目的 克隆腐食酪螨Tyr p 34基因,表达、纯化出重组蛋白,并鉴定其免疫原性。方法 提取腐食酪螨的总RNA,通过反转录聚合酶链式反应获得其cDNA片段。根据引物和cDNA进行PCR扩增,通过酶切连接构建表达载体pET-28a(+)-Tyr p 34,并将鉴定为阳...目的 克隆腐食酪螨Tyr p 34基因,表达、纯化出重组蛋白,并鉴定其免疫原性。方法 提取腐食酪螨的总RNA,通过反转录聚合酶链式反应获得其cDNA片段。根据引物和cDNA进行PCR扩增,通过酶切连接构建表达载体pET-28a(+)-Tyr p 34,并将鉴定为阳性的重组表达质粒转入感受态大肠杆菌中。分别在高温及低温条件下,少量诱导表达重组蛋白Tyr p 34以进行蛋白鉴定;大量诱导表达该重组蛋白并采用亲和层析法纯化,将纯化后的目的蛋白用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行检测并进行免疫印迹分析(Western blot)及生物信息学分析。结果 双酶切结果显示Tyr p 34基因与载体成功连接。少量表达后进行蛋白鉴定,显示目的基因在大肠杆菌中成功表达,Tyr p 34蛋白主要以可溶性蛋白的形式存在,在高温组中含量相对较多。大量表达后纯化的重组Tyr p 34蛋白分子量约为20 kDa。Western blot显示有明显条带,表明该重组基因所表达的蛋白可与腐食酪螨过敏患者的血清反应。生物信息学分析结果显示Tyr p 34基因大小为462 bp,编码153个氨基酸。氨基酸序列与NCBI公布的Tyr p 34氨基酸序列(登录号:ACL36923.1)同源性为99%。Tyr p 34蛋白有5个磷酸化位点,主要由无规则卷曲和α-螺旋构成。结论腐食酪螨Tyr p 34基因成功克隆并表达、纯化出重组蛋白,该蛋白具有免疫原性。通过Tyr p 34的生物信息学分析,有利于深入研究腐食酪螨过敏原的结构和理化性质。展开更多
AIM: To investigate the influence of the CagA diversity in Helicobacter pylori (H. pylori ) strains from Colombia on the host cell biology. METHODS: Eighty-four H. pylori-cagA positive strains with different Glu-Pro-I...AIM: To investigate the influence of the CagA diversity in Helicobacter pylori (H. pylori ) strains from Colombia on the host cell biology. METHODS: Eighty-four H. pylori-cagA positive strains with different Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs patterns, isolated from patients with gastritis (n=17), atrophic gastritis (n=17), duodenal ulcer (n=16), intestinal metaplasia (n=16) and gastric cancer (n=18), were included. To determine the integrity of the cag pathogenicity island (cag PAI) we evaluated the presence of cagA, cagT, cagE, and cag10 genes by polymerase chain reaction. AGS gastric epithelial cellswere infected with each strain and assayed for translo-cation and tyrosine phosphorylation of CagA by western blot, secretion of interleukin-8 (IL-8) by enzyme-linked immuno sorbent assay after taking supernatants from cocultures and cell elongation induction. For cell elongation quantification, coculture photographs were taken and the proportion of "hummingbird" cells (>15 μm) was determined. RESULTS: Overall 72% (60/84) of the strains were found to harbor a functional cag PAI. Levels of phos-phorylated CagA were significantly higher for isolates from duodenal ulcer than the ones in strains from gas-tritis, atrophic gastritis, intestinal metaplasia and gastric cancer (49.1% ± 23.1% vs 21.1% ± 19.5%, P < 0.02; 49.1% ± 23.1% vs 26.2%±14.8%, P<0.045; 49.1% ± 23.1% vs 21.5% ± 19.5%, P<0.043 and 49.1% ± 23.1% vs 29.5% ± 27.1%, P < 0.047 respectively). We observed variable IL-8 expression levels ranging from 0 to 810 pg/mL and from 8.8 to 1442 pg/mL at 6 h and 30 h post-infection, respectively. cagPAI-defective strains did not induce detectable levels of IL-8 at 6 h post-infection. At 30 h post-infection all strains induced IL-8 expression in AGS cells, although cagPAI-defective strains induced significantly lower levels of IL-8 than strains with a functional cagPAI (57.1 ± 56.6 pg/mL vs 513.6 ± 338.6 pg/mL,P < 0.0001). We did not observe differences in the extent of cell elongation induction between strains with a functional or a defective cagPAI in 6 h cocultures. At 24 h post infection strains with functionalcagPAI showed high diversity in the extent of hummingbird phenotype induction ranging from 7% to 34%. cag PAI defective strains induced significantly lower levels of elongation than strains with functional cag-PAI with one or more than one EPIYA-C motif (15.1% ± 5.2%vs 18.9% ± 4.7%,P < 0.03; and 15.1% ± 5.2% vs 20.0% ± 5.1%, P < 0.003 respectively). No differences were observed in cellular elongation inductionor IL-8 expression among H. pylori strains bearing one and more than one EPIYA-C motifs, neither at 6 h nor at 24 h of coculture. There were no associations between the levels of induction of cell elongation or IL-8 expression and number of EPIYA motifs or pathology. CONCLUSION: The present work describes a lack of association between H. pylori CagA protein EPIYA motifs variations from Colombian isolates and disease-associated cellular responses.展开更多
