同尾酶技术在构建疟疾多价重组DNA疫苗中的应用

同尾酶是一类识别不同核苷酸序列但能酶切产生相同粘性末端的限制性内切酶,依靠同尾酶的这种特性,可以根据需要将不同的ＤＮＡ 片段进行灵活组合,获得各种排列顺序的多价表位重组疫苗．将这种方法用于疟疾多价重组ＤＮＡ 疫苗的研制;ＢＡＬＢ／ｃ 小鼠免疫实验对所得重组疫苗ＰＵ２８６的免疫原性进行了测定．     

重组原核表达载体pQE30-HPV58L1的构建及鉴定

目的：构建重组原核表达载体以获得HPV58L1活性蛋白,为进一步研制HPV58基因工程疫苗打下基础。方法：用聚合酶链反应（PCR）扩增HPV58L1完整编码区基因,将PCR扩增产物克隆至pUC19质粒中并测序。利用pQE30质粒载体构建重组原核表达载体pQE30-HPV58L1,并通过酶切电泳验证重组结果的正确性。结果：PCR扩增出1.6Kb特异性片段,经克隆至pUC19后测序表明序列同源性与Gen-Bank报道一致。重组质粒pQE30-HPV58L1酶切后显示其大小约5.1Kb,酶切图谱与预期相同。结论：成功构建了重组原核表达载体pQE30-HPV58L1。     

人乳头瘤病毒58型L1基因重组表达载体的构建及鉴定

目的　人乳头瘤病毒 (HPV)是一种能引起人类皮肤、粘膜的乳头状瘤或疣 ,并可导致恶性病变的病原体。研究表明 ,90 %宫颈癌的发生与人乳头瘤病毒感染有关。近年流行病学研究和文献报道显示 ,HPV5 8型在中国妇女宫颈癌中检出阳性率非常高 ,是仅次于HPV1 6型、1 8型的乳头瘤病毒高发型 ,尤其是东南省份 ,5 8型的感染率有上升趋势 ,与HPV1 6型的检出率相近 ,同时二者在宫颈各病变组的检出强度趋势基本一致。由此可见 ,在我国HPV5 8型感染与宫颈癌关系密切。在HPV5 8基因组中 ,L1和L2基因分别编码主要衣壳蛋白和次要衣壳蛋白 ,其中L1基…     

Anti-HIF-1α-pEGFP重组质粒的构建及表达

目的构建EGFP-HIF-1α反义重组质粒,转染人宫颈癌Hela细胞,用以探讨HIF-1α蛋白在宫颈癌的生长、转移中的作用。方法应用基因重组技术构建EGFP-HIF-1α反义重组质粒,通过脂质体介导将其转染入Hela细胞,倒置荧光显微镜观察转染效果,Western blot检测HIF-1α蛋白的表达。结果酶切鉴定结果显示含EGFP-HIF-1α反义重组质粒构建成功,并能在Hela细胞中封闭HIF-1α蛋白的表达。结果 EGFP-HIF-1α反义重组质粒构建及转染成功,封闭Hela细胞中HIF-1α蛋白的表达,为进一步研究HIF-1α蛋白在宫颈癌的生长、转移中的作用提供试验基础。     

正义、反义MCP-1基因逆转录病毒载体重组质粒的构建和包装

为构建含单核细胞趋化蛋白-1(Monocytechemoattractantprotein-1,MCP-1)基因的重组逆转录病毒pLXSN/MCP-1质粒.用RT-PCR技术从大鼠系膜细胞中扩增出MCP-1全长DNA,将其与Pgem-TE连接,用限制性内切酶EcoRⅠ对pTE-MCP-1和逆转录病毒质粒pLXSN分别进行酶切.在T4连接酶的作用下,构建重组逆转录病毒质粒pLXSN/MCP-1.经BglⅡ,XhoⅠ酶切鉴定MCP-1在质粒中的方向,用脂质体介导的方法把重组质粒DNA转染进入包装细胞PA317中,经过G418筛选出抗性克隆.通过NIH3T3细胞测定病毒液的病毒滴度.结果证实经过RT-PCR技术从大鼠系膜细胞中扩增出MCP-1全长DNA与所需的大小一致.重组质粒经酶切分析与预期的结果一致.G418筛选出抗性克隆,能稳定合成并分泌重组逆转录病毒颗粒.NIH3T3细4胞测定病毒滴度为17×10cfu/mL.     

