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1.
在构建了含伪狂犬病毒(pseudorabies virus，PRV)湖北株部分PK基因和gG基因转移载体的基础上，利用平端连接的方法将绿色荧光蛋白(GFP)的基因表达盒插入到缺失的部分，并在下游引入了1个多克隆位点，构建了重组转移载体KGDF。用限制性内切酶鉴定重组转移载体KGDF。根据质粒EGFP-C1中的GFP基因序列设计1对引物鉴定GFP表达盒插入的正确性。用脂质体转染试剂盒将KGDF和PRVFIB的基因组或病毒共转染BHK-21细胞，在荧光显微镜下将出现病变的荧光斑挑出得到重组病毒。将重组病毒扩大培养后提取基因组鉴定重组病毒中的GFP基因，并通过挑取病变的荧光斑的方法纯化重组病毒。 相似文献
2.
猪伪狂犬病病毒gE基因缺失苗免疫试验 总被引：1，自引：0，他引：1
为了掌握猪伪狂犬病gE基因缺失疫苗免疫效果,并制定合理的免疫程序,选用国产和进口的猪伪狂犬病gE基因缺失弱毒疫苗,用4种不同的免疫程序,对250头母猪和1000头仔猪进行了免疫试验。试验期间,按比例定时采集免疫猪的血液,用ELISA试剂盒进行抗体检测,证明猪伪狂犬gE基因缺失疫苗,无论国产苗或进口苗都可以产生良好的免疫效果;无论跟胎免疫、1年2次普免,每隔4个月定时免疫,效果均良好。种猪免疫抗体合格率达100%,仔猪49日龄前抗体合格率达100%,75日龄后抗体逐渐降低,120日龄后基本消失。为了使猪体免疫力更强,不给野毒入侵的机会,建议仔猪在首免后,适当时间进行二次加强免疫。而只给公猪、母猪春秋两季免疫,不给仔猪免疫组,仔猪在35日龄后抗体降为阴性,不能抵抗野毒的侵袭,这种免疫方法不宜推广。 相似文献
3.
为了克隆和表达编码扩展莫尼茨绦虫无精症缺失(DAZ)基因,试验根据GenBank中扩展莫尼茨绦虫DAZ基因cDNA序列(登录号为GH291478)设计1对特异性引物,以扩展莫尼茨绦虫组织总RNA为模板,RT-PCR扩增DAZ基因,并克隆到pMD19-T载体中进行序列测定,构建重组质粒pET28a-DAZ,转化BL21(DE3)大肠杆菌感受态细胞,以异丙基-β-D-硫代吡喃半乳糖苷(IPTG)进行诱导表达;利用非变性聚丙烯酰胺凝胶(SDS-PAGE)分析表达产物,通过螯合琼脂糖凝胶FF亲和层析柱对重组蛋白进行纯化。结果表明:重组DAZ基因在大肠杆菌中高效表达,表达的融合蛋白分子量约为14 ku。 相似文献
4.
根据犬瘟热病毒（CDV）Onderstepoort株的核苷酸序列，设计合成引物，通过RT-PCR．从CDV Onderstepoort株RNA中扩增出2197bp的CDV全长融合蛋白（F）基因。将其与pGEM-T easy载体连接．通过软件分析其序列中的疏水区后，以pGEM-T／F为模板．PCR扩增出约254bp和1017bp的F蛋白疏水区外编码序列。以2个扩增产物为模板，以OE-PCR（overlap extension-PCR）扩增出约1271bp的缺失疏水区的F基因（dF）．测序结果表明．dF的序列除在疏水区以4个甘氨酸序列替代外，其余部分与F基因的同源性为99.2％。这表明，OE-PCR扩增法已成功地使CDVF基因疏水区缺失。 相似文献
5.
