共查询到20条相似文献，搜索用时 218 毫秒
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猪白细胞介素18全基因的克隆、真核表达质粒的构建及其在猪肾细胞中的表达 总被引：2，自引：1，他引：1
通过RT-PCR方法自猪脾脏淋巴细胞中扩增mpIL-18基因.序列测定表明,pIL-18全基因核苷酸长度为579bp,编码192个氨基酸.将其克隆到真核表达载体pcDNA3.1中,构建重组质粒pcDNA-IL18m,所获重组质粒经过酶切、测序鉴定,证实含有目的片段,且连接、构建正确.阳性克隆鉴定后,在脂质体作用下转染猪肾细胞(PK15),通过提取RNA检测到了mpIL-18在PK15细胞中的表达. 相似文献
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为研制FMD复制缺陷型腺病毒活栽体疫苗，通过RT—PCR方法获得了。型口蹄疫病毒（FMDV）全开放阅读框（oRF）基因片段，将其克隆到pMD18-T栽体后测序，再将oRF编码基因定向克隆入腺病毒穿梭载体pAdTrack—CMV中，构建了重组腺病毒穿梭质粒。经PCR、酶切及测序鉴定，所克隆的oRF编码基因序列与原始强毒株Akesu／58的ORF编码基因比较，L、P1、P2、P3基因的核苷酸同源性分别为98．8％、97．9％、99．0％和97．6％，PCR、酶切鉴定重组腺病毒穿梭质粒均获得了长约6．9kb的目的基因片段。表明，成功获得了含有。型FMDV全ORF编码基因的阳性克隆，并成功构建了含有完整FMDV开放阅读框架的编码基因表达盒的重组腺病毒穿梭质粒。 相似文献
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金荞麦查尔酮合成酶基因CHS的克隆及序列分析 总被引：2，自引：0，他引：2
采用同源克隆的方法获得金荞麦查尔酮合成酶基因(CHS)的保守片段554 bp,进一步采用染色体步移法(genome-walking)和RT-PCR技术克隆到CHS基因的全长DNA序列和cDNA开放阅读框(ORF)序列.序列分析结果表明,金荞麦CHS基因DNA全长1 650 bp,含一个462 bp的内含子;其cDNA编码区全长1 188 bp,编码395个氨基酸,命名为FdCHS,NCBI登录号为GU169470.该氨基酸序列与同为蓼科的掌叶大黄、虎杖CHS的氨基酸序列同源率分别达到94%和93%,且含有CHS活性位点和底物结合口袋位点等保守位点. 相似文献
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根据抗体重链与轻链基因的核苷酸序列设计并合成了1对引物,以猪外周血淋巴细胞基因组mRNA为模板,通过RT_PCR方法获得了一大小约85bp的DNA片段,并将其克隆到pGEM_T载体上进行序列测定,测序结果显示,猪抗体信号肽基因核苷酸序列长度为57bp,编码19个氨基酸,核苷酸及推导的氨基酸序列与已发表的抗体信号肽基因序列一致,同时在信号肽基因3’端下游提供可供肽酶切割的窗口和外源基因的克隆位点。然后将信号肽基因亚克隆到真核表达载体pcDNA3.1( )中,成功构建了一含信号肽序列的真核表达载体3.1_SFc,为外源基因在pcDNA3.1( )中的表达与分泌提供了一有效的信号肽,也为研究基因的功能奠定了基础。 相似文献
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《黑龙江畜牧兽医》2020,(18)
为了研制新型猪β_2-受体激动剂,试验利用PCR扩增猪β_2-肾上腺素能受体(β_2-AR)的编码区基因序列并进行测序,用TMHMM软件对猪β_2-AR蛋白质进行跨膜区段分析,找出第二细胞外环的DNA序列并进行大肠杆菌偏好性密码子优化,设计一段含两拷贝第二细胞外环的优化DNA序列,然后通过基因合成技术合成这段DNA序列,并将其克隆至原核表达载体pET-32a,再将重组质粒转化入大肠杆菌BL21(DE3)中,最后提取重组质粒进行双酶切及测序鉴定。结果表明:PCR扩增得到1 277 bp的β_2-AR全基因序列,其中包含一个1 257 bp的开放阅读框,编码418个氨基酸残基;蛋白跨膜区段分析显示其第二细胞外环(pβ_2-AR-E_Ⅱ)位于第170～200位氨基酸,由93对碱基编码;设计并合成的DNA序列(D-E_Ⅱ)共201 bp;构建的重组质粒pET-32a-D-E_Ⅱ经双酶切后得到两条预期大小的片段,测序显示插入序列与D-E_Ⅱ序列一致。说明猪β_2-AR第二细胞外环基因原核表达载体构建成功。 相似文献
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口蹄疫病毒3ABC基因的克隆与测序 总被引：1，自引：0，他引：1
参考 Gen Bank中发表的猪源 O型口蹄疫病毒 3 ABC的基因序列 ,设计一对特异引物 ,以猪源 FMDV/ O/ CC株基因组 RNA为模板 ,RT-PCR扩增 3 ABC基因 ,并克隆到 p MD1 8-T载体中。测序结果显示 ,FMDV/ O/ CC株 3 ABC基因 c DNA长 1 2 81 bp,编码为 42 7个氨基酸残基组成的多肽。核苷酸序列和推导氨基酸序列同源性比较发现 ,FMDV/ O/ CC株和 FMDV/O/ TW/ 99株 3 ABC基因亲缘关系密切。核苷酸同源性为 97% ,推导氨基酸序列同源性为96.3 %。 相似文献
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根据GenBank中收录的三种猪Ⅰ型干扰素（porcine interferon,pIFN）基因序列,设计了3对特异性引物。运用RT—PCR技术从长白猪外周血淋巴细胞总RNA中扩增得到了三种猪干扰素基因,并将其克隆至pGEM-T Easy载体上。序列测定结果显示,获得的猪干扰素α1基因序列全长570bp,编码189个氨基酸,获得的猪干扰素α2基因序列全长540bp,编码179个氨基酸,获得的猪干扰素β基因序列全长563bp,编码186个氨基酸。三种猪Ⅰ型干扰素基因与已知猪Ⅰ型干扰素核苷酸序列和氨基酸序列的相似性最高可达99％～100％。 相似文献
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试验参照猪SRY基因保守序列设计了1对引物,对香猪基因组DNA进行PCR扩增。将扩增产物通过T—A互补法克隆到质粒pGEM—T载体中,筛选阳性克隆进行DNA测序。测序结果显示,香猪SRY基因开放阅读框全长627bp,编码227个氨基酸。其中核心区域HMG—box长237bp,并且编码79个氨基酸。与白鲸、人、牛、绵羊4种哺乳动物的SRY基因序列相比较,香猪和白鲸的SRY序列具有较高的相似性,相似度达82．1％。序列相似性和邻接法聚类树的结果均显示,在SRY基因中香猪和白鲸有着较近的亲缘关系,表明以白鲸为代表的鲸目和以香猪为代表的偶蹄目有较近的亲缘关系。 相似文献
