重组甲型流感病毒基质蛋白1和2通过ERK信号因子诱导小鼠气管上皮细胞产生IFN-γ

探讨重组甲型流感病毒基质蛋白1和2(rM1和rM2)通过细胞外信号调节蛋白激酶(Extracellular signal regulated kinase,ERK)诱导小鼠气管上皮细胞产生γ-干扰素(Interferon-γ,IFN-γ)作用及机制。以原代小鼠气管上皮细胞为实验模型,实验分为6组(rM1组、rM2组、甲型流感病毒(Influenza A virus,IAV)组、rM1+IAV组、rM2+IAV组和正常对照组)。在各组分干预细胞4h、8h、24h时,采用半定量RT-PCR法检测各组细胞中IFN-γmRNA的表达和免疫印迹法检测各组细胞中IFN-γ、ERK、磷酸化ERK(phospho-ERK,p-ERK)蛋白的表达;用抑制剂阻断ERK信号因子信号传导,观察对各组分诱导小鼠气管上皮细胞产生IFN-γ的影响。各组分干预细胞4h、8h、24h,rM1组、rM2组、IAV组、rM1+IAV组、rM2+IAV组细胞的IFN-γmRNA和IFN-γ蛋白表达水平高于正常对照组(P0.01或P0.05);rM1+IAV组、rM2+IAV组细胞的IFN-γmRNA和IFN-γ蛋白表达水平高于IAV组(P0.01或P0.05)。在干预细胞4h,仅rM2组细胞中ERK磷酸化水平显著高于正常对照组(P0.01),在干预细胞8h、24h,rM1组、rM2组、IAV组、rM1+IAV组、rM2+IAV组细胞中ERK磷酸化水平均显著高于正常对照组(P0.01或P0.05)。加入ERK抑制剂,rM1组、rM2组、rM1+IAV组、rM2+IAV组细胞的IFN-γmRNA和IFN-γ蛋白表达水平显著低于非抑制剂组。本研究数据表明rM1和rM2可通过上调ERK信号因子的磷酸化水平诱导小鼠气管上皮细胞中产生IFN-γ,该诱导作用在干预4h即显著表现,并维持至少24h。     

甲型流感病毒M2e通用疫苗的研究进展

流感严重地影响人们的身体健康和工作生活,给社会带来巨大经济损失。疫苗接种是预防流感的有效措施之一,市场上的流感疫苗包括流感灭活疫苗、减毒活疫苗和亚单位疫苗等。这些流感疫苗只能预防相同亚型流感病毒的感染,无法预防不同亚型流感病毒引起的季节性流感和流感大流行,因此迫切需要研发广谱的能预防不同亚型的甲型流感病毒感染的通用疫苗。在此简要介绍甲型流感病毒M2e通用疫苗的研究进展。     

含禽流感病毒M2e 氨基端抗原表位的重组传染性法氏囊病毒VP2 蛋白的免疫原性鉴定

【目的】构建传染性法氏囊病毒VP2蛋白展示禽流感M2e抗原表位的重组蛋白,研发预防H5或H9亚型禽流感和传染性法氏囊的基因工程疫苗。【方法】根据现有禽流感疫苗株M2e的氨基端12个氨基酸多肽序列(nM2e)序列,结合GenBank中H5和H9亚型禽流感病毒nM2e的比对结果,确定nM2e序列。用融合PCR分别将1拷贝H5或H9的nM2e序列插入IBD B87株VP2基因的PBC区,获得VP2BCnM2e重组基因。将重组基因克隆至杆状病毒表达系统,转染Sf9细胞进行表达。经间接免疫荧光和Western blotting检测Sf9细胞表达重组基因后,扩繁重组病毒,制备疫苗,间隔4周对非免鸡作2次重复免疫,用间接ELISA和鸡胚成纤维细胞中的病毒血清中和试验检测血清中VP2和nM2e的抗体效价。【结果】成功构建含H5或H9 nM2e的VP2BCnM2e重组基因,该重组基因在Sf9细胞中得到表达。经免疫鸡,两重组蛋白均能激发针对VP2和nM2e的抗体,VP2BCnM2eH5组抗体效价高于VP2BCnM2eH9组。【结论】两重组蛋白均具有免疫原性,VP2BCnM2eH5免疫原性更佳。     