鸡gga-miR-7b靶基因VDAC1-3’UTR双荧光素酶报告基因质粒的构建

本研究以鸡gga-miR-7b作为研究对象,为了明确与肿瘤形成相关的关键基因VDAC1(Voltage-dependent anion channel 1)是否为gga-miR-7b的靶基因,深入研究gga-miR-7b对VDAC1基因的调控作用,本研究运用TargetScan和miRBD生物学软件预测了gga-miR-7b与VDAC1 m RNA的3’端非编码区(3’UTR)存在种子结合位点(GTCTTCC),并使用PCR克隆以及同源重组突变的方法构建VDAC1-3’UTR野生型和突变型双荧光素酶报告基因重组质粒,经SacⅠ和XbaⅠ双酶切鉴定及测序结果显示,片段大小符合且序列正确;成功构建了VDAC1-3’UTR野生型和突变型双荧光素酶报告基因重组质粒并命名为pmirGLOVDAC1-wt3’UTR和pmirGLO-VDAC1-mut3’UTR。本研究结果进一步为gga-miR-7b候选靶基因VDAC1的鉴定以及功能研究提供理论依据。     

登革3型病毒prM-E基因重组质粒DNA的构建和真核表达

构建登革 3型病毒 prM E基因的真核表达重组质粒 ,并进行体外表达 ,为登革DNA疫苗的研究奠定基础。用RT -PCR法获得 prM -E基因片段 ,然后将其克隆到真核表达载体中。用电穿孔法将重组质粒DNA转入BHK细胞 ,通过免疫荧光法检测外源基因在真核细胞中的表达。结果 ,通过酶切和序列测定证实了构建的重组质粒DNA含序列正确的 prM- E基因。用免疫荧光法检测到转染了重组质粒DNA的BHK细胞的胞浆中有登革 3型病毒特异蛋白的表达。说明含有登革 3型病毒prM -E基因的真核表达重组质粒可以在BHK细胞中表达 ,该结果为观察该重组质粒的免疫原性奠定了基础。     

宫颈癌中CD1α分子和趋化因子CCL20/MIP-3α的表达

目的:研究CD1α分子,趋化因子人巨噬细胞炎性蛋白-3α(CCL20/MIP-3α)在宫颈癌组织中的表达及其相关性,探讨其在宫颈癌免疫逃逸机制中的作用。方法:应用免疫组化PV-6000通用二步法检测CD1α和趋化因子CCL20/MIP-3α在正常宫颈组织及宫颈癌组织中的表达,并分析两者之间的相关性。结果:CD1α和CCL20/MIP-3α在宫颈癌中的表达均明显降低(P均0.01);CD1α与CCL20/MIP-3α在宫颈癌中的表达显著相关(P0.01)。结论:在宫颈癌组织中,趋化因子的减少导致抗原提呈细胞数量下降,不足以引起肿瘤局部免疫反应而导致了宫颈局部病变的侵袭及发展。     

