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山羊Sox2基因克隆、原核表达和GST融合蛋白纯化 总被引：2，自引：0，他引：2
本研究旨在克隆山羊Sox2基因,构建pGEX-Sox2原核表达重组质粒,从大肠杆菌中获得纯化的GST-Sox2融合蛋白.应用RT-PCR方法从6周龄胎山羊生殖嵴扩增Sox2基因编码全序列,将其克隆到pMD18-T载体,然后再亚克隆到pGEX-KG表达载体.重组质粒pGEX-Sox2转化大肠杆菌BL21(DE3),经IPTG诱导,用SDS-PAGE电泳及Western blotting检测GST-Sox2融合蛋白表达,用谷胱甘肽Sepharose 4B介质分离纯化该融合蛋白.结果表明,山羊Sox2基因编码序列全长为960 bp;在优化的表达条件(1 mmo|·L-1 IPTG,22℃诱导16h)下,重组质粒(pGEX-Sox2)在大肠杆菌得到了高效表达;谷胱甘肽Sepharose 4B颗粒纯化蛋白得到预期大小的融合蛋白(约60 ku).结论,本研究克隆了山羊Sox2基因,获得了纯化的GST-Sox2融合蛋白,为制备其多克隆抗体奠定了基础,为进一步研究山羊iPS细胞(Induced pluripotent stem cells)创造了条件. 相似文献
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为原核表达H3N2亚型犬流感病毒(CIV)HA1蛋白,本研究利用特异性引物扩增CIV H3分离株的HA1基因,将其克隆到pMD18-T载体后进行序列测定。再将其亚克隆于pET-32a(+)中构建重组表达质粒pET-HA1。将该质粒转化于大肠杆菌BL21(DE3)中,经IPTG诱导,SDS-PAGE电泳分析,表达的重组蛋白约为58 ku。纯化的HA1蛋白经western blot和Dot-ELISA鉴定表明,表达的重组HA1蛋白可以与H3N2亚型CIV阳性血清发生特异性反应。 相似文献
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原癌基因c-Myc虽然在许多肿瘤组织中特异性高表达,但它也是机体正常细胞生长与增殖所必需的因子,然而目前家畜诱导多潜能干细胞（iPS）研究中关于多功能转录因子c-Myc的报道很少.为此,研究拟利用蛋白转导区域（PTD）的跨膜作用,构建TAT-bc-Myc-9R融合表达载体,并对其进行原核表达,旨在为转录因子蛋白重编程牛体细胞奠定基础.试验以牛c-Myc基因编码区序列为参考,设计并合成含酶切位点、跨膜转导肽TAT和9R的引物,利用PCR等基因工程方法获得包括牛c-Myc、TAT和9R的重组原核表达载体（pTAT-HA-bc-Myc-9R）;酶切鉴定和DNA测序结果表明,试验成功获得bc-Myc-9R重组序列,成功构建重组质粒pTAT-bc-Myc-9R;不同IPTG浓度和诱导时间条件对该重组载体的诱导表达和SDS-PAGE分析表明,TAT-bc-Myc-9R在0.6 mmol/L IPTG和37℃诱导6h条件下表达约57KDa的目的蛋白.这些结果为PTD介导的转录因子蛋白直接重编程牛体细胞以获得iPS研究积累了科学资料. 相似文献
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为建立利用内源诱导因子诱导绵羊体细胞为多能干细胞(induced pluripotent stem cell,iPS)的方法,对绵羊Sox2基因进行克隆,并与pMXs连接构建逆转录病毒载体,将构建的载体转染293GP细胞以获得假病毒上清,利用假病毒上清侵染绵羊胎儿成纤维细胞以检测细胞中的Sox2表达变化。PCR和酶切鉴定结果显示,成功构建了pMXs-Sox2重组质粒,该质粒具有转染293GP细胞的能力,所获得的假病毒上清侵染绵羊胎儿成纤维细胞后,可诱导细胞表达Sox2基因。本研究为开展绵羊iPS的相关研究提供依据。 相似文献
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构建美利奴绵羊磷脂酶 C zeta（PLCζ）融合蛋白表达载体,并在原核细胞内表达及纯化,为其特异性抗体的制备及生物学功能研究奠定基础。用 PCR 技术扩增出 PLCζ基因片段,亚克隆到原核表达载体pCzn1中,导入 Arctic Express TM （DE3）感受态细胞,IPTG 诱导表达,收样后进行 SDS-PAGE 电泳或Western blot 检验 PLCζ的表达情况。用镍离子亲和层析的方法大量纯化融合蛋白。结果显示,重组质粒经 PCR、酶切和测序鉴定证明载体构建正确。pCzn1表达载体在 Arctic Express TM （DE3）表达菌中能很好地表达融合蛋白。表达纯化后可获得了分子量约74 ku 的融合蛋白,符合预期大小。成功构建 pCzn1-PLCζ原核表达载体,表达并纯化了其融合蛋白,Western blot 试验表明其蛋白分子的完整性良好,这将有利于对 PLCζ蛋白进行深入的研究。 相似文献
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牛分枝杆菌mpb64-ag85b-esat6融合基因的分子克隆及表达 总被引：2，自引：0，他引：2
以牛分枝杆菌valleeⅢ株基因组DNA为模板,PCR扩增mpb64、ag85b和esat6 3个目的基因片段,采用重叠延伸剪接技术（SOE）和基因重组技术获得融合基因mpb64-ag85b,将mpb64-ag85b和esat6串联到同一原核表达载体pET32a（＋）中获得重组质粒pET64-85b-e6。将其转化到大肠杆菌感受态细胞BL21（DE3）中,以IPTG（终浓度1 mmol/L）进行诱导,SDS-PAGE电泳分析表达情况,以Ni^2＋亲和层析柱纯化表达的融合蛋白和Western blot分析融合蛋白的免疫活性。结果表明：表达的融合蛋白大约为76 Ku,与预测大小相符,以Ni^2＋亲和层析柱纯化的融合蛋白能与牛结核病阳性血清发生反应。 相似文献
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构建致倦库蚊(贵阳株)天蚕素B2(CecB2)基因原核表达载体,并原核表达获得重组蛋白。定向克隆CecB2成熟肽序列至原核表达载体pET32a(+)上,将成功构建的pET32a-CecB2重组表达质粒转化大肠埃希菌Rosetta中经IPTG诱导表达,对IPTG诱导浓度和诱导时间优化后表达所得目的蛋白采用镍离子亲和层析纯化,SDS-PAGE和Western blot检测鉴定表达蛋白。结果表明,成功构建原核表达载体pET32a-CecB2,IPTG浓度和时间优化结果为IPTG浓度为0.05mmol/L,诱导时间为3h。SDS-PAGE检测获得大小约25ku的可溶性纯化蛋白,Western blot鉴定纯化蛋白可与鼠抗His-tag单克隆抗体发生抗原抗体结合反应。说明所构建原核表达载体pET32a-CecB2能在大肠埃希菌中可溶表达,为进一步研究其生物学功能奠定了基础。 相似文献
