GP73及其糖基化修饰对肝癌细胞HepG2炎症相关分子信号通路的影响

目的:通过构建高尔基体膜蛋白73(GP73)氨基酸序列109、144位糖基化位点双突变真核表达质粒,研究GP73及其糖基化修饰对肝癌细胞炎症相关分子信号通路的影响。方法:根据GP73的DNA序列,设计合成2对针对GP73氨基酸序列109、144位糖基化位点突变的PCR引物,以本实验室构建的野生型质粒pc DNA3-Flag-GP73为模板,构建GP73的109、144位糖基化位点双突变质粒pc DNA3-Flag-GP73(DM);用脂质体将此双突变质粒转染293T细胞,用糖蛋白染色和免疫印迹检测该质粒在细胞中的表达情况,用双萤光素酶报告基因实验检测GP73及其糖基化修饰对Hep G2细胞中NF-κB转录激活的影响。结果:糖蛋白染色和免疫印迹结果证实构建的双突变质粒pc DNA3-Flag-GP73(DM)能够表达GP73双糖基化位点突变的蛋白,且糖基化位点的突变使GP73的糖基化修饰完全缺失;双萤光素酶报告基因实验结果表明,野生型GP73能够激活Hep G2细胞中NF-κB的转录活性,而双糖基化位点突变会使GP73失去此激活作用。结论:构建了GP73双糖基化位点突变的真核表达质粒pc DNA3-Flag-GP73(DM)。GP73参与激活肝癌细胞炎症信号通路,糖基化修饰对于GP73发挥此作用是必不可少的。     

PDX-1表达质粒的构建及表达

目的:为进一步研究PDX-1基因的功能打下基础.方法:从人的胰腺cDNA中PCR扩增PDX-1基因的阅读框架,构建质粒pAAV-PDX-1.用pAAV-PDX-1转染293T细胞48小时后,用RT-PCR和Western Blot方法检测293T细胞中mRNA和蛋白水平PDX-1的表达.结果:转染了pAAV-PDX-1的293T细胞中有PDX-1的表达,而在转染质粒pAAV-MCS和未转染的293T细胞中均没有检测到PDX-1的表达.结论:构建的pAAV-PDX-1质粒在mRNA和蛋白水平都能表达PDX-1.     

pcDNA3.1/myc-His-DJ-1^M26I重组载体构建及其对NIH3T3细胞增殖和凋亡研究

目的构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组表达载体,为研究DJ-1M26I突变与细胞增殖、凋亡的关系及建立转基因动物模型奠定基础。方法采用突变试剂盒将DJ-1蛋白第26位氨基酸进行突变,分别构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组表达载体,并采用脂质体介导的方法分别将其转染入NIH3T3细胞,500μg/mL G418压力筛选稳定克隆,对2种转染细胞在DNA水平、RNA水平和蛋白质水平进行鉴定,采用MTT染色方法和AnnexinV-FITC试剂盒进行转染阳性克隆细胞的细胞活力与细胞凋亡检测。结果 pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组质粒转染NIH3T3细胞经G418筛选后,PCR方法检测分别获得1个和3个阳性细胞克隆,RT-PCR及western blot方法进行DJ-1-His基因表达检测,结果均证明外源插入基因的表达,MTT实验结果初步证明转染DJ-1M26I基因的NIH3T3阳性细胞组细胞增殖速率低于正常NIH3T3细胞组（P〈0.05）,转染DJ-1基因的NIH3T3阳性细胞组细胞增殖速率与正常NIH3T3细胞相比无明显差别;细胞凋亡检测表明转染DJ-1M26I基因的NIH3T3阳性细胞组细胞凋亡率高于正常NIH3T3细胞,转染DJ-1基因的NIH3T3阳性细胞组细胞凋亡率低于正常NIH3T3细胞（P〈0.05）。结论成功构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1 M26I重组表达载体,成功筛选出稳定表达人DJ-1及DJ-1 M26I的NIH3T3细胞。DJ-1 M26I基因突变更易导致NIH3T3细胞的凋亡。     

PEI-壳聚糖在结肠癌细胞CD133~+梯度表达的转染研究

目的:以PEI-壳聚糖(CP)为载体材料,考察CD133~+呈梯度表达的4种结肠癌细胞上的转染效率,将特异性结合CD133的TR多肽修饰CP材料,探讨材料与转染的关系。方法:根据CD133~+含量将细胞分为高表达组和低表达组,合成法制备CP材料,激光粒度仪测定纳米粒粒径、电位,CCK8实验检测细胞毒性,用质粒p GL3和EGFP考察CP材料的转染效果,Western印记检测CD133~+蛋白的表达。结果:CD133~+高表达组SW480、HCT116的CD133~+含量为96.97%、92.96%,低表达组CaCO_2、SW620的CD133~+含量为2.00%、0.56%;CCK8结果得出,CP材料按质量比N/P=5时,生物安全性较高;质粒p GL3转染高表达组,其CD133~+RLU值低于低表达组;质粒EGFP转染高表达组,绿色荧光弱于低表达组,Western blot检测接TR肽的CP材料CD133~+表达高于CP材料。结论:CP材料在低表达CD133~+组转染效率高,且接TR肽的CP材料针对CD133~+有可能介导材料进入细胞。     

