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1.
结核分枝杆菌Rv2450基因的克隆、表达及纯化   总被引:4,自引:1,他引:3  
薛莹  姜泓  高雪  柏银兰  师长宏  张海  徐志凯  李元 《医学争鸣》2004,25(19):1778-1781
目的:克隆结核分枝杆菌(Mtb)Rv2450基因,序列测定正确后进行融合表达、纯化.方法:采用聚合酶链反应(PCR)从Mtb H37Rv基因组中扩增出Rv2450编码基因,序列测定正确后,亚克隆到融合表达载体pPro-EX HT中,转化大肠杆菌DH5α,目的基因经IPTG诱导,由T7启动子调控表达带6个组氨酸残基的Rv2450蛋白,在变性条件下对目的蛋白进行亲和层析纯化.结果:得到融合6个组氨酸残基的Rv2450蛋白纯度大于90%.结论:构建了Mtb Rv2450基因的重组表达载体,并获得了高纯度的融合表达蛋白.  相似文献   

2.
【目的】构建藤黄微球菌复苏促进因子Rpf结构域突变体基因E54K、E54A的原核表达质粒,在大肠杆菌中表达和纯化。【方法】采用PCR方法从藤黄微球菌基因组中扩增出Rpf结构域E54A、E54K突变体基因,连接进入pMD-18T载体中,测序正确的基因插入到pGEX-4T-1表达载体中,转化大肠杆菌DH5ct,经IPTG诱导,SDS—PAGE鉴定表达蛋白,利用Rpf结构域单克隆抗体进行Western—blotting分析。用GS-4B亲和层析柱纯化目的蛋白。【结果】获得藤黄微球菌Rpf结构域E54K和E54A突变体基因,测序结果与GenBank公布的序列相同,突变体位点与设定一致;表达蛋白相对分子质量与文献报道一致;Western—blotting结果显示,在相对分子量约32KD处有与Rpf结构域单克隆抗体特异性结合带。通过GS-4B系统,得到纯化的GST融合蛋白。【结论】成功表达、纯化了Rpf结构域突变体E54K、E54A融合蛋白,为上述蛋白在结核病中的应用奠定基础。  相似文献   

3.
高辉  柏银兰  王丽梅  徐志凯  薛莹 《医学争鸣》2006,27(18):1633-1636
目的:表达和纯化RpfA融合蛋白.方法:将含有RpfA基因片段的质粒用NdeI与BamHⅠ双酶切,然后将目的基因片段克隆入pcDNA3.1 载体构建重组载体pcDNA3.1 -RpfA,测序正确后再将目的基因片段亚克隆入pET19b原核表达载体并转化E.coli DE3,IPTG诱导表达融合蛋白,Western blot鉴定融合蛋白.在变性条件下Ni2 -NTA 亲和色谱柱纯化目的融合蛋白.结果:目的基因片段测序与Genbank报道一致(切去了5'端99 bp).SDS-PAGE显示,在Mr约为80 ku处有表达条带,Western blot鉴定为(His)6融合蛋白,并与小鼠抗Rv1884和抗Rv2389免疫血清有交叉反应.可溶性分析发现融合蛋白主要以包涵体形式存在.经Ni2 -NTA亲和色谱柱纯化得到了融合有6个组氨酸残基的RpfA融合蛋白.结论:成功构建了pET19b-RpfA表达载体,并在E.coli DE3中高效表达,亲和层析后获得了纯化目的蛋白.  相似文献   

4.
乙型肝炎病毒PreS基因的克隆表达及亲和层析纯化   总被引:8,自引:4,他引:4  
杨敏  刘新平  韩炯  张晓光  苏成芝  陈常庆 《医学争鸣》2001,22(16):1493-1496
目的:获取乙型肝炎病毒前S区基因(HBVpreS),序列测定正确后进行融合表达,为今后研究其机制及应用创造条件。方法:利用乙型肝炎病毒基因组为模板体外扩增HBVPreS基因后进行基因克隆,目的基因经测序正确后克隆入融合表达载体pRSET-C中,转化大肠杆菌JM109()DE3)。目的基因经IPTG诱导,由T7启动子调控表达了氨基端带6个连续组氨酸残基的HBV-PreS蛋白,在变性条件下对目的蛋白进行纯化。结果:成功地扩增到preS区全长基因,得到融合6个His的HBV-PreS蛋白纯度大于90%,结论:构建了乙型肝炎病毒前S区基因的重组表达载体,获得了稳定表达的工程菌,为以后的深入研究奠定了基础。  相似文献   

