H3N2亚型猪流感病毒M1基因克隆及表达特性分析

从H3N2亚型猪流感病毒感染的鸡胚尿囊液中提取病毒基因组RNA,采用RT-PCR方法克隆M1全长基因,将M1基因亚克隆至pET-28a(+)表达载体中,构建重组表达质粒pET-28a-M1,将该重组质粒转化大肠杆菌BL21并经IPTG诱导表达。诱导产物经SDS-PAGE电泳分析显示重组蛋白M1获得大量表达,表达蛋白纯化后免疫Wistar大鼠制备多克隆抗体。Westernblotting检测结果表明制备的抗M1蛋白多克隆抗体可以识别大肠杆菌表达的M1蛋白和病毒感染细胞的病毒M1蛋白。构建M1基因真核重组质粒p3xFLAG-CMV-7.1-M1并转染Vero细胞,Western blotting检测表明抗M1蛋白多克隆抗体可以识别在Vero细胞中得到表达的M1蛋白,成功建立M1基因的真核表达系统。分析了病毒感染过程中M1蛋白的变化及其作为病毒复制指示分子的可能性。鼠源M1蛋白多克隆抗体的制备和M1基因真核重组表达质粒的构建,为进一步研究猪流感病毒的复制机理以及M1蛋白在病毒复制过程中的生物学功能奠定了基础。     

抗CD20嵌合抗体的表达与活性检测

表达了基因重组抗CD20嵌合抗体并对其生物学活性进行了初步鉴定。设计合成轻、重链可变区序列;提取血液RNA,通过RT-PCR得到人κ、IgG1的轻、重链恒定区序列。运用重叠延伸PCR,连接可变区与恒定区,将轻、重链基因连接至pIRES双表达载体。将质粒以阳离子脂质体转染CHO细胞,ELISA挑选阳性克隆,共获得7株表达较高的克隆,表达量约为2mg/L。扩大培养阳性克隆anti-CD20-1B3,收获上清,以蛋白A进行亲和层析纯化表达蛋白。SDS-PAGE检测表明纯化纯度达到95％,蛋白相对分子量与理论值吻合。以CD20₊细胞Raji、Daudi、Ramous检测,表明该抗体能与CD20抗原特异性结合,体外杀伤试验说明抗体能够杀伤CD20₊淋巴瘤细胞。     

人IL35-IgG4(Fc)融合蛋白在CHO/DG44细胞中的稳定表达

本研究利用基因重组技术构建人IL35-IgG4(Fc)融合基因真核表达载体, 稳定转染CHO/DG44细胞并检测重组蛋白的表达。主要采用聚合酶链式反应(PCR)从脂多糖(Lipopolysaccharides, LPS)诱导的人髓性白血病细胞株KG-I cDNA文库中克隆EBI3和IL-12p35 cDNA, 重叠PCR法连接2个片段, 并克隆到IgG4(Fc)- pOptiVEC?-TOPO?载体上,对新构建的IL-35-IgG4 (Fc) pOptiVEC?-TOPO?真核表达载体并进行酶切、测序、PCR鉴定; 脂质体法转染CHO/DG44细胞; RT-PCR检测转染结果, 采用a-MEM-培养基筛选实验组细胞, 对筛选的阳性克隆细胞再进行氨甲喋呤(Methotrexate, MTX)的加压筛选, ProteinG-Agarose纯化阳性克隆培养上清, 免疫印迹检测目的蛋白表达。结果显示IL-35-IgG4 (Fc) pOptiVEC?-TOPO?表达载体稳定转染CHO/DG44细胞并获得阳性克隆; SDS-PAGE电泳得到一条与预期相对分子质量大小相符的蛋白条带; 该蛋白能与羊抗人IgG4抗体特异结合。本实验获得了能够稳定表达具有稳定结构的IL35-IgG4(Fc)融合蛋白的CHO/DG44细胞株。     

