新型冠状病毒RBD蛋白原核表达及多克隆抗体的制备

为原核表达严重急性呼吸综合征冠状病毒2（简称新型冠状病毒,severe acute respiratory syndrome-coronavirus 2,SARS-CoV-2）S蛋白受体结合域（receptor binding domain, RBD）并制备多克隆抗体,利用基因克隆技术将RBD基因连接到原核表达载体pGEX-6p-1和pET-32a(+)上,电转化至大肠杆菌XL1-Blue感受态细胞,利用优化后的表达条件大量表达重组蛋白,经亲和层析纯化后通过SDS-PAGE检测蛋白的表达情况。利用GST-RBD融合蛋白作为免疫抗原免疫小鼠制备多克隆抗体,ELISA和Western blot分析抗血清的效价和特异性。PCR鉴定和序列测定结果显示,成功构建了重组载体pGEX-RBD和pET-RBD,在大肠杆菌中实现了GST-RBD和RBD-His融合蛋白的可溶性高效表达。研究获得的多克隆抗体的滴度达到约1∶3 000,并具有良好的结合特异性。原核表达的可溶性新型冠状病毒RBD重组蛋白具有良好的免疫原性,为后续制备基因工程抗体奠定了实验基础。     

冠状病毒HCoV-229E S1蛋白片段的免疫原性分析

目的表达和纯化HCoV-229E的S1蛋白片段（S1 417-547）,分析其诱导的免疫应答。方法将纯化蛋白免疫小鼠,ELISA检测小鼠血清特异性抗体以及血清IL-4和IFN-γ含量,流式细胞术检测小鼠脾脏T淋巴细胞亚群分布,观察其诱导的免疫应答。结果表达蛋白经金属螯合获得纯化,并诱导小鼠产生了高滴度的抗体。脾脏CD4^＋和CD8^＋比例均升高,CD4^＋/CD8^＋比值下降;免疫鼠血清IFN-γ和IL-4水平显著升高。结论成功构建了HCoV-229E S1蛋白的表达载体,并在BL21（DE3）中得到了高效表达,表达蛋白免疫小鼠后诱导了明显的细胞和体液免疫应答。     

重组痘病毒表达SARS冠状病毒纤突蛋白及其免疫原性分析

对牛痘病毒弱毒株表达的SARS冠状病毒纤突蛋白(Spike protein,S)的免疫原性进行分析与比较.以减毒痘病毒(WR株)为载体重组了SARS冠状病毒全长S基因(rWR-SARS-S).SDS-PAGE和Western blot试验表明,迁移率约为190kD的重组SARS S蛋白可在HeLa细胞中表达,而且可以被鸡抗SARS全病毒高免血清识别,具有特异免疫反应原性.进一步研究表明,rWR-SARS-S感染的细胞在IFA试验中可与鸡抗SARS的高免血清发生特异反应,具有良好的敏感性和特异性.以104PFU的rWR-SARS-S免疫BALB/c小鼠产生的抗体在间接ELISA试验中可以被S蛋白识别,产生特异抗原抗体反应.利用痘病毒表达的SARS冠状病毒S蛋白具有良好的抗原性和生物学活性,可替代SARS冠状病毒全病毒,为研究安全、敏感和特异的重组诊断抗原奠定了重要基础.     

SARS亚基疫苗-突刺蛋白受体结合区RBD在烟草叶绿体中的高效表达

叶绿体表达系统为植物源重组药用蛋白和亚基疫苗的生产提供了一个有效的途径。为验证SARS亚基疫苗在叶绿体中表达的可行性,以及为植物源SARS亚基疫苗的生产提供一套高效、低成本的技术平台,本研究将人工优化合成的SARS-CoV突刺蛋白(S蛋白)受体结合区序列RBD与载体分子CTB融合基因导入烟草叶绿体基因组中。PCR和Southern杂交分析表明,外源融合基因已整合到烟草叶绿体基因组中,并获得同质化。Western杂交分析表明,重组融合蛋白CTB-RBD在叶绿体转基因烟草中获得表达,且主要以可溶性单体形式存在。ELISA分析表明,在不同生长阶段、不同生长部位和不同时间点烟草叶片中,重组融合蛋白CTB-RBD的表达水平呈现明显的变化。重组蛋白在成熟叶片中的表达水平最高可以达到10.2%TSP。本研究通过SARS亚基疫苗RBD在烟草叶绿体中的高效表达,有望为植物源SARS亚基疫苗的生产以及SARS血清抗体的检测提供一个有效的技术平台。     

