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1.
目的 应用基因工程技术表达甲状腺激素反应蛋白1(TRP-1)。方法 应用RT-PCR方法,从新生大鼠脑组织RNA中扩增编码TRP-1的cDNA片段,构建表达型重组质粒,经DNA序列分析确证后,在大肠杆菌中表达,以Western印迹来初步鉴定是否表达了目的融合蛋白,用亲和层析纯化融合目的蛋白,并以SDS-PAGE电泳测定其相对分子质量。结果 所获特异PCR产物正确地重组入Pinpoint Xa-1表达载体中。Western印迹表明经异丙基硫代半乳糖苷诱导的原核细胞表达产生了目的融合蛋白,亲和层析得到了纯度较高、相对分子质量约23400的融合目的蛋白。结论 应用原核细胞表达体系成功地表达了TRP-1融合蛋白,为进一步研究其在脑发育中的所起的作用以及该蛋白的其它功能提供了可能。 相似文献
2.
目的 将贝氏柯克斯体 (Coxiellaburnetii)表面抗原P1蛋白基因与热休克蛋白B(HspB)基因片段融合 ,并使该融合基因在大肠杆菌细胞内表达 ,产生P1 HspB融合蛋白。 方法 采用PCR方法 ,从贝氏柯克斯体新桥株基因组DNA中扩增P1蛋白基因 (p1)和HspB蛋白基因 (hspB)片段 ,将两基因片段分别与原核表达载体 pQE30连接 ,分别构建重组表达质粒pQE30 / p1和pQE30 /hspB。用BamHI和SacIDNA内切酶将 p1基因片段从 pQE30 /p1切下并与 pQE30 /hspB的hspB连接 ,构建重组质粒 pQE30 / p1 hspB。用IPTG诱导pQE30 / p1 hspB转化的大肠杆菌表达融合基因 p1 hspB。 结果 SDS PAGE分析发现 pQE30 /p1 hspB转化的大肠杆菌表达一 83kDa融合蛋白 ;经薄层扫描分析 ,该融合蛋白约占到全菌体蛋白的 12 .1% ;免疫印迹分析发现该融合蛋白能被贝氏柯克斯体感染小鼠血清所识别。结论 构建的 p1 hspB融合基因在大肠杆菌中得到了有效表达 ,表达的P1 HspB融合蛋白能与抗贝氏柯克斯体抗体特异反应 ,P1 HspB融合蛋白为Q热亚单位疫苗的研制提供了一种新型融合蛋白 相似文献
3.
目的 在原核细胞中表达 Sj GST和 Sj14- 3- 3的融合蛋白。 方法 RT- PCR法从日本血吸虫成虫总 RNA中扩增出 Sj14- 3- 3基因 ,亚克隆至原核表达载体 p GEX- 4T- 1,并转化 E.coli BL2 1 ,IPTG诱导表达 ,Western- blot鉴定表达产物。 结果 扩增产物约为 76 5 bp,重组质粒经 Bam H 和 Xho 双酶切后可得到一与 PCR产物大小相同的 DNA片段 ,经 IPTG诱导后在 5 5 ku附近出现一新增蛋白条带 ,Western- blot结果显示 ,表达产物可被兔抗鼠 14- 3- 3ε多克隆抗体识别。 结论 在原核细胞融合表达 Sj GST和 Sj14- 3- 3成功。 相似文献
4.
两种血吸虫病疫苗候选分子在原核细胞的融合表达 总被引:3,自引:1,他引:3
目的 在原核细胞中表达SjGST和Sj14-3-3的融合蛋白。方法 RT-PCR法从日本血吸虫成虫总RNA中扩增出Sj14-3-3基因,亚克隆至原核表达载体pGEX-4T-1,并转化E.coli BL21,IPTG诱导表达,Western-blot鉴定表达产物。结果 扩增产物约为765bp,重组质粒经Bam HI和Xho I双酶切后可得到一与PCR产物大小相同的DNA片段,经IPTG诱导后在55ku附近出现一新增蛋白条带,Western-blot结果显示,表达产物可被免抗鼠14-3-3ε多克隆抗体识别。结论 在原核细胞融合表达SjGST和Sj14-3-3成功。 相似文献
5.
