {"type":"clinical-trial","attrs":{"text":"NCT00292552","term_id":"NCT00292552"}}NCT00292552

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0113-2) contains supplementary material, which is available to authorized users.     

Observations on parasitic gastroenteritis and bronchitis in grazing calves: Untreated calves

Pott J. M., Jpones R. M. and Cornwell R. L. 1978. Observations on parasitic gastroenteritis and bronchitis in grazing calves: untreated calves. International Journal for Parasitology8: 331–339. The epidemiology of parasitic gastroenteritis (p.g.e.) and incidental observations on lungworm infection were studied on two equal-sized paddocks of permanent pasture on a Kent farm in 1970. Groups of 10 Friesian calves were exposed for the first time to each paddock in May and grazed until the end of October. Faeces were monitored, body weights recorded and clinical signs observed. Herbage samples were examined for larvae throughout the year. Five ‘cumulative’ tracer calves exposed at the commencement were removed at monthly intervals and 5 ‘short term’ tracer calves exposed at successive monthly intervals for 1 month were killed for worm counts. A similar pattern of events occurred on both paddocks. Moderate herbage larval levels of Ostertagia and Cooperia fell to almost zero during April/May but rose quickly in July to high levels in August/September. Lower levels continued until the end of the year. Faecal egg excretion began in the test calves at the end of May, showed peaks in July and September and fell to low levels in October. D. viviparus larval excretion occurred in most calves with greater counts on Paddock 2. Clinical signs of p.g.e. complicated by lungworm infection occurred in August/September. Body weight gain which was initially rapid levelled off in June due partly to drought in addition to the parasitic infection. Weight loss occurred in August and there were 2 deaths on Paddock 1 and one on Paddock 2. Survivors gained weight again in September/October.Worm burdens in tracer calves showed quite heavy infections with Ostertagia and Cooperia as early as June/July. Massive infections complicated by lungworm disease accompanied clinical signs and death. There was evidence that aquisition of resistance to Cooperia and Dictyocaulus was acquired more readily than to Ostertagia. Inhibition of development of Ostertagia and Cooperia also became evident at the end of the trial period.     

小儿肺咳颗粒联合头孢呋辛酯治疗急性支气管炎患儿的临床疗效及对免疫功能和炎症指标的影响

摘要 目的：探讨小儿肺咳颗粒联合头孢呋辛酯治疗急性支气管炎患儿的临床疗效及对免疫功能和炎症指标的影响。方法：将我院2019年1月到2020年12月期间接收的88例急性支气管炎患儿以信封抽签法的方式分为对照组（头孢呋辛酯治疗）和观察组（小儿肺咳颗粒联合头孢呋辛酯治疗）,各44例,两组患儿均治疗10 d。观察两组患儿疗效、临床症状消失时间、免疫功能、炎症因子及不良反应。结果：观察组患儿临床总有效率为90.91%（40/44）,高于对照组患儿的70.45%（31/44）（P<0.05）。与对照组相比,观察组的临床症状消失时间（肺部啰音、咳嗽、发热）更短（P<0.05）。治疗10 d后,观察组CD3⁺、CD4⁺、CD4⁺/CD8⁺高于对照组,CD8⁺低于对照组（P<0.05）。治疗10 d后,观察组血清白介素-6（IL-6）、超敏C反应蛋白（hs-CRP）、肿瘤坏死因子-α（TNF-α）水平低于对照组（P<0.05）。两组不良反应发生率组间对比差异无统计学意义（P>0.05）。结论：急性支气管炎患儿在头孢呋辛酯治疗的基础上给予小儿肺咳颗粒治疗,症状缓解时间缩短,同时还可有效降低血清炎症因子水平,提高患儿免疫力,安全可靠。     

氯雷他定对急性支气管炎患儿免疫功能及炎症反应的影响

目的:分析氯雷他定对急性支气管炎患儿免疫功能及炎症反应的影响。方法:收集我院收治的急性支气管炎患儿100例,将其随机分为对照组与观察组,每组50例。对照组患儿给予常规治疗;观察组患儿给予常规治疗联合氯雷他定治疗。比较两组患儿治疗前后动脉氧分压(PaO_2)、二氧化碳分压(PaCO_2)、CD3~+、CD4~+、CD4~+/CD8~+、免疫球蛋白A(IgA)、免疫球蛋白G(IgG)、免疫球蛋白M(IgM)、血清降钙素原(PCT)、C反应蛋白(CRP)及白介素-6(IL-6)水平的变化。结果:治疗后,观察组患儿PaO_2水平明显高于对照组,PaCO_2水平明显低于对照组(P0.05);两组患儿细胞免疫及体液免疫水平较治疗前比较均显著升高(P0.05),且观察组患儿CD3~+、CD4~+、CD4~+/CD8~+、IgA、IgG及IgM水平明显高于对照组患儿(P0.05)。治疗后,两组患儿血清中PCT、CRP及IL-6水平均较治疗前显著降低,且观察组患儿上述炎症因子水平明显低于对照组患儿(P0.05)。结论:氯雷他定治疗小儿急性支气管炎可显著提高患儿的免疫功能,抑制炎症因子释放,缓解炎症反应,临床效果显著。     

Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA

Coronavirus nucleocapsid proteins are basic proteins that encapsulate viral genomic RNA to form part of the virus structure. The nucleocapsid protein of SARS-CoV is highly antigenic and associated with several host-cell interactions. Our previous studies using nuclear magnetic resonance revealed the domain organization of the SARS-CoV nucleocapsid protein. RNA has been shown to bind to the N-terminal domain (NTD), although recently the C-terminal half of the protein has also been implicated in RNA binding. Here, we report that the C-terminal domain (CTD), spanning residues 248-365 (NP248-365), had stronger nucleic acid-binding activity than the NTD. To determine the molecular basis of this activity, we have also solved the crystal structure of the NP248-365 region. Residues 248-280 form a positively charged groove similar to that found in the infectious bronchitis virus (IBV) nucleocapsid protein. Furthermore, the positively charged surface area is larger in the SARS-CoV construct than in the IBV. Interactions between residues 248-280 and the rest of the molecule also stabilize the formation of an octamer in the asymmetric unit. Packing of the octamers in the crystal forms two parallel, basic helical grooves, which may be oligonucleotide attachment sites, and suggests a mechanism for helical RNA packaging in the virus.