A型禽流感病毒M2e重组T7噬菌体疫苗构建及免疫原性研究

构建展示A型禽流感病毒M2e多肽的重组T7噬菌体,检测其对SPF鸡的免疫保护效果。比对GenBank近期发表的A型禽流感病毒M2e基因序列进行人工合成并重复至两拷贝,将其克隆到T7Select 415-1b噬菌体多克隆位点,构建重组噬菌体T7-M2e。经PCR鉴定并序列测定筛选阳性重组噬菌体,SDS-PAGE和Westernblot检测重组噬菌体表面M2e多肽。重组噬菌体以1×1010pfu/只剂量免疫SPF鸡,免疫后不同时间段采血通过ELSIA检测血清中抗M2e抗体,免疫荧光检测血清抗体与H9亚型禽流感病毒的结合能力,并以200个EID50/只剂量进行攻毒保护效率检测。成功构建重组噬菌体T7-M2e,插入两拷贝M2e基因获得表面展示,并与M2e抗体有免疫反应活性。噬菌体疫苗免疫后均产生抗M2e抗体,其抗血清能跟病毒粒子特异性结合,攻毒保护率达4/5(80%)。获得了展示禽流感病毒M2e多肽的重组噬菌体,噬菌体疫苗免疫鸡产生较高血清抗体并提供攻毒保护,为新型通用禽流感疫苗研制提供新思路。     

基于流感病毒HA柄部和M2e融合基因的DNA疫苗

由于流感病毒容易突变,流感通用疫苗的研究势在必行.流感病毒血凝素(HA)柄部和基质蛋白2的胞外域(M2e)都是流感病毒通用疫苗的重要候选靶点.通过重叠PCR的方法用A/PR/8/34(H1N1)(简称PR8)流感病毒的M2e或者4个甘氨酸取代HA的头部,分别获得HAM2e和HA4G,然后将两种重组基因插入真核表达载体pCAGGS-P7中,制得pHAM2e和pHA4G两种DNA疫苗.通过电击免疫的方法对小鼠分别免疫pHAM2e和pHA4G,免疫3次,每次免疫间隔2周.第3次免疫2周后用5LD50的同源流感病毒感染小鼠.结果表明HAM2e组和HA4G组的小鼠均产生了特异性抗体,HAM2e组比HA4G组具有更好的抗PR8流感病毒的能力,这提示可以用M2e替换HA头部用于疫苗研发.     

H9N2禽流感病毒M2 DNA疫苗初免-蛋白加强免疫的保护效果研究

流感病毒M2(基质蛋白2)是A型流感病毒的一个高度保守的蛋白。由于其免疫原性较弱,本研究采用M2 DNA疫苗初免-蛋白加强的策略来考察M2的免疫保护效果。制备A/Chicken/Jiangsu/07/2002(H9N2)流感病毒的M2 DNA疫苗以及经大肠杆菌表达的去除M2跨膜区的M2蛋白即sM2。以SPF级BALB/c小鼠为模型,电击法免疫M2 DNA疫苗,滴鼻法免疫sM2蛋白,免疫间隔三周,并于末次免疫后三周以致死量5LD50流感病毒H9N2攻击小鼠,通过检测小鼠存活率、体重丢失率、肺部病毒滴度及IgG抗体水平等指标来评价免疫的保护效果。实验结果表明,基于M2的疫苗采用DNA疫苗初免蛋白加强免疫二次的免疫程序能诱导较高的特异性抗体,明显减轻小鼠流感病症,提供完全的保护。     

含EB病毒核抗原1重组腺病毒的构建及其在小鼠中免疫效果研究

构建含EB病毒核抗原1(ebna1)重组腺病毒疫苗防治鼻咽癌,并为鼻咽癌疫苗的研究提供药效学资料。PCR法扩增B95-8细胞株中ebna1的C-端部分片段,EcoRI和BglⅡ酶切后插入pDC316穿梭质粒,ebna1片段测序比对正确后,与腺病毒骨架质粒pBHG共转染293细胞。收集病变后细胞,通过流式细胞仪和Western blot检测EBNA1在293细胞中的表达。电镜下观察所获得的病毒颗粒大小,TCID50法确定重组腺病毒滴度。以2×108 VP/只的rAd-ebna1免疫BALB/c小鼠。在免疫结束后的第1、2、4和8周检测小鼠体内EBNA1特异性细胞免疫应答的水平变化。结果显示,经PCR扩增获得约900bp ebna1目的片段,测序结果与标准株序列一致,质粒共转染293细胞后,细胞出现病变,获得含EB病毒核抗原1重组腺病毒(rAd-ebna1),流式细胞仪和Western blot检测结果证实,ebna1在细胞中正确转录表达,收获病毒二代滴度可达108 TCID50/mL。rAd-ebna1免疫小鼠后,能够激活EBNA1的CD4+T细胞特异性细胞免疫应答。本实验成功构建了含ebna1片段的重组腺病毒,并具激活CD4+T细胞免疫应答活性。     