5.
【目的】克隆、表达空肠弯曲菌表面蛋白PEB1,并制备针对该蛋白的多克隆抗体。【方法】采用PCR方法从空肠弯曲菌Penner19标准株基因组中扩增出PEB1蛋白基因,用限制性内切酶消化后插入到pUC-19克隆载体中,测序正确后,再亚克隆到融合表达载体pPro-EXHTb中,转化大肠杆菌DH5α,目的基因经IPTG诱导,由T7启动子调控表达了氨基端带6个连续组氨酸残基的PEB1蛋白,并经Western-blotting证实。然后,在非变性条件下用Ni-NTA亲和色谱柱纯化目的蛋白。用纯化的PEB1蛋白作为免疫原免疫BALB/c小鼠,制备多克隆抗体,用ELISA法检测抗体效价。【结果】在非变性条件下用Ni2+-NTA亲和色谱柱纯化PEB1融合蛋白,纯化获得的蛋白纯度大于90%。用该融合蛋白免疫小鼠后,得到抗PEB1抗体,效价达1:2×105。【结论】成功构建了空肠弯曲菌PEB1基因原核表达载体,并获得了高纯度的PEB1蛋白及其高效价多抗,对进一步制备肠粘膜高亲和力的过敏原重组BCG打下了坚实的基础。  相似文献   

6.
人Era蛋白在大肠杆菌中的高效表达及其纯化   总被引:4,自引:2,他引:2  
目的 在大肠杆菌中对人era基因(简称hera)进行表达、纯化。方法 PCR扩增hera基因,测序正确后克隆入大肠杆菌融合表达载体pRSET-C,重组质粒pRSETC-hera以大肠杆菌BL21(DE3)为宿主菌,用IPTG进行诱导表达。然后利用亲和层析的方法对表达蛋白进行纯化。结果 克隆了hera基因,测序正确;利用pRSET-C载体在大肠杆菌中融合表达了全长hera-cDNA基因,表达量占细菌总蛋白的59.6%。融合蛋白在菌体内主要以包涵体形成存在,在变性条件下用Ni-NTA亲和色谱柱纯化此融合蛋白,其纯度达到了98.0%。结论 利用基因重组技术在大肠杆菌中高表达了hera基因。并对融合蛋白进行了初步纯化。  相似文献   

7.
李坤  王小霞  徐皓  余春艳  张衍国  张惠中 《医学争鸣》2008,29(21):1931-1934
目的:构建人可溶性CD83基因原核表达载体,并在大肠杆菌中表达和纯化.方法:利用反转录PCR方法从正常人外周血单个核细胞中获得可溶性CD83基因,克隆到表达载体pET-32a载体,构成重组质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达6×His融合蛋白,并经SDS-PAGE及Western blot检测和鉴定.表达产物包涵体经Ni-NTA亲和层析纯化.结果:经酶切鉴定及测序证实可溶性CD83蛋白基因的原核表达载体构建正确.经SDS-PAGE及Western blot显示在M,约32 000处出现融合表达条带.融合蛋白的表达量约占菌体蛋白总量的45%.重组蛋白经Ni-NTA亲和层析进行纯化后,得到了高纯度的融合蛋白.结论:成功克隆人外周血单个核细胞来源的可溶性CD83基因片段,并在大肠杆菌B121(DE3)中高效表达,亲和层析纯化后获得高纯度融合蛋白.  相似文献   

8.
目的 原核表达人睾丸生精相关基因SPAG4L,并对重组蛋白进行纯化,为下一步研究SPAG4L的生物学功能奠定基础.方法 运用RT-PCR从人睾丸中扩增编码SPAG4L126-379的基因片段,并将PCR产物克隆至pUCm-T载体.用限制性内切酶EcoRⅠ和HindⅢ消化后,将目的 片段克隆至带有6个组氨酸标签的原核表达载体PQE30中.重组质粒PQE-30-SPAG4L经酶切和测序验证后,转化大肠杆菌JM15,IPTG诱导融合蛋白表达.SPAG4L融合蛋白以westernblot进行鉴定,最后用NI-NTA磁性琼脂糖珠进行纯化.结果 成功构建了重组表达质粒PQE-30-SPAG4L,并能够在大肠杆菌JM15中诱导表达,经Westernblot分析证实,IPTG诱导表达的是SPAG4L融合蛋白.建立小规模的SPAG4L融合蛋白表达、纯化系统,获得了带有6个组氨酸标签的纯化的SPAG4L融合蛋白.结论 成功地对人睾丸生精相关基因SPAG4L进行了体外原核表达,获得了纯化的融合蛋白蛋白,为进一步研究该蛋白在精子发生中的生物功能奠定了基础.  相似文献   

9.
结核分枝杆菌furA基因片段的克隆、表达和分离纯化   总被引:3,自引:2,他引:1  
高雪  薛莹  姜泓  柏银兰  师长宏  张海  李元  徐志凯 《医学争鸣》2004,25(18):1637-1640
目的: 获得结核分枝杆菌furA基因片段并高效表达、纯化. 方法: 用PCR技术从MTB毒株H37Rv-DNA中扩增出相应大小的DNA片段,将片段与pGEM-T-easy 载体连接后测序. 将测序正确的furA按照BamHⅠ和HindⅢ酶切位点克隆入原核表达载体pRSET-A,将连接产物转化入E.coli BL21,挑出阳性克隆,IPTG诱导表达重组的(His)6融合蛋白. 对重组融合蛋白通过镍柱进行纯化. 结果: PCR获得的furA序列与Genbank报道的一致(为453 bp). 重组载体在E.coli BL21中以可溶形式高效表达,融合蛋白经SDS-PAGE和Western blot分析,在Mr为23.0×103处有特异的蛋白条带,目的蛋白表达量占菌体总蛋白量的10%,重组蛋白经镍柱进行纯化后,得到了高纯度的FurA融合蛋白. 结论: 成功克隆结核分枝杆菌furA基因片段,并在E.coli BL21中高效表达,亲和层析纯化后获得高纯度FurA融合蛋白.  相似文献   