果蝇凋亡蛋白Strica的原核表达、多克隆抗体的制备及在放线菌酮诱导下的激活状态检测

【目的】细胞凋亡是真核生物中保守的细胞程序化死亡,由蛋白酶caspase介导。Strica是果蝇Drosophila中一个特殊的caspase,至今未有其商品化抗体且生化功能未知。本研究旨在通过克隆、原核表达纯化果蝇Strica蛋白并制备其多克隆抗体来初步研究其功能。【方法】根据果蝇Strica的基因序列构建原核表达载体并转化至大肠杆菌Escherichia coli BL21(DE3)感受态细胞,表达Strica蛋白;将表达纯化后的Strica蛋白免疫家兔制备多克隆抗体;通过间接ELISA和Western blot分别测定抗体效价和灵敏度;利用前期构建的过表达载体pAc5-V5-Strica进行转染果蝇S2细胞,设计合成siRNA进行RNAi验证该抗体的特异性;利用该抗体通过免疫荧光实验检测Strica在果蝇S2细胞中的亚细胞定位;利用该抗体通过Western blot实验探究放线菌酮(10 μg/mL)处理后果蝇S2细胞中Strica蛋白的激活状态。【结果】获得了果蝇Strica重组蛋白;ELISA检测Strica抗体的效价大于1∶25 600;Western blot鉴定该抗体在1∶10 000的稀释度下能与重组蛋白反应;该抗体可以检测到S2细胞中过表达以及内源性Strica蛋白;Strica蛋白在S2细胞质中呈现散点分布;放线菌酮介导Strica蛋白的切割激活。【结论】成功制备了兔抗果蝇Strica多克隆抗体。Strica响应凋亡刺激物放线菌酮激活,推测Strica参与果蝇的细胞凋亡过程。本研究为进一步深入研究Strica的功能奠定了实验基础。     

细胞周期蛋白B1、D1和E真核表达载体的构建及表达

目的：为研究细胞周期蛋白（cyclin）在肿瘤形成过程中的分子机制,构建带FLAG标签的细胞周期蛋白B1、D1、E的真核表达载体,并检测其在293T细胞中的表达。方法：以乳腺cDNA文库为模板,分别扩增细胞周期蛋白B1、D1、E基因全长编码区序列,克隆到pcDNA3-FLAG真核表达载体上;用脂质体介导的基因瞬时转染法,将重组正确的表达载体转染293T细胞,检测细胞中的FLAG融合蛋白的表达。结果：酶切鉴定和DNA序列分析显示构建了正确的FLAG-Cyclin真核表达载体,Western印迹分析表明克隆的载体都能在真核细胞中表达分子大小相符的重组蛋白。结论：构建了FLAG-CyclinBl、FIAG-CyclinDl、FLAG-CyclinE真核表达载体,为细胞周期蛋白及其相关蛋白的研究奠定了基础。     

真核细胞表达单纯疱疹病毒Ⅰ型包膜糖蛋白B及其抗原性与免疫原性分析

为在真核细胞中表达并纯化I型单纯疱疹病毒(HSV I)包膜糖蛋白gB,并分析其抗原性和免疫原性,化学合成了包膜糖蛋白gB1胞外区基因片段,构建真核表达载体,并转染至HEK293细胞,表达的蛋白用羊抗HSV1+HSV2血清作为一抗,用ELISA检测其抗原性;用纯化的gB1蛋白免疫昆明小鼠,观察诱发抗体产生的时间及其效价,并用ELISA和Western blot检测小鼠抗gB1多克隆抗体特异性识别重组gB1抗原的能力,评价其免疫原性。结果显示在HEK293细胞中成功表达重组gB1蛋白,ELISA证实羊抗HSV1+HSV2多抗能够识别重组gB1蛋白;重组gB1蛋白免疫小鼠7周后,小鼠血清中多克隆抗体效价达到5×103,表明在真核细胞中高效表达并纯化的重组gB1蛋白具有良好的抗原性和免疫原性,为HSV检测试剂和疫苗研究提供了理论基础。     