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Stasny BR De Guise S Rompato G Garmendia AE 《Veterinary immunology and immunopathology》2001,78(1):57-70
A dual expressing (CD4(+)/CD8(+)) porcine lymphoblastoid T-cell line (pIL-2d) generated from peripheral blood mononuclear (MN) cells shown to be highly responsive to exogenous interleukin-2 (IL-2) was characterized. The swine MN cells were initially stimulated with concanavalin A (Con A), and sub-passaged using decreasing amounts of conditioned medium (CM), which was prepared from culture fluids of Con A activated porcine MN cells, until a steady growth was observed. The resulting pIL-2d cells require exogenous IL-2 from CM and are highly responsive to recombinant human IL-2 (rhIL-2). The pIL-2d cells exhibited a specific, dose-dependent proliferative response to stimulation with IL-2. The specificity of this proliferative response was confirmed to be IL-2 induced by its inhibition with an anti-swine IL-2 receptor (alpha-swIL-2R) monoclonal antibody (mAb). Furthermore, the pIL-2d cells are highly responsive to exogenous IL-2 contained in culture fluids derived from antigen-driven blastogenic tests performed with lymphocytes of vaccinated swine. This property makes the pIL-2d cells an ideal functional adjunct to immunochemical or molecular tests that are commonly used to measure total porcine IL-2. Interestingly, the phenotype of the pIL-2d cells after five or more passages was shown by flow cytometric analysis to be CD4(+)/CD8(+)/CD45RA(-)/CD25(+) and to remain unchanged thereafter. Although, the mechanism of selection and maintenance of the CD4(+)/CD8(+) DP cells developed here remains unclear, our data suggest that an oligoclonal or polyclonal expansion and maintenance of cells of this phenotype was mediated by exogenous IL-2. 相似文献
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Guohua Chen Shuang ZengHuaijie Jia Xiaobing HeYongxiang Fang Zhizhong Jing Xuepeng Cai 《Research in veterinary science》2014
In this paper, we investigated the enhancement of adjuvant effects of porcine IL-2 (pIL-2) by packaging it into a solid lipid nanoparticle (SLN) delivery system. SLN–pIL-2 was prepared using hydrogenated castor oil and Polylactide-co-glycolide by double emulsion solvent evaporation methods (w/o/w). In animal trials, BALB/c mice were immunized with inactivated foot and mouth disease virus (FMDV) antigen combined with the SLN–pIL-2 adjuvant on days 0 and 14. Antibody titer, splenocyte proliferation, and secretion of IFN-γ and IL-4 cytokines were determined. Our results showed that SLN–pIL-2 could significantly enhance FMDV-specific antibody level compared with recombinant pIL-2 alone (p < 0.05). In addition, SLN–pIL-2 significantly increased the proliferative responses of antigen-specific spleen cells. Furthermore, SLN–pIL-2 induced the secretion of IFN-γ at a level higher than that induced by recombinant pIL-2 alone. Our results indicate that packaging recombinant pIL-2 in SLNs can be an effective way of boosting the effectiveness of pIL-2 as an adjuvant to enhance immune responses of vaccines. 相似文献