目的 将人癌胚抗原(CEA)分泌信号肽基因片段与人防卫素2(hBO-2)抗菌肽基因片段组成为一个融合基因,并构建该融合基因的拟转录病毒表达载体。方法 采用PCR法等方法将CEA分泌信号肽、肠激酶(EK)识别位点序列、上下游酶切位点及保护性碱基克隆在一个片段上,酶切后,将CEA-EK定向连接在含hBD-2基因片段的BlueseriptSK质粒上中,最后将CEA-EK-hBD2融合基因构建到pLNCX2逆转录病毒表达载体上。结果 重组pLNCX2表达载体经酶切后电泳并回收测序,序列中含有融合基因片段。结论 成功构建人CEA分泌信号肽与hBD-2抗菌肽的融合基因及表达载体。 相似文献
6.
目的 构建幽门螺杆菌空泡毒素 (VacA)毒性片段和细胞毒素相关蛋白 (CagA)的融合基因 (vlc) ,在原核细胞中表达 ,为Hp双价融合蛋白候选疫苗的研究提供材料。 方法 用GeneSOEing技术将vacA毒性亚单位片段 (v)与cagA保守片段 (c)用疏水性多肽接头 (Gly4Ser) 3 进行拼接 ,构建融合基因vlc,将vlc定向插入原核表达质粒pQE30 ,经DNA测序分析确认后 ,转化E .coliDH5a ,IPTG诱导表达 ,Westernblot分析其抗原性。结果 DNA序列分析表明融合基因的连接顺序、方向正确 ,有一个碱基发生了无义突变。工程菌诱导后可表达相对分子量为 5 8× 10 3 的融合蛋白 ,与预期分子量一致 ,约占菌体总蛋白的 8%。Westernblot显示融合蛋白具有良好的抗原性。结论 融合基因vlc构建成功 ,表达的融合蛋白具有良好的抗原性 ,有望进一步进行免疫保护性机制的研究和Hp疫苗的制备。 相似文献
7.
目的:构建HBV X-HCV C融合基因真核表达载体,并获得稳定表达该基因的HepG2细胞株。方法:双酶切质粒pXT1-X,得到完整的HBV X基因片段后,将其插入到质粒PBK-CMV和PBK-HCVC的相应酶切位点,得到重组质粒PBK-X和PBK-X-C;再将质粒RBK-CMV、PBK-X、PBK-HCV C和PBK-X-C分别导入肝癌细胞株HepG2中,G418筛选,RT-PCR、蛋白印迹鉴定HBV X和HCV C蛋白表达。结果:质粒PBK-CMV、PBK-X、PBK-HCV C和PBK-X-C在HepG2细胞中有稳定表达。结论:成功构建HBV X-HCVC融合基因真核表达载体,并获得稳定表达该基因的HepG2细胞株。 相似文献
8.
幽门螺杆菌lpp20基因与麦芽糖结合蛋白基因的融合表达与纯化 总被引:1,自引:0,他引:1
目的 研究幽门螺杆菌(Helicobacter pylori,H.pylori)lpp20基因与麦芽糖结合蛋白基因的融合表达产物的纯化方法,为幽门螺杆菌基因工程疫苗的制备建立基础。方法 采用本研究室分离的HpMEL-HP27菌株提取染色体DNA,用PCR方法从HP染色体DNA上扩增出lpp20基因片段,将目的基因插人到表达载体pMAL-c2X中,用重组质粒转化大肠杆菌(E.coil TB1)。采用IPTG进行诱导表达。用多糖树脂(amylose resin)作为填充料,制备层析柱,将可溶性菌体蛋白进行亲和层析。应用SDS-PAGE方法对纯化产物进行分析。结果 融合蛋白的分子量约为60kDa,融合蛋白的表达量约占全菌总蛋白的34%;纯化后的融合蛋白纯度达90%以上。结论 与麦芽糖结合蛋白基因融合的lpp20基因在E.coli TB1中能够高效表达;用多糖树脂作为填充料,进行亲和层析的方法,具有良好的纯化效果。 相似文献
9.
HIV-1 p24和gp41融合基因表达载体的构建及融合蛋白的表达纯化 总被引:3,自引:0,他引:3
目的用基因重组技术将HIV-1p24基因以及gp41基因具有抗原线性中和表位的部分重组连接,构建重组质粒,并在大肠埃希菌中高效表达融合蛋白。方法设计带有酶切位点的引物,分别PCR扩增p24和gp41两个基因,将它们分别连接到pMD18-T载体中,测序验证后,挑选出含有目的基因的正确克隆。将p24片段酶切后连接到gp41基因所在的pMD18T载体中,再将连接后的两个基因酶切,重新连接到pET21a表达载体中。将表达载体转染大肠埃希菌诱导表达,经Western-blot验证表达正确。结果融合蛋白p24-gp41在大肠埃希菌中高效表达。结论融合蛋白p24-gp41可以在pET21a表达载体中高效表达。 相似文献
10.
