SARS-CoV GDH株N蛋白全长基因克隆及其可溶性表达

目的构建SARS冠状病毒N蛋白的原核表达质粒,诱导重组蛋白表达并纯化,鉴定其抗原性。方法以我国SARS冠状病毒GDH株总RNA为模板,采用RT-PCR技术扩增N蛋白的全长基因,TA克隆后测序。构建pET-23d的N基因表达载体,用IPTG诱导目的蛋白表达,利用硫酸铵沉淀、分子筛层析及离子交换层析纯化重组蛋白,免疫印迹鉴定重组蛋白。结果RT-PCR扩增出1269bpSARS冠状病毒N蛋白的基因片段,其序列分析结果与SARS-CoVGD01、BJ01株的同源性为99.92%;该基因在大肠杆菌表达系统中高效表达,占可溶性蛋白的33.57%,表达产物为非融合的可溶性蛋白,Westernblot结果显示重组N蛋白具有良好的抗原性;纯化后重组N蛋白纯度为92.9%。结论成功构建了SARS冠状病毒N蛋白的重组表达质粒,并在大肠杆菌中以非融合蛋白的形式得到高效的可溶性表达,为SRAS的诊断和疫苗的研制奠定了基础。     

Evidence against a role of a pertussis toxin-sensitive guanine nucleotide-binding protein in the alpha1-adrenoceptor-mediated positive inotropic effect in the heart

Summary Pertussis toxin, which specifically inactivates guanine nucleotide-binding proteins (N-proteins) involved in the signal transduction in various receptor systems, did not influence the positive inotropic effect of the alpha₁-adrenoceptor agonist phenylephrine in rat isolated left auricles. This indicates that the alpha₁-adrenoceptor-mediated positive inotropic effect does not involve a pertussis toxin-sensitive N-protein. Send offprint requests to W. Schmitz at the above addressSupported by the Deutsche Forschungsgemeinschaft     

Effect of thyroid status on beta-adrenergic receptor, adenylate cyclase activity and guanine, nucleotide regulatory unit in rat cardiac and erythrocyte membranes

The effect of thyroid hormone on the β-receptor coupled adenylate cyclase in rat crude cardiac membranes was analysed by measuring the number of DHA-binding sites, adenylate cyclase activity and the amount of cholera toxin catalysed ADP-ribosylation of a protein with a molecular weight of 42,000 in cardiac and erythrocyte membranes. In crude rat cardiac membranes, the number of DHA-binding sites (78 ± 15 fmole/mg protein in the euthyroid state) is increased to 158 ± 20 fmole/mg protein in the hyperthyroid state and decreased to 51 ± 6 fmole/mg protein in the hypothyroid state; the affinity of the binding sites remained unchanged (K_D 2.9?4.3 nM). l-Isoprenaline (10^?4 M)-stimulated adenylate cyclase activity varied in parallel to the number of DHA-binding sites in hyper- and euthyroidism. Thyroid hormone, however, did not influence GppNHp (10^?4 M)-stimulated adenylate cyclase activity. Cholera toxin catalysed ADP-ribosylation of normal crude cardiac membranes resulted in a 1.8 fold increase in adenylate cyclase activity in the presence of GTP (10^?4) and l-isoprenaline (10^?4M), presumably as a result of inhibition of GTPase. In crude cardiac membranes cholera toxin catalyses the ADP-ribosylation of one major protein, which comigrates on sodium dodecylsulfate-polyacrylamide gel electrophoresis with the putative regulatory component of adenylate cyclase (mol. wt 42,000). In different thyroid states the amount of the regulatory component (as determined by cholera toxin dependent labelling) was equal (112 fmole/mg protein in euthyroid crude cardiac membranes). Basal activity of adenylate cyclase showed a significant difference between activity in euthyroid (3.7 ± 0.2 pmole cAMP/mg protein/min) and hypothyroid (5.4 ± 0.2 pmole cAMP/mg protein/min), but not in hyperthyroid crude cardiac membranes (3.4 ± 0.2 pmole cAMP/mg protein/min). Our results indicate, that thyroid hormone regulates the number of DHA-binding sites and basal activity (in hypothyroidism) in crude cardiac membranes and thereby causes different results in l-isoprenaline-induced adenylate cyclase activity.     

Long-Term Humoral Immune Response against SARS-CoV-2 after Natural Infection and Subsequent Vaccination According to WHO International Binding Antibody Units (BAU/mL)

The new WHO reference standard allows for the definition of serum antibodies against various SARS-CoV-2 antigens in terms of binding antibody units (BAU/mL) and thus to compare the results of different ELISA systems. In this study, the concentration of antibodies (ABs) against both the S- and the N-protein of SARS-CoV-2 as well as serum neutralization activity were evaluated in three patients after a mild course of COVID-19. Serum samples were collected frequently during a period of over one year. Furthermore, in two individuals, the effects of an additional vaccination with a mRNA vaccine containing the S1-RBD sequence on these antibodies were examined. After natural infection, the antibodies (IgA, IgG) against the S1-protein remained elevated above the established cut-off to positivity (S-IgA 60 BAU/mL and S-IgG 50 BAU/mL, respectively) for over a year in all patients, while this was not the case for ABs against the N-protein (cut-off N-IgG 40 BAU/mL, N-IgA 256 BAU/mL). Sera from all patients retained the ability to neutralize SARS-CoV-2 for more than a year. Vaccination resulted in a rapid boost of antibodies to S1-protein but, as expected, not to the N-protein. Most likely, the wide use of the WHO reference preparation will be very useful in determining the individual immune status of patients after an infection with SARS-CoV-2 or after vaccination.     