羊口疮024基因的表达、多抗制备及亚细胞定位

目的克隆表达羊口疮病毒(Orf virus,ORFV)024基因,制备多克隆抗体,检测其免疫原性,并对其进行亚细胞定位分析。方法根据GenBank中羊口疮病毒024基因序列设计引物,进行PCR扩增。将其亚克隆至表达载体,构建原核重组质粒pGEX-6P-1-024以及真核重组质粒pEGFP-N1-024。将原核重组质粒转化Rosetta (DE3)细胞,IPTG诱导表达,SDS-PAGE鉴定024蛋白的表达情况。纯化后的024蛋白免疫BALB/c雌鼠,获得血清并制备多克隆抗体,进行Western-blot分析。将构建的真核质粒转染MDBK细胞,24h后通过免疫荧光观察其在细胞中的表达及亚细胞定位。结果 SDS-PAGE结果表明,羊口疮病毒024基因在Rosetta (DE3)细胞中正确表达,大小为59kDa。Western-blot结果表明024蛋白能与制备的多抗发生特异性反应,具有良好的反应原性。荧光显微镜下结果表明024蛋白转染MDBK细胞后主要在细胞质中表达。结论本实验为后续深入研究ORFV 024基因功能和致病机制奠定了基础。     

真核细胞表达单纯疱疹病毒I型包膜糖蛋白B及其抗原性与免疫原性分析

为在真核细胞中表达并纯化I型单纯疱疹病毒（HSV I）包膜糖蛋白gB,并分析其抗原性和免疫原性,化学合成了包膜糖蛋白gB1胞外区基因片段,构建真核表达载体,并转染至HEK293细胞,表达的蛋白用羊抗HSV1+HSV2血清作为一抗,用ELISA检测其抗原性;用纯化的gB1蛋白免疫昆明小鼠,观察诱发抗体产生的时间及其效价,并用ELISA和Western blot检测小鼠抗gB1多克隆抗体特异性识别重组gB1抗原的能力,评价其免疫原性。结果显示在HEK293细胞中成功表达重组gB1蛋白,ELISA证实羊抗HSV1+HSV2多抗能够识别重组gB1蛋白;重组gB1蛋白免疫小鼠7周后,小鼠血清中多克隆抗体效价达到5×103,表明在真核细胞中高效表达并纯化的重组gB1蛋白具有良好的抗原性和免疫原性,为HSV检测试剂和疫苗研究提供了理论基础。     

结核分枝杆菌38kD蛋白真核表达载体的构建及其在HEK293T细胞中的分泌性表达

目的:构建含结核分枝杆菌38kD蛋白基因的真核表达载体并转染HEK293T细胞,高效表达分泌性38kD蛋白。方法:设计合成的结核分枝杆菌38kD基因被克隆到T载体,然后亚克隆到真核表达载体pcDNA3.0,经酶切鉴定正确后,PEI转染法导入293T细胞,换不含血清的DMEM培养基培养3 d后收集细胞及上清,采用Western印迹检测38kD蛋白的表达。结果:酶切结果显示,获得正确的含38kD基因的重组表达载体;Western印迹结果显示表达载体导入293T细胞中后能在细胞及上清中检测到38kD蛋白表达。结论:构建了含重组结核分枝杆菌38kD蛋白基因的真核表达载体pcDNA3.0-38kD,该载体可在哺乳动物细胞HEK293T中高效分泌性表达38kD蛋白,为结核病诊断试剂盒研发奠定了基础。     