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c-Myc原癌基因与胚胎干细胞(ES细胞)和诱导的多能性干细胞(iPS细胞)生物学特性密切相关,因此制备其相应的多克隆抗体尤为必要。本研究以pMD18T-Myc质粒为模版,PCR扩增c-Myc片段,然后将其亚克隆到pSE380表达载体上,获得pSE380-Myc重组质粒。该质粒转化工程菌BL21(DE3),IPTG诱导表达His-Myc融合蛋白,随后用SDS-PAGE电泳及Western blot加以验证。在变性条件下用Ni-NTA argrose介质分离纯化His-Myc融合蛋白,并以此为抗原免疫新西兰大白兔。经过4次免疫,采集血液、分离血清,用Western blot检测抗体特异性。结果表明:(1)pSE380-Myc重组质粒在工程菌BL21(DE3)得到了高效表达;(2)得到了高纯度高含量的His-Myc融合蛋白;(3)经Wstern blot检测发现,c-Myc多克隆抗体与His-Myc融合蛋白能够特异性结合。本研究获得的特异性山羊c-Myc多克隆抗体,为进一步研究山羊ES细胞和iPS细胞的自我更新机制奠定了基础。 相似文献
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构建大片吸虫硫氧还蛋白过氧化物酶(Fg.TPx)原核表达质粒,经大肠埃希菌Rosetta表达融合蛋白(Fg.TPx/His)并对其进行纯化和初步鉴定。PCR扩增Fg.TPx的基因片段,亚克隆至pET32a(+)中构建重组表达载体pPET32a-Fg.TPx。IPTG诱导表达,经镍离子亲和层析分离纯化后,进行SDS-PAGE和Western blot分析。重组质粒经限制性内切酶双酶切和测序分析表明构建成功,表达产物以可溶性和包涵体形式存在,纯度为90%,Wsetern blot证实该蛋白可与抗His单克隆抗体发生特异性结合反应,分子质量为TPx和His分子质量之和,表明是融合蛋白。重组蛋白可以被大片吸虫感染水牛阳性血清特异性识别。免疫家兔产生的抗体效价最高可达1∶4 000。成功构建了p ET32a-Fg.TPx原核表达质粒,重组蛋白得到高浓度表达,具有抗原性,为进一步研究其在大片吸虫病诊断中的应用奠定了基础。 相似文献
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试验通过候选基因法,选定卵泡抑素(follistatin,FS)作为影响水牛繁殖性能的主要候选基因,通过基因工程的方法,用XhoⅠ、SacⅡ双酶切广西大学动物遗传育种与繁殖实验室克隆的添加有ACC的Kozaka 序列的pMD-bFS质粒和pEGFP-N1,构建pEGFP-bFS真核表达质粒,将其与阳离子脂质体混匀后,分别转染体外培养的水牛胎儿成纤维(BFF)细胞和仓鼠肾(BHK)细胞系,经过48 h培养后,在相差荧光显微镜下观察绿色荧光蛋白的表达水平,用RT-PCR和Western blotting方法对转入质粒表达进行定性鉴定。结果显示,本研究成功构建了添加有ACC的Kozaka 序列的pEGFP-bFS真核表达质粒,该重组质粒在BFF和HBK两种细胞均表达,但在HBK细胞系的表达量稍高。本研究结果将为下一步制备具有生物活性的bFS工程疫苗,研究bFS对水牛繁殖性能的影响提供参考。 相似文献
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NE Gade MD Pratheesh A Nath PK Dubey Amarpal B Sharma G Saikumar G Taru Sharma 《Reproduction in domestic animals》2013,48(3):358-367
Immune privileged mesenchymal stem cells (MSCs) can differentiate into multiple cell types and possess great potential for human and veterinary regenerative therapies. This study was designed with an objective to isolate, expand and characterize buffalo bone marrow‐derived MSCs (BM‐MSCs) at molecular and cellular level. Buffalo BM‐MSCs were isolated by Ficoll density gradient method and cultured in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum (FBS). These cells were characterized through alkaline phosphatase (AP) staining, colony‐forming unit (CFU) assay, mRNA expression analysis (CD 73, CD 90, CD 105, Oct4 and Nanog), immunolocalization along with flow cytometry (Stro 1, CD 73, CD 105, Oct4, Sox2 and Nanog) and in situ hybridization (Oct4 and Sox2). Multilineage differentiation (osteogenic, adipogenic and chondrogenic) was induced in vitro, which was further assessed by specific staining. Buffalo BM‐MSCs have the capacity to form plastic adherent clusters of fibroblast‐like cells and were successfully maintained up to 16th passage. These cells were AP positive, and further CFU assay confirmed their clonogenic property. RT‐PCR analysis and protein localization study showed that buffalo BM‐MSCs are positive for