编码山羊补体C3d与口蹄疫病毒VP1融合蛋白重组质粒的构建及表达

旨在构建含分子佐剂山羊补体C3d基因的O型口蹄疫病毒VP1基因真核表达质粒。克隆山羊C3d基因, 通过linker(G4S)2将3拷贝C3d基因串联; 克隆羊源O型口蹄疫病毒VP1基因, 通过linker(G4S)2与3拷贝C3d基因相连, 构建重组质粒pUC19-VP1-C3d3。将VP1-C3d3融合基因亚克隆入含有分泌表达信号肽tPA序列的pcDNA3.1(+)CMV启动子下游, 构建重组真核表达质粒pcDNA3.1-tPA-VP1-C3d3。在脂质体介导下, 将pcDNA3.1-tPA -VP1-C3d3转染HeLa细胞。间接免疫荧光分析表明, VP1- C3d3在HeLa细胞中获得了瞬时表达, Western blot分析证实转染的阳性细胞能分泌预期大小(133 kD)的融合蛋白。重组质粒pcDNA3.1-tPA-VP1-C3d3为研制以羊补体C3d为分子佐剂的口蹄疫新型疫苗奠定了基础。     

一种构建多拷贝串联小分子多肽基因的方法

目的:构建蛇毒锯鳞蝰血抑环肽(Ecs)基因串联多拷贝重组质粒。方法:采用重叠延伸PCR克隆Ecs基因,将第28位Met的密码子突变为Leu,利用其序列特点及酶切位点,在表达载体pET30a上将Ecs基因以同向串联方式连接,在E.coliBL21(DE3)中表达产物。结果:工程菌表达的串联Ecs与预期结果相符,实现了Ecs基因的串联表达。结论:为活性小肽的体外表达提供了新的思路和方法。     

Copy number variations of <Emphasis Type="Italic">CCL3L1</Emphasis> and long-term prognosis of HIV-1 infection in asymptomatic HIV-infected Japanese with hemophilia

We set up a cohort of HIV-infected, asymptomatic Japanese patients with hemophilia for follow-up study in 1995. All subjects who had been infected with HIV-1 for more than 10 years met the criteria for long-term nonprogressors (LTNPs) at the time of entry; however, some of them later developed lymphopenia and required antiretroviral treatment during five more years of observation. In this study, we investigated the impacts of the CCL3L1 dose on the long-term prognosis in the subjects with chronic HIV-1 infection. We collected genomic DNA from 95 long-term survivors including 48 nonprogressors and 47 subjects receiving antiretroviral treatment. The distributions of CCL3L1 copy number significantly differed between the 95 HIV-1-infected subjects with hemophilia and 205 controls. Average copy number of CCL3L1 in the HIV-1-infected subjects was significantly lower than in control (5.00 ± 0.22 vs 3.35 ± 0.24, p < 0.001). Moreover, the subjects possessing two or less copies of CCL3L1 had significantly higher risk of acquiring HIV-1. However, CCL3L1 copy number variations had no significant effect on the disease progression among the LTNP subjects who had been afflicted with chronic HIV-1 infection for more than 15 years, when compared between nonprogressors and patients under treatment (3.68 ± 0.37 vs 3.02 ± 0.29, ns). Furthermore, variations in the CCL3L1 copy number had little effect on the levels of HIV-1 load among them. We conclude that variation in the CCL3L1 copy number is apparently not a factor that determines the prognosis of chronic HIV-1 infection, even though it is linked to HIV-1 susceptibility.     

一种基于二代测序辨识短串联重复序列长度变异的新方法及其在人类遗传疾病研究中的应用

二代测序技术的涌现推动了基因组学研究,特别是在疾病相关的遗传变异研究中发挥了重要作用．虽然大多数遗传变异类型都可以借助于各种二代测序分析工具进行检测,但是仍然存在局限性,比如短串联重复序列的长度变异．许多遗传疾病是由短串联重复序列的长度扩张导致的,尤其是亨廷顿病等多种神经系统疾病．然而,现在几乎没有工具能够利用二代测序检测长度大于测序读长的短串联重复序列变异．为了突破这一限制,我们开发了一个全新的方法,该方法基于双末端二代测序辨识短串联重复序列长度变异,并可估计其扩张长度,将其应用于一项基于全外显子组测序的运动神经元疾病临床研究中,成功地鉴定出致病的短串联重复序列长度扩张．该方法首次原创性地利用测序读长覆盖深度特征来解决短串联重复序列变异检测问题,在人类遗传疾病研究中具有广泛的应用价值,并且对于其他二代测序分析方法的开发具有启发性意义．     