家蚕浓核病毒中国株非结构蛋白1(NS1)的表达

利用PCR技术扩增出BmDNV-3 NS1基因,将目的基因与原核表达载体pET-30a进行连接,转化BL21 star菌并在该菌中表达,经Western blot鉴定表达的产物为BmDNV-3 NS1蛋白,纯化NS1蛋白并制备兔多克隆抗体.同时BmDNV-3 NS1基因亚克隆到杆状病毒转移载体pFastBae-HTb-eGFP中,转化BmDH10BAC感受态细胞,提取的重组Bacmid通过脂质体包埋转染家蚕BmN细胞,再以收获的重组病毒感染家蚕幼虫.家蚕BmN细胞和幼虫感染重组病毒2d后均观察到绿色荧光,经SDS-PAGE分析真核表达的产物与预测的NS1-eGFP融合蛋白大小不一致,说明NS1-eGFP融合蛋白被昆虫内源性的蛋白酶降解.降解的产物用NS1蛋白抗体进行Western blot鉴定为BmDNV-3 NS1蛋白.     

Expression analysis of Foxp3 in T cells from bovine leukemia virus infected cattle

In the present study, we monitored Foxp3⁺ T cells in bovine leukemia virus (BLV)‐infected cattle. By flow cytometric analysis, the proportion of Foxp3⁺CD4⁺ cells from persistent lymphocytotic cattle was significantly increased compared to control and AL cattle. Interestingly, the proportion of Foxp3⁺CD4⁺ cells correlated positively with the increased number of lymphocytes, virus titer and virus load, whereas it inversely correlated with IFN‐γ mRNA expression, suggesting that Foxp3⁺CD4⁺ T cells in cattle have a potentially immunosuppressive function. Further studies are necessary to elucidate the detailed mechanism behind the increased Treg during BLV infection.     

双歧杆菌对溃疡性结肠炎小鼠外周血和肠系膜淋巴结CD4~＋ CD25~＋ Foxp3~＋调节T细胞表达的影响

目的探讨双歧杆菌治疗溃疡性结肠炎与CD4＋ CD25＋ Foxp3＋调节T细胞的相关可能机制。方法采用DSS制作UC小鼠结肠炎模型,随机分成3组：正常对照（NC）组,模型（MD）组,双歧杆菌治疗（BbT）组。造模7 d后,给予双歧杆菌后续治疗7 d。评估小鼠疾病活动指数（DAI）,结肠行HE染色及病理学评分（HDS）;流式细胞仪检测外周血和肠系膜淋巴细胞中表达CD4＋ CD25＋ Foxp3＋的Treg细胞的百分比率。结果 BbT组的DAI明显低于模型组（P〈0.05）;MD组HDS明显高于正常组（P〈0.05）;BbT组的HDS明显低于模型组（P〈0.05）;模型组外周血和肠系膜淋巴细胞CD4＋ CD25＋ Foxp3＋Treg细胞占CD4＋T细胞的百分率明显低于正常组（P〈0.05）;BbT组结肠外周血和肠系膜淋巴细胞CD4＋ CD25＋ Foxp3＋Treg细胞占CD4＋T细胞的百分率明显高于模型组（P〈0.05）。结论双歧杆菌可以提高CD4＋ CD25＋ Foxp3＋Treg数量,调节机体和肠道免疫功能,对UC发挥了一定的治疗作用。     

Effect of HIF1α on Foxp3 expression in CD4+CD25− T lymphocytes

The aim of the present study was to investigate the effect of HIF1α on Foxp3 expression in CD4⁺CD25^? T lymphocytes. CD4⁺CD25^? T lymphocytes were sorted from PBMC using a CD4⁺CD25⁺ regulatory T cell isolation kit. Lentivirus containing lentiviral vector that overexpressed HIF1α (HIF‐lenti) and those containing empty expression vector (control‐lenti) were produced. Meanwhile, lentivirus that contained lentiviral vector that suppressed HIF1α expression (siHIF‐lenti) and those containing control vector (sicontrol‐lenti) were also generated. The sorted CD4⁺CD25^? T lymphocytes were infected with HIF‐lenti, control‐lenti, siHIF‐lenti, and sicontrol‐lenti, respectively. Approximately 72 hr after transduction, real‐time PCR and Western blot were carried out to analyze the RNA and protein expression level of HIF1α and Foxp3. CD4⁺CD25^? T lymphocytes cultured under 21% O₂, 5% CO₂ (normoxia) and 1% O₂, 5% CO₂ (hypoxia) were used as control. Our results showed that overexpression of HIF1α increased both mRNA and protein expression of Foxp3 and, meanwhile, suppression of HIF1α expression by RNAi could reverse high Foxp3 expression in CD4⁺CD25^? T lymphocytes caused by hypoxic culture. These results suggested that hypoxia could stimulate Foxp3 expression by increasing HIF1α expression in CD4⁺ T lymphocytes which may promote CD4⁺ T lymphocytes to convert to Treg.

