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??OBJECTIVE To investigate the expression and purification of protease inhibitor OH-TCI in Escherichia coli and its protective effect and mechanism in myocardial ischemia-reperfusion injury model. METHODS The recombinant plasmid of Pet32a-OH-TCI was transformed into E. coli and induced by IPTG, soluble fusion protein was expressed in the constructed expression system and isolated by a nickel affinity chromatography column. Then, the products was digested by TEV enzyme and was purified by the nickel affinity chromatography column. The adult male rats were divided into two groups randomly:normal saline group and OH-TCI group. At 5 min before the operation, rats were treated with OH-TCI by intraperitoneal injection in the experimental group and was given the same volume of saline in the control group. In all animal experiments, rats left anterior descending coronary artery were ligated for 30 min to construct the model. After 120 min of reperfusion, samples were taken to determine the contents of malondialdehyde (MDA), tumor necrosis factor-?? (TNF-??) and interleukins-6 (IL-6) and the activities of lactic acid dehydrogenase (LDH), creatine kinase (CK) and superoxide dismutase (SOD). Finally, the protective effects of OH-TCI against myocardial ischemia-reperfusion injury in rat model were evaluated. RESULTS The recombinant protease inhibitor OH-TCI was expressed in the E.coli expression system in a soluble pattern and successfully purified by two steps of nickel affinity chromatography. Comparing with those in the control group, the recombinant protease inhibitor OH-TCI could significantly decrease the levels of LDH, MDA and IL-6 (P<0.01)as well as inflammatory factor TNF-?? (P<0.05). CONCLUSION The recombinant protease inhibitor OH-TCI can be expressed successfully in E. coli. Purified recombinant OH-TCI has natural protease inhibitory activity and shows protective effects against ischemia and reperfusion myocardium injury.     

独行菜幼苗叶片乙酰CoA酰基转移酶基因克隆、序列分析与原核表达

目的:研究独行菜强心苷生物合成途径的相关基因,从独行菜幼苗叶片中克隆强心苷生物合成途径关键酶乙酰CoA酰基转移酶(acetyl-CoA C-acetyltransferase,AACT)基因,进行序列分析和原核表达。方法:根据独行菜转录组数据中LaAACT基因序列设计特异性引物,克隆得到LaAACT基因的cDNA序列,构建pET-32a-LaAACT原核表达载体,诱导表达LaAACT重组蛋白。结果:LaAACT基因ORF全长为1 212 bp,编码403个氨基酸。序列分析结果显示LaAACT蛋白没有跨膜区,不含信号肽,位于细胞质中,含有硫解酶家族保守结构域。系统进化结果显示LaAACT蛋白与十字花科植物的AACT蛋白亲缘关系较近。通过IPTG诱导,在大肠埃希菌中成功表达LaAACT重组蛋白,并得到了纯化的LaAACT重组蛋白。结论:首次从独行菜幼苗叶片中克隆了LaAACT基因,建立其稳定的原核表达体系,为LaAACT蛋白抗体的制备,进一步研究LaAACT基因在独行菜强心苷生物合成途径中的功能奠定了基础。     

北葶苈子GGPS基因的克隆、序列分析与原核表达

目的:获得北葶苈子(Lepidium apetalum)中牻牛儿基牻牛儿基焦磷酸合成酶(geranylgeranyl pyrophosphate synthase,GGPS)的编码基因,进行生物信息学分析,在大肠杆菌中表达该蛋白。方法:根据北葶苈子转录组测序结果中的GGPS基因序列,设计特异性引物,通过PCR扩增得到北葶苈子GGPS的c DNA序列,进行TA克隆、测序及序列分析,构建原核表达载体并于大肠杆菌中表达北葶苈子GGPS蛋白。结果:得到北葶苈子GGPS c DNA全长1 146 bp,编码381个氨基酸;序列分析表明北葶苈子GGPS基因编码的氨基酸序列包含类异戊二烯合成酶结构域,氨基酸序列进化关系表明北葶苈子GGPS与拟南芥(Arabidopsis thaliana)和白芥(Sinapis alba)亲缘关系最近;成功构建了p ET-32a-La GGPS原核表达载体,并在大肠杆菌BL21菌株中成功诱导表达。结论:首次克隆得到北葶苈子GGPS基因,并在大肠杆菌中成功表达了北葶苈子GGPS蛋白,为纯化该蛋白并研究其结构和功能奠定了基础。     

独行菜磷酸甲羟戊酸激酶LaPMK基因克隆、生物信息学分析及原核表达

目的从独行菜Lepidium apetalum中克隆萜类合成途径关键酶磷酸甲羟戊酸激酶(phosphomevalonate kinase,PMK)基因,进行序列分析和原核表达。方法根据独行菜转录组数据中La PMK基因序列设计特异性引物,克隆得到La PMK基因的开放阅读框(open reading frame,ORF),构建p ET32a-La PMK原核表达载体,在大肠杆菌BL21(DE3)中用IPTG诱导表达La PMK融合蛋白。结果 La PMK基因ORF全长为1 518 bp(Genbank注册号KT004541),编码505个氨基酸。生物信息学分析结果显示La PMK蛋白没有跨膜区,不含信号肽,位于细胞质中,含有GHMP激酶家族特异的N末端和C末端的保守结构域。系统进化树结果显示La PMK蛋白与芜菁的PMK蛋白具有92%的序列相似性,亲缘关系较近。构建p ET32a-La PMK原核表达载体,在大肠杆菌中成功表达La PMK融合蛋白。结论克隆了La PMK基因,建立其稳定的原核表达体系,为La PMK蛋白纯化、抗体的制备,进一步研究La PMK基因在独行菜萜类合成途径中的作用奠定了基础。     