目的:构建钙调磷酸酶(CaN)融合基因的原核表达载体,并使其获得表达,为研究该蛋白的功能,在病理生理过程中的变化及相关疾病的治疗奠定基础。方法:利用OLIGO6.0生物学软件设计一对特异性引物,并在引物中引入适当的酶切位点,采用逆转录聚合酶链反应(RT-PCR),扩增出CaN基因,经BamHI和XhoI双酶切后,将其克隆于上游融合了GST标签的pGEX-6P-1表达载体中,选取鉴定为阳性的重组表达载体,转化于受体菌BL21中,并利用1mMIPTG对其进行诱导,收获表达产物进行SDS-PAGE电泳分析和Western印迹(blot)鉴定,以确定融合表达的CaN基因的反应活性。结果:RT-PCR后扩增得到了大小约为700bp的目的片段,克隆于表达载体pGEX-6P-1,经测序分析后,证实所获得的与Genebank提供的序列相一致,CaN基因融合表达产物经SDS-PAGA电泳分析发现该重组蛋白大小约为45kD,利用CaNAβ抗体进行Western blot分析,结果证实该表达的融合蛋白具有较好的免疫反应性。结论:钙调磷酸酶Aβ以融合蛋白的形式获得了表达,而且融合表达的蛋白具有反应活性。 相似文献
11.
目的:应用筑巢式RT—PCR(RT—nest-PCR)法检测慢性髓细胞性白血病(CML)患者非亲缘异基因骨髓移植(URD)后微小残留病变(MRD),并探讨它与复发的相关性。方法:分别采用RT-nest—PCR法和常规细胞遗传学方法检测bcr/abl融合基因和Ph染色体。结果:20例CML行URD患者术前Ph染色体和bcr/abl融合基因检测均为阳性,移植术后30dPh染色体皆转为阴性,bcr/abl融合基因完全转阴时间为1~3个月,中位时间2个月;在随防的18例CML患者中,有16例患者bcr/abl融合基因完全转阴后没再转为阳性。在1例复发的CML患者中可见到血型、Ph染色体和bcr/abl融合基因动态变化,另1例CML患者在移植成功后的21个月和36个月检测到bcr/abl融合基因,经随访未见临床和血液学复发征象。结论:检测bcr/abl融合基因是目前观察CML患者非亲缘异基因骨髓移植后微小残留病变最敏感的方法之一,非亲缘异基因骨髓移植能最大限度地消除CML患者体内残留病变,患者能长期无病生存。 相似文献
12.
Activation of a novel Bcr/Abl destruction pathway by WP1130 induces apoptosis of chronic myelogenous leukemia cells 总被引:1,自引:0,他引:1
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Bartholomeusz GA Talpaz M Kapuria V Kong LY Wang S Estrov Z Priebe W Wu J Donato NJ 《Blood》2007,109(8):3470-3478
Imatinib mesylate (Gleevec) is effective therapy against Philadelphia chromosome-positive leukemia, but resistance develops in all phases of the disease. Bcr/Abl point mutations and other alterations reduce the kinase inhibitory activity of imatinib mesylate; thus, agents that target Bcr/Abl through unique mechanisms may be needed. Here we describe the activity of WP1130, a small molecule that specifically and rapidly down-regulates both wild-type and mutant Bcr/Abl protein without affecting bcr/abl gene expression in chronic myelogenous leukemia (CML) cells. Loss of Bcr/Abl protein correlated with the onset of apoptosis and reduced phosphorylation of Bcr/Abl substrates. WP1130 did not affect Hsp90/Hsp70 ratios within the cells and did not require the participation of the proteasomal pathway for loss of Bcr/Abl protein. WP1130 was more effective in reducing leukemic versus normal hematopoietic colony formation and strongly inhibited colony formation of cells derived from patients with T315I mutant Bcr/Abl-expressing CML in blast crisis. WP1130 suppressed the growth of K562 heterotransplanted tumors as well as both wild-type Bcr/Abl and T315I mutant Bcr/Abl-expressing BaF/3 cells transplanted into nude mice. Collectively, our results demonstrate that WP1130 reduces wild-type and T315I mutant Bcr/Abl protein levels in CML cells through a unique mechanism and may be useful in treating CML. 相似文献
13.