一种新的基于STR的染色体三体性疾病诊断策略中遗传标记的评估

背景:在此前发表的文章中,我们提出了一种新的基于短串联重复序列(Short Tandem Repeats,STR)的染色体三体性疾病的诊断策略.当应用这种策略来检测特定染色体拷贝数时,需要从人类基因组众多的STR中选择适宜的染色体特异性STR基因座构建一个诊断系统,根据系统中的STR基因座是否检测到三种不同的等位基因产物来判断个体是否为三体患者.目的:本研究拟进一步提出并验证对单个STR基因座及由多个STR基因座构成的诊断系统的评估方法,旨在帮助选择适宜的STR基因座构建一个高效能的诊断系统.方法:我们提出一个新的参数--三等位基因栓出率,并推导出该参数的计算公式,用于定量评估一个STR基因座在这种诊断策略中的效能.在此基础上,推导出另一个数学公式,计算一个非整倍体诊断系统能够在一个三体性患者?哌 检测到三个不同等位基因的概率,根据这个概率的大小来衡量系统诊断效能的高低.最后,我们将所提出的两个公式用于评估我们在先前研究中构建的一个21三体的诊断系统.结果:这个21三体诊断系统由9个21号染色体特异性STR基因座构成.根据我们所提出的两个公式,这些STR基因座的三等位基因检出率在0.203-0.638之间,该系统在21三体患者能够检测到三个不同等位基因的概率大于0.95.结论:我们所提出并验证的公式可以对单个STR基因座和系统的诊断效能进行定量评估,帮助选择适宜的STR遗传标记,并确定一个高效能诊断系统所需的STR基因座的数量,从而为这种诊断策略的广泛应用提供基础.     

脂多糖诱导成骨细胞中CHI3L1表达的分子机制研究

慢性骨髓炎因其病程漫长、易出现并发症以及复发率高成为临床上棘手的难题,其主要致病原因是金黄色葡萄球菌等革兰氏阴性菌感染．脂多糖(LPS)是革兰氏阴性细菌细胞壁的重要成分,用LPS在体外刺激骨组织相关细胞在一定程度上可以模拟骨髓炎患者的病理特征．实时荧光定量PCR和Western blot等试验结果表明,在骨髓炎患者的骨组织和LPS刺激的成骨细胞中,几丁质酶家族成员CHI3L1的表达均有明显升高．核因子κB (NF-κB) 萤光素酶报告载体检测结果显示,LPS能诱导细胞的NF-κB活化,NF-κB活化抑制剂Bay11-7082能抑制LPS诱导的CHI3L1表达升高．用抗肿瘤坏死因子α(TNF-α)的抗体预处理细胞,或采用siRNA干扰的方法抑制TNF-α受体的表达,都能明显抑制LPS诱导的CHI3L1表达上调．同时,NF-κB活化抑制剂Bay11-7082预处理细胞能抑制LPS对TNF-α表达的诱导作用．结果提示,LPS通过激活NF-κB诱导TNF-α分泌上调,刺激CHI3L1表达．提出骨髓炎及脂多糖刺激条件下CHI3L1表达上调,并在细胞水平上初步探讨了脂多糖诱导CHI3L1表达的分子机制．     

一种构建重组杆状病毒的简易方法

对传统构建重组杆状病毒的方法作了如下改进：先用磷酸钙共沉淀法单独将质粒DNA转进昆虫Sf细胞中,其中重组质粒采用聚乙二醇沉淀法纯化,12～24h后再用低剂量的病毒攻击细胞．改进后的方法简便、省时、经济、重组率高,适于一般实验室使用．     