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??OBJECTIVE To prepare green fluorescent protein-hemagglutinin A-cervical cancer(GFP-HA-PTP) fusion protein trageting HPV transformed cervical cancer cells with endosome escape capability, and further investigate its penetrating ability for cervical cancer cell lines. METHODS pET15b-GFP-HA-PTP expression plasmid was constructed, and E. coli BL21 (DE3) was transformed with the plasmid, then the yielded GFP-HA-PTP fusion protein was purified by Ni-NTA His-Bind resin and conformed by Western blotting assay. Specific penetrating analysis of GFP-HA-PTP was performed under fluorescence microscopy. RESULTS GFP-HA-PTP fusion protein was highly expressed in BL21 cells. Western blotting analysis result showed that the fusion protein was expressed correctly. The fusion protein can selectively penetrate into cervical cancer cell lines Siha and Caski with endosome escape efficiently. CONCLUSION GFP-HA-PTP can specificly penetrate into cervical cancer cell lines after endosome escaping with high efficiency.
     

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??OBJECTIVE To investigate apoptosis induction ability of hPP10-Apoptin fusion protein to mouse B16 melanoma cell. METHODS pET15b-hPP10-Apoptin expression plasmid was constructed, and E. coli BL21 (DE3) was transformed with the plasmid, then the yielded hPP10-Apoptin fusion protein was purified by Ni-NTA His-Bind Resin and confirmed by Western blotting assay. Melanoma cell apoptosis induced by hPP10-Apoptin fusion protein was analyzed by TUNEL assay, and the antitumor effect was examined in melanoma cell-bear mouse model. RESULTS hPP10-Apoptin fusion protein was highly expressed in BL21 cells, Western blotting analysis result showed that fusion protein was expressed correctly. The fusion protein can induce melanoma cell apoptosis in vivo and in vitro. CONCLUSION The results confirm that hPP10-Apoptin fusion protein could penetrate into melanoma cell and also has antitumor effect.     

丹参二萜合酶基因CPS4的原核表达体系优化及活性蛋白纯化

目的:优化丹参柯巴基焦磷酸合酶基因家族新基因CPS4的原核表达体系并对重组蛋白进行纯化。方法:对2种原核表达菌株进行比较。对诱导条件,包括异丙基-β-D-硫代半乳糖苷(IPTG)诱导浓度、诱导时宿主菌的密度(A600)和诱导时间进行正交试验。利用HisTrap HP蛋白纯化柱对重组蛋白进行纯化。结果:Escherichia coli Tuner(DE3)表达菌株能诱导产生更多的可溶重组蛋白。最佳诱导表达条件为诱导时宿主菌的A600值0.7,IPTG浓度0.2 mmol·L-1,诱导表达时间10 h。HisTrap HP蛋白纯化柱纯化时梯度洗脱能得到纯度高的重组蛋白。结论:利用此优化体系可得到足量用于后续研究的重组蛋白,为其他二萜类合酶基因的原核表达提供参考。     

地黄RgGGPPS2基因克隆、生物信息学分析及表达分析

目的为了研究地黄萜类化合物生物合成途径的功能基因,从地黄中克隆了一条新的地黄GGPPS基因(RgGGPPS2),进行生物信息学分析,原核表达、纯化以及基因表达模式分析。方法根据地黄转录组数据中RgGGPPS2基因的序列信息,设计特异性引物,克隆了RgGGPPS2基因的开放阅读框(ORF)序列,构建pET32a-RgGGPPS2原核表达载体,用IPTG在大肠杆菌BL21(DE3)中诱导表达RgGGPPS2重组蛋白,荧光定量PCR检测RgGGPPS2基因的组织特异性表达。结果 RgGGPPS2基因的ORF为867 bp,编码289个氨基酸。生物信息学分析结果发现RgGGPPS2蛋白含有2个富含天冬氨酸的基序FARM(first aspartate-rich motif)和SARM(second aspartate-rich motif),RgGGPPS2与芝麻、长春花等双子叶植物中GGPPS蛋白的同源性较高。通过构建pET-32a-RgGGPPS2原核表达载体在大肠杆菌中成功表达RgGGPPS2重组蛋白,利用Ni2+亲和色谱得到了纯化的RgGGPPS2重组蛋白。荧光定量PCR结果显示RgGGPPS2基因在根中表达量最高,其次是叶,茎中最低。结论克隆了RgGGPPS2基因,获得了纯化的RgGGPPS2重组蛋白,为研究RgGGPPS2基因在地黄萜类化合物生物合成途径中的功能奠定了基础。