various cell surface markers and pluripotency markers. Cytoplasmic distribution of mRNA for pluripotency markers in buffalo BM‐MSCs and multilineage differentiation were induced in vitro, which was further assessed by specific staining. To the best of our knowledge, this is the first report of buffalo BM‐MSCs, which suggests that MSCs can be derived and expanded from buffalo bone marrow and can be used after characterization as a novel agent for regenerative therapy. 相似文献
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Chunfeng Wu Lixian Liu Jinlong Huo Dalin Li Yueyun Yuan Feng Yuan Yongwang Miao 《Tropical animal health and production》2014,46(1):33-41
Insulin-induced genes (INSIGs) are recently discovered genes that are involved in the metabolism of cholesterol and lipogenesis in animal tissues. In this study, two INSIG genes (INSIG1 and INSIG2) were isolated and characterized in 11 buffalo. The full-length coding sequence (CDS) of the buffalo INSIG1 consists of 831 bp which encodes a 276 amino acid protein with molecular mass 29.55 kD. And the INSIG2 CDS is 678 bp in length which encodes a 225 amino acid protein with molecular mass 24.87 kD. No polymorphisms were found in the CDSs of the buffalo INSIGs, but seven and two nucleotide differences were found in the CDSs between buffalo and other bovine species. Phylogenetic analyses based on the INSIG amino acid sequences showed that buffalo was grouped with other members in the Bovidae family. Four types of putative modification sites were detected in buffalo INSIG proteins. And two predicted microRNA target sites were found respectively in the CDSs of buffalo INSIG1 and INSIG2. The tissue expression analyses by quantitative PCR (qPCR) revealed that the buffalo INSIG1 was expressed in ten tissues tested. Among these tissues, the liver and mammary gland showed high expression levels. And the INSIG2 was only expressed in the brain, mammary glands, pituitary, abomasum, heart, and liver. Among these tissues, the mammary gland, brain, and pituitary demonstrated a high expression levels. These data provide the primary foundation for further insights into the buffalo INSIG genes. 相似文献
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In this study, buffalo AQP8 gene was cloned and its eukaryotic expression vector was constructed, the expression pattern of AQP8 gene in buffalo ovary tissue was also assayed. The results showed that the CDS length of cloned buffalo AQP8 gene was 732 bp, and it shared 100% homology of amino acid sequence with cattle and mouse. AQP8 protein was detected in different developmental stages of buffalo follicles, it had significantly higher expression in the secondary follicles than that of in the primordial and the primary follicles (P＜0.05), and it mainly expressed in the granulosa cells of the secondary follicles. Clear EGFP green fluorescent was observed in transfected cell groups with transfection of the pEGFP-N1-AQP8 eukaryotic expression plasmid into HEK293T cells by LipofectamineR LTX and PLUSTM reagent. The above results lay foundation to further investigate the function of AQP8 gene in the buffalo follicle development and granulosa cell apopotosis. 相似文献