基因Ⅰ型乙型脑炎病毒亚单位疫苗候选抗原的免疫原性比较

为了筛选出免疫原性最佳的基因Ⅰ型乙型脑炎病毒亚单位疫苗候选抗原,将基因Ⅰ型JEVGS株的prMEIII融合基因、polytope复合表位基因和prMEIII-polytope融合基因分别克隆构建到原核表达载体pET-30a上,经诱导表达纯化获得重组蛋白。将制备的重组蛋白免疫小鼠,通过ELISA监测体液免疫反应、通过噬斑减少中和试验滴定中和抗体滴度、通过细胞因子表达丰度和淋巴细胞增殖实验分析细胞介导的免疫反应,比较分析制备的乙型脑炎病毒亚单位疫苗候选抗原的免疫原性。结果表明:获得的分子量分别为35kDa(prMEIII)、28kDa(polytope复合表位抗原)和57 kDa (prMEIII-polytope)的重组蛋白均能诱导免疫小鼠产生较强的体液免疫和细胞免疫反应。与prMEIII-polytope和polytope重组蛋白免疫组相比,prMEIII蛋白可诱导免疫小鼠产生更高的IL-2和IFN-γ表达丰度和淋巴细胞增殖水平(P0.05)。prMEIII蛋白免疫小鼠诱导产生的中和抗体滴度接近于商品化乙脑减毒疫苗SA14-14-2 (P0.05)。上述研究结果表明,prMEIII重组蛋白可以作为乙型脑炎病毒亚单位疫苗的备选蛋白。     

含分子内佐剂的SARS-CoV-2 RBD域重组蛋白制备及免疫效果评价北大核心CSCD

制备含破伤风毒素肽(tetanus toxin,TT)、促吞噬肽(tuftsin)和新型冠状病毒刺突蛋白(spike,S蛋白)受体结合域(receptor-binding domain,RBD)的融合蛋白,探讨分子内佐剂对RBD蛋白体液免疫和细胞免疫效果的影响。将破伤风毒素肽、促吞噬肽与S蛋白RBD区域通过柔性多肽串联,密码子优化后构建重组载体,原核表达纯化制备重组S-TT-tuftsin蛋白,与铝佐剂混合后免疫BALB/c小鼠,对其体液及细胞免疫效果进行评价。重组S-TT-tuftsin蛋白以包涵体形式表达,离子交换层析纯化后采用梯度透析进行复性,复性蛋白经Dot blotting鉴定,可与新冠亚单位疫苗(安徽智飞公司)免疫后人血清发生反应。小鼠免疫实验结果表明,免疫35 d时抗体水平到达平台期,含分子内佐剂重组蛋白(铝佐剂)免疫小鼠后血清ELISA抗体效价高达1︰66240,显著高于S-RBD蛋白(铝佐剂)免疫小鼠抗体效价(P<0.05)。同时,含分子内佐剂重组蛋白刺激小鼠产生更强的淋巴细胞增殖能力,刺激指数可达4.71±0.15,相较于S-RBD蛋白的刺激指数1.83±0.09具有显著性差异(P<0.0001)。分子内佐剂破伤风毒素肽和促吞噬肽可显著增强新冠S蛋白RBD域的体液免疫和细胞免疫效果,可为新冠亚单位疫苗和其他病毒亚单位疫苗的研制提供理论基础和参考。     