Acetic acid activates PKD1L3-PKD2L1 channel—A candidate sour taste receptor

The polycystic kidney disease (PKD) 1L3-PKD2L1 channel is a candidate sour taste receptor expressed in mammalian taste receptor cells. Various acids are reported to activate PKD channels after the removal of the acid stimuli, but little information is available on the activation of these channels by acetic acid. It was difficult to analyze the PKD channel activation by acetic acid using Ca²⁺ imaging experiments because this acid induces a transient and nonspecific response in cultured cells. Here, we developed a novel method to evaluate PKD channel activation by acetic acid. Nonspecific responses were observed only over a short period after the application of acetic acid. In contrast, PKD channel activation evoked by acetic acid as well as citric acid was detected even at a later time point. This method revealed that PKD1L3-PKD2L1 channel activation by acetic acid was pH-dependent and occurred when the ambient pH was <3.1.     

Off-response property of an acid-activated cation channel complex PKD1L3-PKD2L1

Ligand-gated ion channels are important in sensory and synaptic transduction. The PKD1L3-PKD2L1 channel complex is a sour taste receptor candidate that is activated by acids. Here, we report that the proton-activated PKD1L3-PKD2L1 ion channels have the unique ability to be activated after the removal of an acid stimulus. We refer to this property as the off-response (previously described as a delayed response). Electrophysiological analyses show that acid-induced responses are observed only after the removal of an acid solution at less than pH 3.0. A small increase in pH is sufficient for PKD1L3-PKD2L1 channel activation, after exposure to an acid at pH 2.5. These results indicate that this channel is a new type of ion channel-designated as an 'off-channel'-which is activated during stimulus application but not gated open until the removal of the stimulus. The off-response property of PKD1L3-PKD2L1 channels might explain the physiological phenomena occurring during sour taste sensation.     

Novel method to differentiate 3T3 L1 cells in vitro to produce highly sensitive adipocytes for a GLUT4 mediated glucose uptake using fluorescent glucose analog

Adipocytes play a vital role in glucose metabolism. 3T3 L1 pre adipocytes after differentiation to adipocytes serve as excellent in vitro models and are useful tools in understanding the glucose metabolism. The traditional approaches adopted in pre adipocyte differentiation are lengthy exercises involving the usage of IBMX and Dexamethasone. Any effort to shorten the time of differentiation and quality expression of functional differentiation in 3T3 L1 cells in terms of enhanced Insulin sensitivity has an advantage in the drug discovery process. Thus, there is a need to develop a new effective method of differentiating the pre adipocytes to adipocytes and to use such methods for developing efficacious therapeutic molecules. We observed that a combination of Dexamethasone and Troglitazone generated differentiated adipocytes over fewer days as compared to the combination of IBMX and Dexamethasone which constitutes the standard protocol followed in our laboratory. The experiments conducted to compare the quality of differentiation yielded by various differentiating agents indicated that the lipid droplet accumulation increased by 112 % and the GLUT4 mediated glucose uptake by 137 % in cells differentiated with Troglitazone and Dexamethasone than in cells differentiated traditionally. The comparative studies conducted for evaluating efficient measurable glucose uptake by GOPOD assay, radioactive ³H-2-deoxy-D-glucose assay and by non-radioactive 6-NBDG (fluorescent analog of glucose) indicated that the non-radioactive method using 6-NBDG showed a higher signal to noise ratio than the conventional indirect glucose uptake method (GOPOD assay) and the radioactive ³H-2-deoxy-D-glucose uptake method. Differentiated 3T3 L1 cells when triggered with 2.5 ng/mL of Insulin showed 3.3 fold more glucose uptake in non-radioactive method over the radioactive ³H-2-deoxy-D-glucose uptake method. The results of this study have suggested that a combination of Dexamethasone and Troglitazone for 3T3 L1 cell differentiation helps in better quality differentiation over a short period of time with increased sensitivity to Insulin. The application of these findings for developing new methods of screening novel Insulin mimetics and for evaluating the immunological responses has been discussed.