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试验旨在克隆河流型水牛热休克蛋白70（heat shock protein 70,HSP70）基因的CDS区序列,构建HSP70原核表达载体,诱导表达HSP70融合蛋白,进一步研究HSP70蛋白对水牛精液抗冻性的影响。以摩拉水牛精子基因组DNA为模板,采用PCR方法扩增HSP70基因CDS区序列,将PCR产物与pET30a质粒连接构建重组原核表达质粒,诱导表达HSP70融合蛋白,将获得的表达蛋白作为稀释液组分添加到水牛精液中制作冷冻精液并评定其活力。结果显示,试验成功扩增获得HSP70基因CDS区长为2 155 bp的片段;SDS-PAGE及Western blotting分析显示,在70 ku处出现一条明显条带,且表达产物可与相应的抗体发生反应;对添加有HSP70蛋白的水牛冷冻精液活力评定结果显示,终浓度为2、4、8 mg/mL的HSP70能提高冷冻精液活力,且8 mg/mL HSP70组冻精活力最高,但各组间差异均不显著（P>0.05）。综上所述,本试验成功表达了水牛HSP70蛋白,获得了高纯度、有活性的HSP70融合蛋白,外源添加HSP70蛋白可提高水牛冷冻精液的活力,为进一步研究水牛HSP70蛋白的抗冻性保护机理奠定了理论基础。 相似文献
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研究猪圆环病毒2型(PCV2)Cap蛋白的抗原性,为开发PCV2的检测方法奠定基础。以PCV2CAU0673毒株的DNA为模板,扩增获得702bp的目的片段,扩增产物克隆入pET30a(+)原核表达载体,构建pET30a-PCV2-ORF2重组质粒,转入大肠埃希菌BL21(DE3);在37℃以1mmol/L IPTG诱导表达6h;采用Ni-NTA树脂亲和层析纯化重组蛋白,并用不同浓度的尿素对纯化蛋白进行复性。SDSPAGE分析表明,该ORF2编码基因在大肠埃希菌中得到表达,蛋白大小约为34ku;Western blot检测结果表明,该重组Cap蛋白与PCV2阳性血清发生特异性反应,与NA-PRRSV和PPV1血清不发生交叉反应。成功构建了PCV2-ORF2原核表达载体,实现了在大肠埃希菌中的表达,纯化后的复性蛋白具有较好的反应原性,为猪圆环病毒2型检测方法建立或试剂盒的开发奠定了基础。 相似文献
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MA Xiao-ya PANG Chun-ying LIANG Sha-sha LU Xing-rong ZHU Peng DUAN An-qin LIANG Xian-wei DENG Ting-xian 《中国畜牧兽医》2017,44(10):2829-2836
This study was aimed to clone the buffalo Δ6-fatty acid desaturases (FADS2) gene using in-Silico cloning and analyze its genetic struction with bioinformatics, which provide a foundation for investigating the milk performance in buffaloes. Primers were designed according the sequence of FADS2 gene in dairy cow (GenBank accession No.:NM_001083444.1). The FADS2 gene was amplified by RT-PCR, and its sequence was analyzed by bioinformatics. Sequence analysis revealed that the buffalo FADS2 gene had 36 600 bp in length and consisted of 12 exons and 11 introns, containing an open reading frame (ORF) of 1 335 bp which encoding 444 amino acids. Sequence homology analysis indicated that the buffalo FADS2 protein gene showed 98.88%, 98.88%, 89.66%, 90.79%, 90.85%, 92.35% and 87.11% identity with that of Bos mutus, Bos taurus, Homo sapiens, Sus scrofa, Oryctolagus cuniculus, Orcinus orca and Rattus norvegicus, respectively. Protein prediction analysis showed that the molecular weight and isoelectric point (pI) of buffalo FADS2 were 52.51 ku and 8.75, respectively, and the FADS2 protein was weak alkali and the hydrophobicity protein without signal peptide. Phylogenetic tree analysis showed that FADS2 gene was highly conserved in different species and evolutionary processes,buffalo was close to Bos mutus and Bos taurus, and was far from Rattus norvegicus. This study suggested that FADS2 gene was successfully cloned in buffalo, which laid a foundation for clarifying the mechanism of milk performance in buffalo. 相似文献