     

IL-2 controls the stability of Foxp3 expression in TGF-beta-induced Foxp3+ T cells in vivo

Stimulation of naive mouse CD4(+)Foxp3(-) T cells in the presence of TGF-β results in the induction of Foxp3 expression and T suppressor function. However, Foxp3 expression in these induced regulatory T cells (iTreg) is unstable, raising the possibility that iTreg would not be useful for treatment of autoimmune diseases. To analyze the factors that control the stability of Foxp3 expression in iTreg, we generated OVA-specific iTreg from OT-II Foxp3-GFP knockin mice. Following transfer to normal C57BL/6 mice, OT-II GFP(+) cells maintained high levels of Foxp3 expression for 8 d. However, they rapidly lost Foxp3 expression upon stimulation with OVA in IFA in vivo. This unstable phenotype was associated with a strong methylation of the Treg-specific demethylated region within the Foxp3 locus. Administration of IL-2/anti-IL-2 complexes expanded the numbers of transferred Foxp3(+) iTreg in the absence of Ag challenge. Notably, when the iTreg were stimulated with Ag, treatment with IL-2/anti-IL-2 complexes stabilized Foxp3 expression and resulted in enhanced demethylation of the Treg-specific demethylated region. Conversely, neutralization of IL-2 or disruption of its signaling by deletion of Stat5 diminished the level of Foxp3 expression resulting in decreased suppressor function of the iTreg in vivo. Our data suggest that stimulation with TGF-β in vitro is not sufficient for imprinting T cells with stable expression of Foxp3. Administration of IL-2 in vivo results in stabilization of Foxp3 expression and may prove to be a valuable adjunct for the use of iTreg for the treatment of autoimmune diseases.     

MicroRNA‐126 regulates the induction and function of CD4+ Foxp3+ regulatory T cells through PI3K/AKT pathway

Recent evidence showed that limited activation of PI3K/Akt pathway was critical for induction and function sustainment of CD4⁺Foxp3⁺ regulatory T cells (Tregs). However, the underlying mechanism remains largely unknown. In this study, we reported that miR‐126 was expressed in mouse and human Tregs. Further study showed that silencing of miR‐126 using miR‐126 antisense oligonucleotides (ASO) could significantly reduce the induction of Tregs in vitro. Furthermore, miR‐126 silencing could obviously reduce the expression of Foxp3 on Tregs, which was accompanied by decreased expression of CTLA‐4 and GITR, as well as IL‐10 and TGF‐β, and impair its suppressive function. Mechanistic evidence showed that silencing of miR‐126 enhanced the expression of its target p85β and subsequently altered the activation of PI3K/Akt pathway, which was ultimately responsible for reduced induction and suppressive function of Tregs. Finally, we further revealed that miR‐126 silencing could impair the suppressive function of Tregs in vivo and endow effectively antitumour effect of CD8⁺T cells in adoptive cell transfer assay using a murine breast cancer model. Therefore, our study showed that miR‐126 could act as fine‐tuner in regulation of PI3K‐Akt pathway transduction in the induction and sustained suppressive function of Tregs and provided a novel insight into the development of therapeutic strategies for promoting T‐cell immunity by regulating Tregs through targeting specific miRNAs.