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参考GenBank发表猪IL-15mRNA序列设计引物,用RT-PCR方法扩增猪IL-15cDNA,并克隆到pMD18-T载体中,通过PCR、酶切和测序验证克隆正确,再亚克隆到真核表达载体pcDNA-3.1（＋）上,得到重组质粒pcD-NA-pIL-15。在脂质体介导下,重组质粒pcDNA-pIL-15转染AD-293细胞。以鼠抗猪IL-15为一抗,用间接免疫荧光分析表明猪IL-15基因均在AD-293细胞中成功进行了瞬时表达。小鼠免疫试验表明,pcDNA-pIL-15作为免疫佐剂,能够有效提高小鼠的脾T细胞增殖,加强pcDNA-ORF2（PCV2）质粒免疫过程中的特异性中和抗体的产生,为进一步研制猪IL-15基因佐剂疫苗及进行临床实验奠定基础。 相似文献
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OBJECTIVE: To examine the effects of DNA dose, site of vaccination, and coadministration of a cytokine DNA adjuvant on efficacy of H1-subtype swine influenza virus hemagglutinin (HA) DNA vaccination of pigs. ANIMALS: 24 eight-week-old mixed-breed pigs. PROCEDURE: 2 doses of DNA were administered 27 days apart by use of a particle-mediated delivery system (gene gun). Different doses of HA DNA and different sites of DNA administration (skin, tongue) were studied, as was coadministration of porcine interleukin-6 (pIL-6) DNA as an adjuvant. Concentrations of virus-specific serum and nasal mucosal antibodies were measured throughout the experiment, and protective immunity was assessed after intranasal challenge with homologous H1N1 swine influenza virus. RESULTS: Increasing the dose of HA DNA, but not coadministration of pIL6 DNA, significantly enhanced virus-specific serum antibody responses. Pigs that received DNA on the ventral surface of the tongue stopped shedding virus 1 day sooner than pigs vaccinated in the skin of the ventral portion of the abdomen, but none of the vaccinated pigs developed detectable virus-specific antibodies in nasal secretions prior to challenge, nor were they protected from challenge exposure. Vaccinated pigs developed high virus-specific antibody concentrations after exposure to the challenge virus. CONCLUSIONS AND CLINICAL RELEVANCE: Co-administration of pIL-6 DNA did not significantly enhance immune responses to HA DNA vaccination or protection from challenge exposure. However, HA DNA vaccination of pigs, with or without coadministration of pIL-6 DNA, induced strong priming of the humoral immune system. 相似文献
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应用cDNA微阵列芯片技术筛选猪蛔虫性别差异表达基因的研究 总被引：6，自引：0，他引：6
采用cDNA微阵列芯片技术，从所构建的猪蛔虫雌、雄成虫cDNA消减文库分别挑取1044和1119个克隆，PCR扩增其插入片段，经纯化后点样于预先处理好的基片上（双点杂交），制备成cDNA微阵列芯片。将分别标记荧光素Cy3-dUTP和Cy5-dUTP的雌虫和雄虫cDNA探针，与制备好的cDNA芯片杂交（平行进行反标杂交试验）。根据每个点杂交后的Ratio值，筛选出双点杂交和正反标中都同时具有表达差异的基因克隆共1559个。将表达差异最明显的前831个克隆进行测序，获得720个有效序列，经生物信息学分析发现，雄虫特异表达的主要精于蛋白和雌虫特异表达的卵巢信息蛋白的基因序列多数与新杆属线虫存在同源性，有31个可能是新的ESTs。性别差异表达基因及其相关生物信息的获得为下一步研究基因功能奠定了基础。 相似文献
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Production and characterization of monoclonal antibodies against porcine interleukin-4 总被引：1，自引：0，他引：1
Ishikura Y Kato H Hashimoto T Nishimura Y Iwata H 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2005,67(5):503-508
Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system. 相似文献
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