DJ-1~（L166P）与DJ-1~（M26I）基因对NIH3T3细胞增殖和凋亡的比较

目的在细胞学水平比较DJ、DJ-1M26 I、DJ-1L166 P基因对NIH 3T3细胞增殖速率与凋亡的关系,为建立转基因动物模型及帕金森疾病发病机制研究奠定基础。方法分别将pcDNA3.1/myc-His-DJ-1、pcDNA3.1/myc-His-DJ-1L166 P和pcDNA3.1/myc-His-DJ-1M26 I重组质粒脂质体方法转染NIH 3T3细胞,500μg/ml G418压力筛选稳定克隆,对3种转染细胞在DNA水平、RNA水平和蛋白质水平进行鉴定,采用MTT染色方法和Annexin V-FITC试剂盒进行转染阳性克隆细胞的细胞活力与细胞凋亡检测。结果 pcDNA3.1/myc-His-DJ-1、pcDNA3.1/myc-His-DJ-1L166 P和pcDNA3.1/myc-His-DJ-1M26 I重组质粒转染NIH 3T3细胞经G418筛选后,PCR方法检测分别获得1个、4个、3个阳性细胞克隆,RT-PCR及Western blot方法进行DJ-1-His基因表达检测,结果均证明外源插入基因的表达,Caspase-3 RNA水平检测DJ-1L166 P和DJ-1M26 I组表达高于正常NIH 3T3细胞组,而DJ-1组caspase-3转录水平相对最低,MTT实验结果初步证明转染DJ-1L166 P和DJ-1M26 I基因的NIH3T3阳性细胞组细胞增殖速率均低于DJ-1组和正常NIH 3T3细胞组（P〈0.05）,转染DJ-1基因的NIH 3T3阳性细胞增殖速率与正常NIH 3T3细胞相比无明显差别;细胞凋亡检测表明转染DJ-1L166 P和DJ-1M26 I基因的NIH3T3阳性细胞凋亡率均高于正常NIH 3T3细胞,转染DJ-1基因的NIH 3T3阳性细胞凋亡率低于正常NIH 3T3细胞（P〈0.05）。结论 DJ-1L166 P和DJ-1M26 I基因突变均降低NIH3T3细胞增殖速率,DJ-1L166 P和DJ-1M26 I基因突变更易导致NIH 3T3细胞的凋亡,DJ-1L166 P和DJ-1M26 I基因突变对NIH3T3细胞增殖速率和细胞凋亡影响是相似的。     

稳定表达小鼠CD40L的NIH3T3细胞系的建立及其在B细胞培养和激活中的应用

该研究构建小鼠CD40L真核表达重组质粒pcDNA3.1-mCD40L,通过电转法将重组质粒转至NIH3T3细胞中。利用G418对转染后细胞进行压力筛选,获得稳定转染细胞株。提取稳定转染细胞株RNA,通过RT-PCR法检测Neo基因的mRNA表达情况。分离稳定转染细胞上清,利用ELISA法检测小鼠CD40L蛋白水平的表达情况。RT-PCR结果显示,Neo基因能够在稳定转染细胞中表达,ELISA结果显示,获得的稳定转染细胞株NIH3T3-mCD40L细胞上清中CD40L的表达量高达1.286 ng/mL。进一步活性研究表明,该细胞系能够在体外与IL-2和IL-21共同作用培养B细胞至14天,并刺激B细胞产生特异性抗体。该细胞系的成功构建,为利用体外B细胞分离培养和活化法分离特异性单克隆抗体奠定了良好的基础。     

STK15的表达对NIH3T3细胞的影响

目的构建pcDNA3.1-STK15表达质粒,探讨STK15基因对小鼠成纤维细胞（NIH3T3）的影响。方法构建pcDNA3.1-STK15质粒,将其转染NIH3T3,应用RT-PCR、免疫细胞化学和Western印迹方法检测STK15的表达;MTT法检测细胞增殖能力;Transwell检测细胞侵袭能力。结果转染pcDNA3.1-STK15质粒的NIH3T3细胞在48 h有STK15的表达,而且该细胞的增殖速度和穿透Matrigel胶的细胞数均明显高于对照组（P〈0.05）。结论STK15基因具有增加细胞增殖和细胞侵袭力的功能,进而形成肿瘤。     