人冠状病毒NL63受体结合区蛋白的原核表达纯化与鉴定

人冠状病毒NL63受体结合区蛋白是其免疫学诊断和疫苗研究的主要靶点,在受体吸附、病毒进入细胞及膜融合中起关键作用。本研究在E.coli系统中进行人冠状病毒NL63受体结合区(RBD)大、小蛋白的表达纯化,并对其进行免疫学鉴定。首先密码子优化设计合成了HCoV-NL63的RBD大片段(RL:232-684aa)与小片段(RS:476-616aa)的编码基因,并将其克隆进硫氧还蛋白表达载体pM48,构建了人冠状病毒NL63的受体结合区蛋白(RBD)大(RL)、小(RS)片段与硫氧还蛋白的融合表达质粒;转化E.coli BL21pLys S,利用IPTG进行诱导表达,用镍亲和层析对蛋白进行纯化,并以表达HCoV-NL63RL与RS蛋白的重组痘苗病毒免疫小鼠血清对重组蛋白进行免疫印迹鉴定。结果表明在37℃,0.8mM IPTG诱导4h时,蛋白表达量达到最高,融合蛋白主要以包涵体形式表达,纯化后纯度可达95%以上。Western blot显示,两个融合蛋白均与痘苗病毒(天坛株)表达的HCoV-NL63RL与RS蛋白免疫的小鼠血清发生特异性反应。本研究首次在国内用原核系统表达纯化并鉴定了人冠状病毒NL63受体结合区大小蛋白(RL和RS),为人冠状病毒NL63感染的免疫学检测与疫苗研究提供了基础。     

SARS-CoV S蛋白受体结合区的重组表达及免疫原性分析

为了表达SARS-CoV的S蛋白的受体结合区并对其免疫原性进行分析,用PCR方法扩增S蛋白的受体结合区基因片段,克隆至原核表达质粒pET-F32a＋并在大肠杆菌中表达,应用Western—blot鉴定表达的目的蛋白,而后以该蛋白作为诊断抗原包被酶联卡反来检测20份SARS病人血清和28份健康人血清,结果原核表达的S蛋白能够和所用的SARS病人血清反应。这提示表达的S重组蛋白具有良好的抗原性。将变性纯化的重组蛋白和复性蛋白分别皮下免疫小鼠,第三次免疫一周后收集抗血清,用ELISA测定抗体和同时测定中和抗体活性。用变性的抗原免疫的小鼠血清均无中和活性;而用复性的蛋白免疫的小鼠产生了中和抗体。实验表明,S蛋白受体结合区无线性中和表位,中和抗体的产生是由构象表位诱导的。提示该蛋白有可能应用于亚单位疫苗的研究。     

重组Ⅱ型登革病毒NS1的表达及其免疫原性的研究

目的:表达制备重组Ⅱ型登革病毒非结构蛋白NS1,检测该重组表达蛋白的免疫原性,为检测试剂盒的研制提供基础。方法:根据Ⅱ型登革病毒NS1蛋白基因序列(GenBank登录号:NC_001474),对基因序列进行编码密码子优化后进行全基因合成,随后,经双酶切将目的片段克隆到表达载体pET28a上,通过IPTG诱导表达;表达产物经Western blot鉴定后,进一步进行蛋白纯化和透析复性,制备获得重组Ⅱ型登革病毒NS1蛋白。收获的NS1蛋白免疫小鼠,制备多克隆抗体,通过间接酶联免疫学方法测定其效价,检测其免疫原性。结果:成功构建了Ⅱ型登革病毒NS1蛋白的表达载体。Western blot显示表达的重组蛋白能够被NS1单抗特异性的结合,纯化复性后的重组蛋白在小鼠体内表现出良好的免疫原性。结论:重组表达的NS1蛋白有良好的免疫原性,为以后的NS1单克隆抗体的制备和登革病毒检测试剂盒的研制提供了良好的基础。     