重组狂犬病病毒糖蛋白活酵母疫苗经小鼠口服给药后的免疫效果

目的:研究以活酵母为输送载体的狂犬病疫苗对小鼠的免疫保护能力和免疫疗程。方法:小鼠首先灌食高浓度空白活酵母INVSI,并于灌胃后8h和12h分别采集小鼠空肠和回肠组织并提取小肠浸出液培养,计算活酵母经肠胃环境后的存活率;分别取狂犬病糖蛋白(glycoprotein,G)分泌型表达菌株pYes-InG和胞内表达型菌株pYes-G灌胃小鼠,灌胃结束后12h采集小鼠血清和小肠组织,采用免疫组织化学方法检测抗原物质G在小肠上皮细胞的分布,采用ELISA检测小鼠血清中和性抗体的滴度。结果:活酵母经灌食消化8h后在小肠中的存活率最高达36.11%,12h后降至0.59%;口服分泌型pYes-InG重组酵母的小鼠小肠组织和血清中能检测到抗原物质G和低量的中和性抗体,ELISA分析显示,小鼠经过3~4次免疫接种,免疫效果基本恒定,而口服胞内表达型pYes-G重组酵母的小鼠小肠组织和血清中均未检测到目标物。结论:分泌型重组酵母pYes-InG经多次口服可对狂犬病起到一定的预防作用,但它诱导产生的中和性抗体浓度低,免疫应答慢,虽不适合用于控制突发性狂犬病的传染以及治疗狂犬病患者,但从免疫机制、免疫方式、安全性以及生产成本等因素考虑,仍具有良好的研究价值。     

共表达H5N1流感病毒M1和HA基因的DNA疫苗与腺病毒载体疫苗的小鼠免疫评价

为评价在小鼠体内表达流感病毒M1和HA基因诱导的免疫反应,制备共表达H5N1亚型禽流感病毒 (A/Anhui/1/2005) 全长基质蛋白1 (M1) 基因和血凝素 (HA) 基因的重组DNA疫苗pStar-M1/HA和重组腺病毒载体疫苗Ad-M1/HA,将其按初免-加强程序免疫BALB/c小鼠,共免疫4次,每次间隔14 d。第1、3次用DNA疫苗,第2、4次用重组腺病毒载体疫苗,每次免疫前及末次免疫后14 d采集小鼠血清用于检测体液免疫应答,末次免疫后14 d采集小鼠脾淋巴细胞用于检测细胞免疫应答。血凝     

Self-assembly polymerization enhances the immunogenicity of influenza M2e peptide

The extracellular domain of Influenza M2 protein (M2e) was considered as a promising target for universal influenza vaccine development. Several M2e-based influenza vaccines have been developed and many of them used a mutant M2e peptide, in which the two conserved cysteine residues were substituted by serine residues. In this paper, we compared the antigenicity and immunogenicity of wild type and cysteine-mutant M2e peptides. We found that the cysteine substitution slightly affected the antigenicity of M2e epitope, but greatly reduced the immunogenicity of M2e peptide. The cysteine substitution also disabled the M2e peptide from inducing protection against influenza virus challenge in mice. Further analysis revealed that the immunogenicity of M2e peptide was enhanced by the self-assembly of the peptide through inter-peptide disulfide bonds. These results provide new information to improve the design of M2e-based vaccines against potential influenza pandemics.     