10.
目的 获取糖皮质激素诱导亮氨酸拉链蛋白(GILZ)的His融合蛋白,为下阶段深入研究GILZ的结构、功能及筛取与其相互作用的蛋白打下基础.方法 利用基因工程技术,BarnH I/Xho I酶切真核表达质粒HA-GILZ/PcDNA3获得GILZ cDNA,重组于能表达6个组氨酸残基的原核表达质粒pET-30a中.然后用该亚克隆重组质粒转化大肠杆茵BL21(DE3),用IPTG诱导表达,用Ni2+-NTA柱纯化.结果 亚克隆获得CILZ/pET-30a高效原核表达重组载体,诱导表达的融合蛋白占细菌总蛋白高达40%以上,纯化的蛋白分子量约为24 kD.结论 成功构建His-GILZ融合蛋白原核表达载体,并高效表达、纯化,为进一步研究其结构功能关系和临床应用奠定了基础.  相似文献   

11.
Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P< 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P< 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.  相似文献   

12.
Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P < 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (>0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P <0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P < 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.  相似文献   

13.
Objective: To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods: SD rats served as both donors and recipients. 4℃ sodium lactate Ringer's was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava (IVC) inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results: Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion: This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.  相似文献   

14.
Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.  相似文献   

15.
Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.  相似文献   

16.
A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care(~'4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.  相似文献   

17.
Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.  相似文献   

18.
Journal of Nanjing Medical University (English Edition) JNMU, sponsored by Nanjing Medical University, was established in 1987. It is a bimonthly comprehensive English medical journal published locally and abroad.Since 2007, Journal of Nanjing Medical University (English Edition )was granted Elsevier the full publishing and distribution rights worldwide for the Electronic Edition, excluding the People's Republic of China.  相似文献   

19.
Objective To measure Derpl and Blot5 allergen levels in asthmatics' homes in Hongkong.Methods Seventy houses were enrolled for a mite indoor environment study. Dust samples were obtained from two sites of each patients' house: bed and floor. Derpl and Blot5 levels were quantified by a two-site monoclonal antibody-based ELISA technique.Results The levels of Derpl allergens found in bed (geometric mean (GM) 3.43 μg/g of dust; 95%CI, 1.89-4.96 μg/g)and on the floor (GM 1.12 μg/g of dust; 95%CI, 0.71-1.53 μg/g) indicated significant differences (P=0.005). However, the levels of Blot5 allergens found in bed (GM 19.00 μg/g of dust; 95%CI, 0.89-38.90 μg/g) and on the floor (GM 6.14 μg/g of dust; 95%CI, 0.40-11.90 μg/g) showed no statistically significant difference. In addition, in regards to the exposure index for Derpl and Blot5 allergens found in bed and on the floor, 17.6% in bed and 8.6% on the floor had levels of Blot5 ≥ 10 μg/g of dust, higher than those obtained for Derp1 (7.2% and 0% in bed and on the floor respectively, P< 0.05); higher percentages in bed and on the floor (25.0% and 35.7%) were observed for levels of Blot5 =0 μg/g of dust as compared with Derpl in bed and on the floor (4.3% and 14.5% respectively, P< 0.05).Conclusions Derpl and Blot5 are the major allergens found in this regional study, Blot5 is a more potent allergen in Hongkong, probably reflecting the high level of exposure to Blomia tropicalis (Bt). Bt and Dermatophagoides pteronyssinus (Dp) allergens should be included for precise diagnosis and effective immuno-therapeutic treatment of mite allergy in Hongkong.  相似文献   

20.
ANTI-PROLIFERATION EFFECT OF ORIDONIN ON HL-60 CELLS AND ITS MECHANISM   总被引:4,自引:0,他引:4  
Objcetive To investigate the anti-proliferation effect of oridonin on leukemic HL-60 cells and its mechanism. Methods HL-60 cells in vitro in culture medium were given different concentrations of oridonin. The inhibitory rate of cells were measured by microculture tetrazolium (MTT) assay, cell apoptotic rate was detected by flow cytometry (FCM), morphology of cell apoptosis was observed by hoechst 33258 fluorescence staining, and the activity of telomerase was detected using telomere repeat amplification protocol (TRAP) PCR-ELISA before and after apoptosis occurred. Results Oridonin could decrease telomerase activity, inhibit growth of HL-60 cells, and cause apoptosis significantly. The suppression was both in time- and dose-dependent manner. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly by hoechst 33258 fluorescence staining especially after cells were treated 48-60 hours by oridonin. Conclusions Oridonin has apparent anti-proliferation and apoptotic effects on HL-60 cells in vitro, decreasing telomerase activity of HL-60 cells may be one of its most important mechanisms. These results will provide strong laboratory evidence of oridonin for clinical treatment of acute leukemia.  相似文献   

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