AIM: To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on the apoptosis, oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation (A/R). METHODS: The H9c2 cells were used to establish a model of A/R. The H9c2 cells were transfected with PTEN small interfering RNA (siRNA) and negative control. After A/R, the expression of PTEN at mRNA and protein levels was determined by RT-PCR and Western blot, respectively. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Xanthine oxidase method was used to determine superoxide dismutase (SOD) activity. The content of malondialdehyde (MDA) was detected by thiobarbituric acid method. The lactate dehydrogenase (LDH) activity in the supernatant was evaluated by 4-dinitrophenylhydrazine method. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in culture supernatant were examined by ELISA. The protein levels of cleaved caspase-3, Bax and FasL in the cells were determined by Western blot. RESULTS: After A/R, the expression of PTEN at mRNA and protein levels was significantly increased in the H9c2 cells (P<0.05). The mRNA and protein levels of PTEN were decreased significantly after transfection with PTEN siRNA (P<0.05). The viability of H9c2 cells was decreased after A/R, while the apoptotic rate was increased. The protein levels of cleaved caspase-3, Bax and FasL were increased in the cells. The MDA level was elevated, the activity of SOD was decreased, and the levels of LDH, TNF-α, IL-1β and IL-6 in the culture supernatant were increased (P<0.05). Down-regulation of PTEN partly antagonized the effects of A/R on the viability, apoptotic rate, MDA content, SOD activity, and the levels of LDH, TNF-α, IL-1β and IL-6 in culture supernatant. CONCLUSION: Down-regulation of PTEN attenuates oxidative damage induced by A/R, reduces apoptosis and secretion levels of TNF-α, IL-1β and IL-6 in the H9c2 cells. 相似文献
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为了构建羊布鲁菌16M(简称16M)的DK63-887基因缺失株(16MΔDK63-887),探讨该基因与16M介导自噬的关系。利用同源重组和抗性替换的方法,以卡那基因替换DK63-887基因,获得突变株16MΔDK63-887。将亲本株16M、疫苗株M5-90、突变株16MΔDK63-887在相同条件下振荡培养,观察其生长趋势变化;将各菌株置于不同外界环境中,观察其生存率;将各菌株侵染小鼠巨噬细胞,比较它们在宿主细胞内的生存能力及RT-qPCR检测自噬相关基因的表达。成功获得了布鲁菌DK63-887基因缺失株且在20代内未发生回复性突变现象。与亲本株相比,16MΔDK63-887在体外培养生长趋势与亲本株相似,只是细菌的浓度存在一定差异;突变株在外界应激条件下生存能力低于亲本株;侵染4h后缺失株胞内细菌数量明显下降;RT-qPCR检测到突变株的ULKI、Beclin1表达量均显著降低(P0.01),结果表明,布鲁菌Ⅳ型分泌系统效应蛋白与16M介导的细胞自噬密切相关,为16M胞内寄生机制的研究奠定了基础。 相似文献
7.
基于转录组测序筛选鼠伤寒沙门菌hfq基因缺失菌的差异表达基因 总被引：2，自引：2，他引：0
旨在鼠伤寒沙门菌hfq基因缺失株转录组测序中分析适应环境变化及细菌分泌通路，筛选相关基因。通过使用实时荧光定量PCR对鼠伤寒沙门菌hfq基因缺失株转录组测序结果进行验证，从而对鼠伤寒沙门菌hfq基因缺失株的细菌趋化性通路、细菌双组分通路、细菌分泌通路进行深入分析，筛选相关的重要调控基因。结果显示：在细菌趋化性通路中筛选出yiaD、STM3138、STM3216和malE等4个共表达基因；在细菌分泌通路中筛选出spaP、invA、prgH、invE、spaS、invG和ssaV等7个共表达基因；在细菌双组分通路中筛选出ybfM、htrA、pagO、STM3138、STM3031、ttrB、hilD、pstS、STM1530、hilA、pgtC、hydH、pocR、STM3216、ttrR和fljB等16个共表达基因。在鼠伤寒沙门菌hfq基因缺失株的趋化性通路、细菌双组分通路、细菌分泌通路中筛选出差异表达基因，hfq能够对这些基因进行调控，从而影响如运动性、毒力等相关作用，为沙门菌中的sRNA与hfq后续研究及沙门菌的防治奠定一定基础。 相似文献
8.