目的 研究b3a2型反义bcr-abl寡核苷酸(ASO)体外对慢性髓细胞性白血病(CML)细胞株K562的抑制作用,为反义技术用于CML患者体内基因治疗和体外骨髓净化提供依据。方法 采用体外细胞培养技术及四唑盐(MTT)比色法、免疫组织化学染色法观察b3a2-ASO体外对K562细胞的生长、克隆形成及P210 bcr-abl蛋白表达的影响。结果 经b3a2-ASO(110μm/ml)作用40h后,K562细胞生长抑制率达66.12%,集落抑制率为65.44%;作用15h后P210bcr-abl蛋白合成抑制率可达60%。而无义寡核苷酸(NSO)对前述三个指标均无显著影响;b3a2-ASO及NSO对bcr-abl阴性细胞株HL60的细胞生长和存活率亦无显著影响。结论 b3a2-ASO对K562细胞有序列特异性抑制作用,提示其可以作为CML反义基因治疗,尤其是骨髓净化的有力措施之一。 相似文献
14.
IL-24融合蛋白表达载体的构建及其对人小肠癌细胞的生长抑制作用 总被引:2,自引:1,他引:1
目的:构建人白细胞介素-24(IL-24)基因的表达载体并在大肠杆菌中表达.研究IL-24融合蛋白对人小肠癌细胞(HIC)的生长抑制作用.方法:用PCR从质粒TRAP-hIL-24中扩增人 IL-24 cDNA片段,并将该片段插入DGEX-KG 原核表达载体中,实现插入基因的融合表达.用SDS-PAGE和Western blot对表达产物进行鉴定.提取并纯化IL-24融合蛋白,以不同浓度与 HIC细胞共培养72 h,同时设立相应浓度梯度的GST蛋白组、HIC细胞对照组及空白孔,采用MTT法检测各组人小肠癌细胞的体外增殖结果:酶切结果证实,成功地构建了pGEX- KG-IL-24原核表达载体,并在大肠杆菌中获得稳定的表达,表达产物的相对分子质量(Mr) 同预期值相一致,融合蛋白为Mr53 000,GST 蛋白为Mr29 000.IL-24融合蛋白以浓度依赖性的关系抑制HIC细胞的生长,并且在20和40 mg/L时的抑制率明显高于GST蛋白.结论:成功地构建了重组表达载体pGEX-KG- IL-24,并在E.coli BL21中表达.IL-24融合蛋白对HIC细胞生长有抑制作用. 相似文献
15.
Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines 总被引:20,自引:34,他引:20
The tyrosine kinase activity of the Bcr/Abl oncogene is required for transformation of hematopoietic cells. The tyrosine kinase inhibitor STI571 (formerly called CGP57148B, Novartis Pharmaceuticals) inhibits BCR/ABL, TEL/ABL, and v-ABL kinase activity and inhibits growth and viability of cells transformed by any of these ABL oncogenes. Here we report the generation of 2 BCR/ABL-positive cell lines that have developed partial resistance to STI571. BCR/ABL-transformed Ba/F3 hematopoietic cells and Philadelphia-positive human K562 cells were cultured in gradually increasing concentrations of STI571 over a period of several months to generate resistant lines. Resistant Ba/F3.p210 cells were found to have an increase in Bcr/Abl messenger RNA, amplification of the Bcr/Abl transgene, and a greater than tenfold increase in the level of BCR/ABL protein. In contrast to Ba/F3.p210 cells, drug-resistant K562 cells did not undergo detectable amplification of the BCR/ABL gene, although they displayed a 2-fold to 3-fold increase in p210BCR/ABL protein. The addition of STI571 to both resistant Ba/F3. p210 and K562 cells resulted in a rapid reduction of tyrosine phosphorylation of cellular proteins, similar to that observed for nonresistant cells. However, the inhibition of kinase activity was transient and partial and was not accompanied by apoptosis. The results suggest that resistance to STI571 may be multifactorial. Increased expression of the target protein BCR/ABL was observed in both lines, and resulted from oncogene amplification in one line. However, altered drug metabolism, transport, or other related mechanisms may also contribute to drug resistance. 相似文献
16.