FNDC5真核表达载体的构建及其N-连接糖基化修饰位点的预测

为构建小鼠FNDC5的真核表达载体p EYFP-N1-FNDC5,预测FNDC5的N-连接糖基化修饰位点。在质粒p EYFP-N1的多克隆位点插入小鼠FNDC5构建真核表达载体p EYFP-N1-FNDC5,用脂质体转染He La细胞。转染48 h后,用免疫印迹法检测融合蛋白的表达,使用激光共聚焦显微镜观察FNDC5融合蛋白的亚细胞定位。利用Net NGlyc 1.0 Server程序,预测FNDC5蛋白N-连接糖基化修饰位点。结果显示:与对照组相比,真核表达载体p EYFP-N1-FNDC5能够表达FNDC5融合蛋白,且FNDC5融合蛋白定位于细胞膜。FNDC5的Ⅲ型纤连蛋白结构域区存在两个N-连接糖基化修饰位点。上述结果表明FNDC5真核表达载体构建及表达成功,FNDC5存在N-连接糖基化修饰位点。     

乙型肝炎病毒e抗原基因与绿色荧光蛋白基因的融合及其表达

质粒pS65T含有T7启动子驱动的绿色荧光蛋白突变型gfpS65T基因,经修饰后,在其中插入乙型肝炎病毒e抗原（HBeAg）基因,使两基因同框成为融合基因。在大肠杆菌BL21中由于T7启动子的控制,高效表达了具有双功能（抗原性和发光性）的融合蛋白（GFP－HBeAg）。融合蛋白MW为52kDa,N端有6个组氨酸残基,因而用Ni金属螯合层析柱对融合蛋白进行了分离纯化,利用诊断HBV的ELISA试剂盒检测了融合蛋白的抗原性,在荧光显微镜下观察到了融合蛋白的绿色荧光。并探讨了用其组装成新型免疫诊断试剂的可能性。     

用表达T7RNA聚合酶细胞系拯救麻疹病毒微复制子

构建稳定表达T7RNA聚合酶的细胞系,用于提高拯救麻疹病毒微复制子效率.PCR扩增T7RNA聚合酶基因,克隆到真核表达载体,转染Vero细胞,用G418筛选到稳定表达的细胞株Vero/pcDNA3-T7.用Westernblotting证明了T7RNA聚合酶在细胞中的表达.将T7启动子控制绿色荧光蛋白表达的质粒转染该细胞后,绿色荧光蛋白在细胞株中得到表达.反向插入报告基因的微复制子转染感染麻疹病毒的Vero/pcDNA3-T7细胞后,细胞中能够检测到报告基因的表达.用细胞系取代痘苗病毒系统,可以提高拯救效率.     

以T细胞受体为基础的免疫疗法研究进展

T细胞是机体抗肿瘤免疫的核心,以T细胞功能调控为基础的免疫检查点疗法已经在多种肿瘤的临床治疗中取得了重大突破,以基因工程化T细胞为基础的过继性免疫细胞疗法在血液瘤治疗中取得了重要进展,免疫治疗已经对肿瘤的临床治疗产生了深刻变革,成为肿瘤临床治疗策略的重要组成部分。T细胞受体（T cell receptor,TCR）赋予了T细胞识别肿瘤抗原的特异性,能够识别由主要组织相容性复合体（major histocompatibility complex,MHC）呈递的包括胞内抗原在内的广泛肿瘤抗原,具有高度的抗原敏感性,因而具有广泛的抗肿瘤应用前景。2022年第一款TCR药物的上市开启了TCR药物开发的新纪元,多项TCR药物临床研究表现出潜在的肿瘤治疗价值。本文综述了以TCR为基础的免疫治疗策略研究进展,包括T细胞受体工程化T细胞（T cell receptor-engineered T cell,TCR-T）和TCR蛋白药物,以及基于TCR信号的其他免疫细胞疗法,以期为以TCR为基础的免疫治疗策略开发提供参考。     

Lysis protein T of bacteriophage T4

Summary Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.     

Self-nonself discrimination by T lymphocytes

Tolerance of T lymphocytes to self-antigens is mainly achieved at the level of the primary lymphoid organ, the thymus, and probably to a lesser extent in the secondary lymphoid tissues. Whether self-reactive lymphocytes ignore their target autoantigen, or are tolerized by the various mechanisms discussed, depends on the circumstances.     