甲型H5N1流感病毒M2与HA双基因载体疫苗在小鼠体内的免疫学评价

高致病性H5N1亚型禽流感病毒 (AIV) 严重威胁到人类健康,因此研制高效、安全的禽流感疫苗具有重要意义。以我国分离的首株人H5N1亚型禽流感病毒 (A/Anhui/1/2005) 作为研究对象,PCR扩增基质蛋白2 (M2) 和血凝素 (HA) 基因全长开放阅读框片段,构建共表达H5N1亚型AIV膜蛋白基因 M2和HA的重组质粒pStar-M2/HA。此外,还通过同源重组以293细胞包装出表达M2基因的重组腺病毒Ad-M2以及表达HA基因的重组腺病毒Ad-HA。用间接免疫荧光 (IFA) 方法检测到了各载体上插入基因的表达。按初免-加强程序分别用重组质粒pStar-M2/HA和重组腺病毒Ad-HA+Ad-M2免疫BALB/c小鼠,共免疫4次,每次间隔14 d。第1、3次用DNA疫苗,第2、4次用重组腺病毒载体疫苗,每次免疫前及末次免疫后14 d采集血清用于检测体液免疫应答,末次免疫后14 d采集脾淋巴细胞用于检测细胞免疫应答。血凝抑制 (HI) 实验检测到免疫后小鼠血清中的HI活性。ELISA实验检测到免疫后小鼠血清中抗H5N1亚型流感病毒表面蛋白的IgG抗体。ELISPOT实验检测到免疫后小鼠针对M2蛋白和HA蛋白的特异性细胞免疫应答。流感病毒M2与HA双基因共免疫的研究,为研究开发新型重组流感疫苗奠定了基础。     

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c（Hsp70359–610） fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70（mHsp70） is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte（CTL） response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2（M2e）, was fused to the C-terminus of Mycobacterium tuberculosis Hsp70（Hsp70c）, to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c（Hsp70359–610）. And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.     

共表达甲型流感病毒M1和HA抗原的重组杆状病毒的构建

为构建同时表达流感病毒M1和HA抗原的重组杆状病毒,采用PCR扩增流感病毒A/PR/8/34株的M1基因和去除信号肽的HA基因,将两基因克隆到杆状病毒转座载体pFastBac Dual的两个启动子下游的多克隆位点,筛选出阳性重组转座载体pFastBac Dual-M1-HA。将其转化含有杆状病毒穿梭载体(Bacmid)的DH10Bac感受态细胞,通过抗生素、蓝白斑筛选和PCR鉴定获得重组杆状病毒穿梭载体rBacmid-M1-HA,在脂质体介导下转染Sf9昆虫细胞,获得重组杆状病毒rBac-M1-HA。提取重组病毒基因组,通过PCR鉴定外源基因插入成功。间接免疫荧光和Western-blot检测表明,该重组杆状病毒在Sf9昆虫细胞中成功地表达了M1和HA。应用杆状病毒/昆虫细胞系统成功共表达流感病毒M1和HA抗原,为研究流感病毒VLP的形成机制和开发新型流感疫苗奠定了基础。     

Multi-isotype Glycoproteomic Characterization of Serum Antibody Heavy Chains Reveals Isotype- and Subclass-Specific N-Glycosylation Profiles

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Highlights

• •nLC-MS/MS method to analyze immunoglobulin (Ig) N-glycopeptides from human serum.
• •Multi-isotype, site-specific characterization of immunoglobulin N-glycosylation.
• •IgA2 sequence and glycosylation-site variant analyses.
• •Platform to define disease-specific N-glycan signatures for different Ig isotypes.

     

Betel quid extract promotes oral cancer cell migration by activating a muscarinic M4 receptor-mediated signaling cascade involving SFKs and ERK1/2

Betel quid (BQ) is a widely accepted etiological factor for oral squamous cell carcinoma (OSCC) in Southeast Asia, but how BQ chewing leads to oral carcinogenesis remains to be elucidated. We have previously demonstrated that the activation of Src family kinases (SFKs) is critical for BQ-induced oral cancer cell motility. Here we investigate whether this biological effect is mediated by specific membrane receptors in oral cancer cells. We found that BQ-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cell migration could be inhibited by atropine, suggesting the involvement of the muscarinic receptor family. The enhanced activities of ERK1/2 and cell migration were significantly counteracted by PD102807, the selective antagonist of muscarinic M4 receptor. Moreover, cold BQ extract effectively competed with a known ligand, [³H]-N-methyl scopolamine, for binding to muscarinic M4 receptor in vitro, thereby implying that BQ could activate motility-promoting signaling pathways through direct interaction with the receptor. The requirement of muscarinic M4 receptor for BQ-induced oral cancer cell migration was demonstrated by knockdown of the receptor using RNA interference (RNAi). Remarkably, ectopic expression of muscarinic M4 receptor in two oral cancer cell lines, Ca9-22 and SCC-9, further augmented BQ-induced cell migration by 83% and 99%, respectively. Finally, we verified that BQ-induced oral cancer cell migration was mediated through a muscarinic M4 receptor → SFKs → ERK1/2 signaling pathway. Thus, our findings have identified a novel signaling cascade mediating BQ-induced oral cancer cell motility, which could be a therapeutic target for BQ-related oral malignancies.