研究报告重组Ⅱ型登革病毒NS1的表达及其免疫原性的研究

目的：表达制备重组Ⅱ型登革病毒非结构蛋白NS1,检测该重组表达蛋白的免疫原性,为检测试剂盒的研制提供基础。方法：根据Ⅱ型登革病毒NS1蛋白基因序列（GenBank 登录号：NC_001474）,对基因序列进行编码密码子优化后进行全基因合成,随后,经双酶切将目的片段克隆到表达载体pET28a上,通过IPTG诱导表达;表达产物经Western blot鉴定后,进一步进行蛋白纯化和透析复性,制备获得重组Ⅱ型登革病毒NS1蛋白。收获的NS1蛋白免疫小鼠,制备多克隆抗体,通过间接酶联免疫学方法测定其效价,检测其免疫原性。结果：成功构建了Ⅱ型登革病毒NS1蛋白的表达载体。Western blot显示表达的重组蛋白能够被NS1单抗特异性的结合,纯化复性后的重组蛋白在小鼠体内表现出良好的免疫原性。结论：重组表达的NS1蛋白有良好的免疫原性,为以后的NS1单克隆抗体的制备和登革病毒检测试剂盒的研制提供了良好的基础。     

siRNA沉默t-bet基因对CD4、CD8T淋巴细胞亚群IFN-γ产生的影响

利用化学合成针对人t-bet基因的小干扰RNA（siRNA）,转染体外培养的人外周血单个核细胞,利用流式细胞仪分选纯化CD4^＋、CD8^＋T淋巴细胞,半定量RT-PCR检测CD4^＋、CD8^＋T淋巴细胞中t-betmRNA的表达变化,流式细胞术检测转染前后IFN-γ产生的变化情况。探讨转录因子t-bet对人CD4^＋、CD8^＋T淋巴细胞亚群IFN-γ产生的调控作用。与对照组相比,转染后的人CD4^＋、CD8^＋T淋巴细胞t-betmRNA表达水平明显下降;转染siRNA后,CD4＋T淋巴细胞中IFN-γ＋细胞比例为（18.46±6.86）%,与对照组（50.20±5.91）%比较有显著性差异（P〈0.01）;CD8＋T淋巴细胞IFN-γ＋细胞比例为（74.18±9.33）%,和对照组（76.51±6.49）%比较差异不明显（P〉0.05）。体外转录合成的siRNA可有效降低人CD4^＋、CD8^＋T淋巴细胞t-bet的基因表达;转录因子t-bet对人不同淋巴细胞亚群IFN-γ的产生所起作用不同。     

合成利巴韦林的关键酶瞟呤核苷磷酸化酶的原核表达及活性分析

目的：在枯草芽孢杆菌中表达嘌呤核苷磷酸化酶（PNPase）并分析其活性。方法：将PNPase的编码基因deoD克隆入pDG148表达载体,构建原核穿梭型表达载体pDG148-deoD,采用电转化方法将表达载体转入枯草芽孢杆菌WB600后诱导表达重组PNPase;研究重组PNPase的活性。结果与结论：获得的重组PNPase活性较对照提高了193．9％,其最适催化条件为65℃、pH7．5、500μmol／L底物浓度和1％1,2,4-三氮唑-3-羧甲酰胺;对重组菌的发酵条件进行了初步优化,IPTG诱导6h后在发酵液中添加0．5％的Tween-80能大幅度提高重组PNPase的酶活力。     