创伤小鼠血清,巨噬细胞,Ts细胞对反抑制T细胞的影响

研究了创伤小鼠反抑制T细胞(Tcs)比例、功能的变化及创伤血清、巨噬细胞、抑制性T细胞(Ts)对正常小鼠Tcs细胞的影响。结果表明,创伤小鼠脾细胞中VVL~ 细胞百分率于伤后一过性减少,Tcs细胞在T淋转、IL-2、IL-2R检测系统中的反抑制活性均明显受抑;创伤小鼠血清、巨噬细胞、Ts细胞在体外对正常Tcs细胞反抑制活性(T淋转、IL-2、IL-2R检测系统)均具有不同程度的抑制作用,创伤后4天小鼠血清在体内对正常小鼠脾脏VVL~ 细胞百分率无明显影响,但可明显降低正常小鼠Tcs细胞的反抑制活性。表明创伤可致Tcs细胞比例及功能发生改变,创伤后血清、巨噬细胞、Ts细胞参与介导了Tcs细胞功能的受抑过程。     

心衰病人的血清T3,T4测定及其意义

目的探讨心衰患者血清Ｔ３、Ｔ４的含量及其临床意义。方法应用放射免疫法测定心衰患者血清Ｔ３、Ｔ４水平５０例,并与３０例病因基本相同心功能代偿者作对照。结果重度心衰者Ｔ３、Ｔ４水平明显下降,与对照组对比差异有显著性（Ｐ＜０．０５）。结论血清Ｔ３、Ｔ４水平与病人预后密切相关,可作为判断病人病情严重程度及临床疗效的综合指标之一。     

Structural chromosome differentiation between Triticum timopheevii and T. turgidum and T. aestivum

 Chromosome pairing at metaphase-I was analyzed in F₁ hybrids among T. turgidum (AABB), T. aestivum (AABBDD), and T. timopheevii (A^tA^tGG) to study the chromosome structure of T. timopheevii relative to durum (T. turgidum) and bread (T. aestivum) wheats. Individual chromosomes and their arms were identified by means of C-banding. Homologous pairing between the A-genome chromosomes was similar in the three hybrid types AA^tBG, AA^tBGD, and AABBD. However, associations of B-G were less frequent than B-B. Homoeologous associations were also observed, especially in the AA^tBGD hybrids. T. timopheevii chromosomes 1A^t, 2A^t, 5A^t, 7A^t, 2G, 3G, 5G, and 6G do not differ structurally from their counterpart in the A and B genomes. Thus, these three polyploid species inherited translocation 5AL/4AL from the diploid A-genome donor. Chromosome rearrangements that occurred at the tetraploid level were different in T. turgidum and T. timopheevii. Translocation 4AL/7BS and a pericentric inversion of chromosome 4A originated only in the T. turgidum lineage. The two lines of T. timophevii studied carry four different translocations, 6A^tS/1GS, 1GS/4GS, 4GS/4A^tL, and 4A^tL/3A^tL, which most likely arose in that sequence. These structural differences support a diphyletic origin of polyploid wheats. Received: 15 June 1998 / Accepted: 19 August 1998     

Vdelta1+ T cells are crucial for repertoire formation of gammadelta T cells in the lung

The pulmonary resident T lymphocytes (RPLs) expressing a nearly invariant T cell receptor γδ heterodimer (γδTCR) migrate from fetal thymus to the lung epithlium, followed by RPL subsets expressing diverse sets of γδTCRs after birth. However, it remains unclear whether the fetal type Vγ6/Vδ1⁺ RPLs are essential for γδ T cell repertoire formation in the lung epithelium. In this study, we found a marked decrease in the number of γδRPLs at 4 weeks of age in Vδ1^−/− mice and they predominantly expressed Vγ6 and Vδ4 genes. The skewed diversity towards the Vδ4-(Dδ1)-Dδ2-Jδ2 junctional region was observed only in γδ RPLs from 4-week-old Vδ1^−/− mice, compared with those from 8-week-old Vδ1^−/− mice and the both ages of wild-type mice. These results suggest that the invariant Vδ1⁺ T cells are crucial not only for optimal γδ T cell expansion but also for affecting the migration or microenvironment for other γδ T cells in the lung epithelium.     

One step engineering of T7-expression strains for protein production: increasing the host-range of the T7-expression system

The T7-expression system has been very useful for protein expression in Escherichia coli. However, it is often desirable to over-express proteins in species other than E. coli. Here, we constructed an inducible broad-host-range T7-expression transposon, which allows simple one-step construction of T7-expression strains in various species, providing the option to over-express proteins of interest in a broader host-range. This transposon contains the T7 RNA polymerase driven by the lacUV5 promoter, which is repressed by the lac-repressor. Leaky expression is prevented by the presence of T7-lysozyme on this construct. The complete T7-expression system is flanked by mariner transposon repeats of the suicidal R6Kgammaori plasmid, pBT20-Deltabla. Stable integration of the whole system is possible by a one-step selection for a Flp-excisable Gm(R)-marker. We showed the engineering of E. coli, Pseudomonas aeruginosa, Erwinia carotovora, Salmonella choleraesuis, Agrobacterium tumefaciens, and Chromobacterium violaceum strains with this construct and demonstrated the expression of the Burkholderia pseudomallei Asd protein in these hosts, by induction with isopropyl-beta-d-thiogalactopyranoside (IPTG).