Persisting Neutralizing Activity to SARS-CoV-2 over Months in Sera of COVID-19 Patients

The relationship between the nasopharyngeal virus load, IgA and IgG antibodies to both the S1-RBD-protein and the N-protein, as well as the neutralizing activity (NAbs) against SARS-CoV-2 in the blood of moderately afflicted COVID-19 patients, needs further longitudinal investigation. Several new serological methods to examine these parameters were developed, validated and applied in three patients of a family which underwent an ambulatory course of COVID-19 for six months. The virus load had almost completely disappeared after about four weeks. Serum IgA levels to the S1-RBD-protein and, to a lesser extent, to the N-protein, peaked about three weeks after clinical disease onset but declined soon thereafter. IgG levels rose continuously, reaching a plateau at approximately six weeks, and stayed elevated over the observation period. Virus-neutralizing activity reached a peak about 4 weeks after disease onset but dropped slowly. The longitudinal associations of virus neutralization and the serological immune response suggest immunity in patients even after a mild clinical course of COVID-19.     

SARS冠状病毒N蛋白致大鼠肺部炎症及糖皮质激素对其的作用

目的探讨严重急性呼吸综合征(SARS)冠状病毒N蛋白所致大鼠肺部炎症反应及糖皮质激素(以下简称激素)对其的调节作用。方法24只SD大鼠随机分为4组,每组6只;A组大鼠气管内滴入无菌生理盐水0.2ml,B组(6h)、C组(24h)大鼠气管内滴入SARS病毒N蛋白溶液0.2ml,D组大鼠气管内滴入SARS病毒N蛋白溶液0.2ml的同时腹腔内注射10mg/kg地塞米松。测4组大鼠外周血、支气管肺泡灌洗液(BALF)中WBC总数及分类;测4组大鼠肺组织湿/干(W/D)比值;观察4组大鼠肺组织病理学变化;ELISA测4组大鼠血清及BALF中IL6、IL10、转化生长因子(TGF)β1水平。结果(1)外周血淋巴细胞比例C组较A组低(P<0.05),D组较A、C组均低(P值均小于0.01);D组WBC总数较A、C组均低(P<0.01)。BALF中WBC总数C组较A组高(P<0.05),D组较C组低(P<0.05);B、C组BALF中肺泡巨噬细胞占98%～99%。(2)肺脏W/D比值B、C组较A组高(P<0.05),D组较C组低(P<0.01)。(3)肺组织病理学变化:B、C组大鼠肺泡间隔明显增宽,有较多的炎性细胞渗出,包括中性粒细胞、淋巴细胞、单核细胞、成纤维细胞等,血管充血、淤血,有的支气管腔内有炎性细胞渗出,C组变化较B组无明显加重;D组大鼠肺组织炎性反应较B、C组减轻,肺泡间隔变薄,炎性细胞渗出减少。(4)血清及BALF中IL6、IL10、TGFβ1水平B组较A组高(P<0.01),C组进一步升高(P<0.01),D组较C组低(P<0.01)。结论SARS冠状病毒N蛋白具有致病性,能够引起大鼠肺部炎症反应和(或)急性肺损伤,肺损伤与促炎性细胞因子、抗炎性细胞因子的升高及失衡有关;激素可有效地减轻SARS冠状病毒N蛋白所致的肺部炎症反应。     

Differential characteristics of the early stage of lung inflammation induced by SARS-CoV Nucleocapsid protein related to age in the mouse

Objective:  Severe acute respiratory syndrome (SARS) is an acute infectious disease of the respiratory system which has newly emerged. Interestingly, it appears to be a disease that predominantly affects adults while the mortality in children is extremely low. However, the pathogenesis of SARS in relation to different characteristics relevant to age remains unclear. Material and Methods:  To better understand the role of cytokines in the immunopathological processes of SARS, weanling (4 weeks old), young (6 weeks old) and adult (10 weeks old) male BALB/C mice were inoculated intranasally with N-protein of SARS-CoV in this study. Serum or lung homogenate levels of some cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) along with acute injury lung index and histology were also analyzed. Results:  Histopathological analysis of adult male BALB/C mice after N-protein infection showed progressive inflammatory reactions, especially pulmonary edema, in accordance with a moderately (~13%) elevated level of W/D ratio at 24 h. Although adult groups underwent a progressive lung inflammation in the acute phase accompanied by raised levels of TNF-α in serum, no significant changes in lung TNF-α level were reported simultaneously. Moreover, adult SARS infected BALB/c mice showed elevated levels of IFN-γ while IFN-γ levels in weanling and young groups had no obvious association with lung inflammation. Conclusion:  Our study supports the observation that adult mice do have progressively greater immune reactions than weanling and adolescent ones over time. The relative immaturity of the immune system in weanlings may confer benefit leading to less impairment of lung function. However, the measurement of TNF-α and IFN-γ levels was not indicative of the severity of lung injury at the early stage of disease. Received 25 March 2008; returned for revision 2 July 2008; received from final revision 24 November 2008; accepted by Graham R. Wallace 23 December 2008