J Nishimura K Shibata S Sadamura H Takahira J Hirata T Umemura H Nawata J Okamura 《International journal of hematology》1992,55(3):227-233
We analysed differences between the populations of tyrosine phosphorylated proteins in two cell lines, K-562 and MR-87, which express two different bcr-abl fusion gene products, using both immunoprecipitation and Western blotting with an anti-phosphotyrosine antibody. K-562 cells preferentially expressed P210bcr/abl (P210), while MR-87 expressed P190bcr/abl (P190). Tyrosine phosphorylated proteins with a molecular mass of 150 kDa (p150) and 115 kDa (p110) were found in both K-562 and MR-87. A 36 kDa protein (p36) was tyrosine phosphorylated in vivo only in K-562 cells, while proteins with a molecular mass of 140 kDa (p140) and 62 kDa (p62) were found only in MR-87 cells. Moreover, several proteins in the detergent-insoluble cell fraction were differently tyrosine phosphorylated in vitro in K-562 and MR-87 lysates. These results suggest that P210 and P190 may have different substrates, and thus, different signal transduction pathways for cell proliferation, although the differential association of such cellular proteins with the two bcr/abl products remains to be clarified. 相似文献
17.
Wang W Yao LB Liu XP Feng Q Shang ZC Cao YX Sun BZ 《World journal of gastroenterology : WJG》2005,11(14):2130-2135
AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line. METHODS: p27 gene was obtained by RT-PCR, and its sequence was approved to be correct. Then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line. p27-pcDNA3.1-K562 cell clone was screened by G418 after transfection, p27 protein was identified by Western blot. MTT was used to detect the survival rate of the cell. Flow cytometry was used to detect cell cycle and apoptosis index. RESULTS: The expression of p27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. A strong inhibition of cell proliferation was observed in p27-pcDNA3.1 -K562 cells as compared with that of the control (pcDNA3.1 -K562 cells). The cells at G0/G1 phase were significantly increased, and cells at S phase were greatly declined. The apoptosis index was increased greatly after p27-pcDNA3.1-K562 cells were treated with STI571, and survival rate of the cell was markedly declined (0.35-0.58, P<0.05-0.048 vs STI571-K562 cell,0.35-0.72,P<0.01-0.001 vs p27-K562 cell). CONCLUSION: p27 and STI571 have a synergistic action on inhibition of proliferation and induction of apoptosis on K562 cells. 相似文献
18.
Lisa C. Crowley Tracey R. O'Donovan Mary R. Cahill Sharon L. McKenna 《American journal of hematology》2013,88(6):455-462
Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr‐Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr‐Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr‐Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr‐Abl is sequestered into vesicular structures that co‐localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1/ATG7). Pharmacological inhibition of autophagy also reduced Bcr‐Abl/LC3 co‐localization in both K562 and CML patient cells. Bcr‐Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr‐Abl protein levels to those of untreated cells. This ability to down‐regulate Bcr‐Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib. 88:455–462, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
19.
Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification 总被引:23,自引:38,他引:23
le Coutre P Tassi E Varella-Garcia M Barni R Mologni L Cabrita G Marchesi E Supino R Gambacorti-Passerini C 《Blood》2000,95(5):1758-1766
The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenic bcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/abl inhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 microM) 10-fold higher than the IC50 (0.1 microM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3-like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro. (Blood. 2000;95:1758-1766) 相似文献
20.
Involvement of reactive oxygen species in adaphostin-induced cytotoxicity in human leukemia cells 总被引:7,自引:4,他引:3
Chandra J Hackbarth J Le S Loegering D Bone N Bruzek LM Narayanan VL Adjei AA Kay NE Tefferi A Karp JE Sausville EA Kaufmann SH 《Blood》2003,102(13):4512-4519
Adaphostin (NSC 680410), an analog of the tyrphostin AG957, was previously shown to induce Bcr/abl down-regulation followed by loss of clonogenic survival in chronic myelogenous leukemia (CML) cell lines and clinical samples. Adaphostin demonstrated selectivity for CML myeloid progenitors in vitro and remained active in K562 cells selected for imatinib mesylate resistance. In the present study, the mechanism of action of adaphostin was investigated in greater detail in vitro. Initial studies demonstrated that adaphostin induced apoptosis in a variety of Bcr/abl- cells, including acute myelogenous leukemia (AML) blasts and cell lines as well as chronic lymphocytic leukemia (CLL) samples. Further study demonstrated that adaphostin caused intracellular peroxide production followed by DNA strand breaks and, in cells containing wild-type p53, a typical DNA damage response consisting of p53 phosphorylation and up-regulation. Importantly, the antioxidant N-acetylcysteine (NAC) blunted these events, whereas glutathione depletion with buthionine sulfoximine (BSO) augmented them. Collectively, these results not only outline a mechanism by which adaphostin can damage both myeloid and lymphoid leukemia cells, but also indicate that this novel agent might have a broader spectrum of activity than originally envisioned. 相似文献