同种异基因骨髓移植GVHD动物模型的建立及其病理生理学机制初探

目的：建立稳定的异基因骨髓移植GVHD（移植物抗宿主病）动物模型,并初步了解其病理生理学机制。方法：以BALB／c（H-2^d）雌性小鼠为受者,接受8Gy致死剂量的。Co全身照射后,输注雄性C57BL／6（H-2^b）供鼠的脾细胞和骨髓细胞,观察受鼠的体征、造血功能恢复及生存时间的变化,并进行病理学、嵌合体和T细胞亚群及相关细胞因子的检查。结果：模型组小鼠在移植后出现了典型的GVHD症状;肠、肝、脾、皮肤的病理学分析均属于Ⅳ度GVHD;嵌合体植入成功;以7、14和21d为检测时间点,发现模型组鼠体内T细胞亚群移植后较移植前CD3^＋CD4^＋T细胞数量减少,CD3＋CD8＋T细胞显著升高,CD4^＋和CD8^＋T细胞比例严重倒置,随着时间变化比值会逐渐升高,但仍然处于较显著的倒置水平;血清中IFN-γ、TNF-α在移植后＋7d表达显著增高,尤其是IFN-γ的表达在＋7d达峰值;IL-4和IL-10的水平在移植前后几乎没有变化。结论：建立了稳定的GVHD动物模型;此模型发病过程中,CD8^＋T细胞介导的CTL细胞毒性作用可能要大于CD4＋Th介导的细胞因子效应;IFN-γ、TNF-α炎症因子在GVHD的早期发挥重要作用;IL-4、IL-10的低水平分泌与急性GVHD的高发病率有关。     

CD8＋CD122＋T细胞在脑缺血过程中作用的研究

目的：探讨CD8＋CD122＋T细胞在脑缺血过程中的作用及其机制。方法：线栓法建立小鼠大脑中动脉栓塞模型（MCAO）;激光共聚焦显微镜检测小鼠脑缺血组织中CD8＋CD122＋T细胞浸润情况;流式细胞仪检测脑缺血组织中CD8＋CD122＋T细胞／CD3＋T细胞的比例及脾和胸腺中CD8＋CD12TT细胞数量变化;RT—PCR方法检测CD8＋CD122＋T细胞对氧糖剥夺（Oxygen—glucosedeprivation,OGD）条件下星形胶质细胞表达TNF-α,IL-1β,IFN-γ的影响。结果：各时间点脑缺血组织中均有CD8＋CD122＋T胞浸润,且随脑缺血时间延长,缺血侧脑组织中CD8＋CD122＋T细胞／CD3＋T细胞比例逐渐增加,5d和7d组差异显著,与非缺血侧相比,P5d〈0．05,P7d〈0．05;MCAO小鼠脾及胸腺中CD8＋CD122＋T细胞呈现先增高后降低的趋势。星形胶质细胞经OGD处理后,与对照组相比,IFN-γ、TNF-α、IL—1β表达显著增高,PIFN-γ〈0．01、PTNF-α〈0．001、PIL-1β〈0．01;CD122-blocked组与CD8＋组相比,IFN-γ、TNF-α、IL-1β表达明显增高,PIFN-γ〈0．05、PINF-α〈0．05、PIL-1β〈0．01;CD8＋组与HBSS组相比,IFN-γ表达降低,P〈0．05;IL-1β表达有降低的趋势。结论：CD8＋CD122可细胞在脑缺血过程中发挥保护性作用,其保护作用通过CD122抑制星形胶质细胞TNF-α,IL-1β,IFN-γ炎症因子表达实现的。     

Bacterial surface display of GFP(uv) on bacillus subtilis spores

To analyze a cotG-based Bacillus subtilis spore display system directly, GFP(uv) was expressed on the surface of Bacillus subtilis spores. When GFP(uv) was fused to the C-terminal of the cotG structural gene and expressed, the existence of a CotG-GFP(uv) fusion protein on the B. subtilis spore was confirmed by flow cytometry confocal microscopic analysis. When the cotG anchoring motif was deleted, no fluorescence emission was observed under flow cytometry and confocal microscopic analysis from the purified spore, confirming the essential role of CotG as an anchoring motif. This GFP(uv) displaying spore might be used for another signaling application triggered by intracellular or extracellular stimuli.     