GUO Jian LIU Xiao-juan ZHANG Guo-zun SUN Hui-cong LIU Lin-li GUO Jin-bo LIU Lei YAO Dong-mei SONG Mei ZHANG Xiao-lan 《园艺学报》2012,28(9):1627-1632
AIM:To investigate the down-regulation of phosphatase and tensin homolog deleted on chromosome 10(PTEN) gene by adenovirus-mediated short hairpin RNA(shRNA) on proliferation and apoptosis of activated hepatic stellate cells(HSCs) in vitro and the related signaling transduction pathways. METHODS:The activated HSCs were cultured in vitro and transfected with recombinant adenovirus expressing shRNA targeting PTEN. The proliferation of HSCs was measured by MTT assay and the apoptosis was assessed by TUNEL and flow cytometry. Western blotting was used to detect the protein levels of PTEN, Bax, Bcl-2, Akt, p-Akt, ERK1/2 and p-ERK1/2 in HSCs, and real-time fluorescent quantitative PCR was applied to detect the mRNA expression of Akt and ERK1. RESULTS:The recombinant adenovirus expressing shRNA targeting PTEN was successfully transfected into activated HSCs in vitro, and significantly promoted the proliferation of HSCs in a time-dependent manner within a certain extent. The apoptotic rate of HSCs was significantly decreased 72 h after transfection(P<0.05). Meanwhile, reduced expression of Bax and elevated expression of Bcl-2 were induced 72 h after transfection(P<0.05). Furthermore, the expression of p-Akt and p-ERK1/2 were increased significantly(P<0.05), while no significant difference in the expression of Akt and ERK1 at mRNA and protein levels was observed(P>0.05). CONCLUSION:Down-regulation of PTEN by adenovirus-mediated shRNA dramatically promotes the proliferation of activated HSCs, and inhibits the apoptosis through Bcl-2/Bax pathway. In addition, the phosphorylation of Akt and ERK1/2 is increased, indicating that PI3K/Akt and ERK1/2 signal transduction pathways may play an important role in the regulation of proliferation and apoptosis of HSCs. 相似文献
9.
AIM: To investigate the role of extracellular signal-regulated kinase 5 (ERK5) in platelet aggregation in vitro and arterial thrombosis in vivo. METHODS: The expression and phosphorylation levels of ERK5 in human platelet were detected by Western blot. The effects of ERK5 selective inhibitor XMD8-92 on platelet aggregation and dense granule secretion were detected by Chrono-Log aggregometer. The effect of ERK5 on in vivo thrombosis was analyzed using an FeCl3 artery thrombosis model. The effects of XMD8-92 on protein kinase B (PKB/Akt) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) phosphorylation levels were determined by Western blot. RESULTS: ERK5 was stably expressed in human platelets and its phosphorylation level increased significantly after platelet activation (P<0.05). XMD8-92, a selective inhibitor of ERK5, inhibited platelet aggregation and dense granule secretion in response to several platelet stimulators (P<0.05). The results of Western blot showed that XMD8-92 inhibited Akt phosphorylation level by down-regulating PTEN Ser370 phosphorylation and enhancing PTEN activity. The pathway was further confirmed using platelet specific PTEN deficiency mice. The first occlusion time was obviously extended in the mice intravenously given XMD8-92 in the FeCl3-induced carotid artery injury model. CONCLUSION: ERK5 plays a role in platelet activation and arterial thrombosis by influencing PTEN and Akt phosphorylation. 相似文献
10.
猪IL-2与IL-6的原核表达及其对伪狂犬病基因缺失疫苗的佐剂效应研究 总被引：8，自引：0，他引：8
将猪白细胞介素2(pIL-2)与猪白细胞介素6(pIL-6)的cDNA序列克隆到大肠杆菌表达载体pET-28a及pGEX-KG上,转化BL21(DE3)表达菌,经IPTG诱导产生重组蛋白pIL-2与pIL-6,表达量分别占菌株总蛋白的43.45％和28.37％,经提纯后含量可分别达到97.06％和86.37％。将提纯后不同剂量的两种重组蛋白以单独或组合的方式,加入伪狂犬病(Ea株)TK~-/gG~-双基因缺失疫苗中,使每头份疫苗分别含2、5和10μg·ml~(-1)的pIL-2或(和)pIL-6,用此疫苗免疫伪狂犬病抗体阴性猪。同时设试验对照组。初次免疫30d后加强免疫,测定中和抗体,观察试验猪的日增重情况,进行统计分析。发现各试验组的抗体水平均高于不含重组蛋白的基因缺失疫苗组;其中,含2μg·ml~(-1)pIL-2试验组与含10μg·ml~(-1)pIL-2/pIL-6试验组抗体水平和基因缺失疫苗对照组之间呈显著差异(P=0.019)与极显著差异(P=0.009)。结果表明,大肠杆菌表达的pIL-2、pIL-6对伪狂犬病鄂A株基因缺失疫苗(TK~-/gG~-/LacZ~+)有较强的佐剂效应,且呈剂量相关性。不同试验组日增重比较结果表明,免疫效果高低与猪的生长速度呈正相关。本试验为重组pIL-2与pIL-6作为新型疫苗佐剂应用提供了重要的试验依据。 相似文献