Antigenicity and immunogenicity of SARS-CoV S protein receptor-binding domain stably expressed in CHO cells

The receptor-binding domain (RBD) of SARS coronavirus (SARS-CoV) spike (S) protein contains multiple conformation-dependent epitopes that induce neutralizing antibody responses. Here we used CHO-K1 cells to establish a cell line for stable expression of a 193-mer (residues 318-510) RBD (RBD193-CHO) and determined its antigenicity and immunogenicity. We found that RBD193-CHO reacted strongly with a panel of six monoclonal antibodies recognizing various conformational and linear epitopes in RBD, suggesting that this recombinant protein maintains intact conformation and good antigenicity. Immunization of mice with RBD193-CHO resulted in induction of high titers of RBD-specific neutralizing antibodies and potent IL-4-expressing T cell responses. RBD193-CHO induced immunity that protected a majority of the vaccinated mice from SARS-CoV challenge. These results suggest that the recombinant RBD produced in an established stable cell line maintains strong immunogenicity with high potential for use as an effective and economic subunit SARS vaccine.     

Bacterial surface display of a co-factor containing enzyme, ω-transaminase from Vibrio fluvialis using the Bacillus subtilis spore display system

To improve the conventional bacterial surface display systems and to display a co-factor containing enzyme, ω-transaminase from Vibrio fluvialis, which needs pyridoxal phosphate (PLP) for efficient transamination, Bacillus subtilis spore display system with cotG, as an anchoring motif was used. Flow cytometry of the B. subtilis spore-expressing ω-transaminase proved its surface localization on the spore. The enzymatic activity of the spore expressing ω-transaminase was more than 30 times higher than that of the host spore. Protease treatment of the ω-transaminase displaying spores resulted in decreased transaminase activity, which is in keeping with the surface location of the fusion protein, CotG-ω-transaminase.     

Comparative antigenicity of spore coat proteins from Bacillus species using antibody to spore coat proteins of Bacillus megaterium

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross-reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross-reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross-reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross-reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.     

The study of heterogeneity of plasmid-bearing and plasmid-f ree populations of Bacillus subtilis under different environmental conditions

The population heterogeneity of recombinant and plasmid-free Bacillus subtilis strains introduced into aquatic microcosms was studied. After introduction, the population of the plasmid-free strain B. subtilis 2335 in microcosms has long been represented by both vegetative cells and spores, whereas, already ten days after introduction, the population of the recombinant strain B. subtilis 2335/105 (Km[symbol: see text]nf+) was represented only by spores. The number of plasmid copies in the spore isolates of the recombinant strain was the same as before introduction, but the plasmid abundance in the vegetative isolates of this strain decreased. The isolates of B. subtilis 2335/105 obtained from microcosms and the variants of this strain obtained by ten successive subcultures on M9 and 0.1 x M9 media with and without kanamycin (Km) differed in the number of plasmid copies, Km resistance, and maximum biomass yield during batch cultivation. Irrespective of the presence of Km, more than 50% of the variants subcultured on M9 medium showed reduced plasmid abundance. At the same time, about 70% of the variants subcultured on 0.1 x M9 medium with Km and 90% of the variants subcultured on the same medium without Km retained the initial number of plasmid copies. The variants subcultured on media with Km retained the initial biomass level. In more than 70% of the variants isolated from media without Km, the biomass yield increased.     

A polysaccharide deacetylase gene (pdaA) is required for germination and for production of muramic delta-lactam residues in the spore cortex of Bacillus subtilis

The predicted amino acid sequence of Bacillus subtilis yfjS (renamed pdaA) exhibits high similarity to those of several polysaccharide deacetylases. Beta-galactosidase fusion experiments and results of Northern hybridization with sporulation sigma mutants indicated that the pdaA gene is transcribed by E(sigma)(G) RNA polymerase. pdaA-deficient spores were bright by phase-contrast microscopy, and the spores were induced to germination on the addition of L-alanine. Germination-associated spore darkening, a slow and partial decrease in absorbance, and slightly lower dipicolinic acid release compared with that by the wild-type strain were observed. In particular, the release of hexosamine-containing materials was lacking in the pdaA mutant. Muropeptide analysis indicated that the pdaA-deficient spores completely lacked muramic delta-lactam. A pdaA-gfp fusion protein constructed in strain 168 and pdaA-deficient strains indicated that the protein is localized in B. subtilis spores. The biosynthetic pathway of muramic delta-lactam is discussed.