The aim of the present work was to investigate the effects of osmoconditioning on chilling injury in chilling-sensitive soybean （Glycine max （L.） Merr. Zhonghuang No. 22） seeds during imbibition. Low temperatures ...The aim of the present work was to investigate the effects of osmoconditioning on chilling injury in chilling-sensitive soybean （Glycine max （L.） Merr. Zhonghuang No. 22） seeds during imbibition. Low temperatures reduced the germination rate and no seed germinated at 1 ℃. Osmoconditioning of seeds at 20℃ with a polyethylene glycol-8000 （PEG8000） solution at 1.5 MPa for 72 h followed by drying back to their initial moisture content （MC） reduced their chilling sensitivity. The phenylarsine oxide （PAO）, an inhibitor of protein tyrosinephosphatases, was used to investigate the possible involvement of phosphorylation-dephosphorylation of Tyr residues in the plasma membrane composition and function when seeds were osmoconditioned. The results showed the germination of osmoconditioned seeds decreased significantly when PAO was added in PEG solution after chilling treatment. PAO inhibited changes in composition of plasma membrane phospholipids and fatty acid induced by osmocondition, indicated that tyrosine protein phosphorylation is involved in the regulatory mechanisms of osmocondition-responsive chilling in soybean seeds. Western blot result further indicated that osmocondition treatment improved the activity of plasma membrane H^＋-ATPase after chilling treatment, but this effect was abolished by PAO. The possible regulation mechanism by Tyr protein phosphorylation is discussed.展开更多
目的克隆腐食酪螨第13组变应原基因(Tyr p 13),表达纯化其重组蛋白,并进行免疫学特性鉴定,利用生物信息学软件分析该过敏原抗原表位等信息。方法挑取腐食酪螨,提取螨总RNA。逆转录-聚合酶链式反应(RTPCR)扩增cDNA,根据已知的Tyr p 13基...目的克隆腐食酪螨第13组变应原基因(Tyr p 13),表达纯化其重组蛋白,并进行免疫学特性鉴定,利用生物信息学软件分析该过敏原抗原表位等信息。方法挑取腐食酪螨,提取螨总RNA。逆转录-聚合酶链式反应(RTPCR)扩增cDNA,根据已知的Tyr p 13基因序列(GeneBank登录号AY710432.1)设计引物,PCR大量扩增目的基因。构建原核表达载体pET-32a(+)-Tyr p 13,转化感受态细胞E. coli Rosetta(DE3),用IPTG(异丙基-β-D-硫代半乳糖苷)诱导目的蛋白表达;层析纯化表达产物,免疫印迹(Western Blot)法检测纯化后的表达产物免疫学活性;通过生物信息学软件推测其抗原表位、构建进化树。结果克隆得到的序列经基因测序可知其长度约400 bp,其表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分子量约为14 kD,呈可溶性表达。免疫印迹结果显示,Tyr p 13能够与过敏患者血清IgE特异性结合,表明其具有过敏原性;表位分析进一步证实该过敏原具有免疫原性。结论经克隆表达、纯化后得到纯度较高的腐食酪螨Tyr p 13,为进一步开展临床特异性诊断和治疗提供参考。展开更多
文摘目的 克隆腐食酪螨Tyr p 34基因,表达、纯化出重组蛋白,并鉴定其免疫原性。方法 提取腐食酪螨的总RNA,通过反转录聚合酶链式反应获得其cDNA片段。根据引物和cDNA进行PCR扩增,通过酶切连接构建表达载体pET-28a(+)-Tyr p 34,并将鉴定为阳性的重组表达质粒转入感受态大肠杆菌中。分别在高温及低温条件下,少量诱导表达重组蛋白Tyr p 34以进行蛋白鉴定;大量诱导表达该重组蛋白并采用亲和层析法纯化,将纯化后的目的蛋白用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行检测并进行免疫印迹分析(Western blot)及生物信息学分析。结果 双酶切结果显示Tyr p 34基因与载体成功连接。少量表达后进行蛋白鉴定,显示目的基因在大肠杆菌中成功表达,Tyr p 34蛋白主要以可溶性蛋白的形式存在,在高温组中含量相对较多。大量表达后纯化的重组Tyr p 34蛋白分子量约为20 kDa。Western blot显示有明显条带,表明该重组基因所表达的蛋白可与腐食酪螨过敏患者的血清反应。生物信息学分析结果显示Tyr p 34基因大小为462 bp,编码153个氨基酸。氨基酸序列与NCBI公布的Tyr p 34氨基酸序列(登录号:ACL36923.1)同源性为99%。Tyr p 34蛋白有5个磷酸化位点,主要由无规则卷曲和α-螺旋构成。结论腐食酪螨Tyr p 34基因成功克隆并表达、纯化出重组蛋白,该蛋白具有免疫原性。通过Tyr p 34的生物信息学分析,有利于深入研究腐食酪螨过敏原的结构和理化性质。
基金Supported by National Cancer Institute, Bogotá, Colombia,Grant No. 41030310 to Bravo MM and Sciences Faculty, LosAndes University, Bogotá, Colombia
文摘AIM: To investigate the influence of the CagA diversity in Helicobacter pylori (H. pylori ) strains from Colombia on the host cell biology. METHODS: Eighty-four H. pylori-cagA positive strains with different Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs patterns, isolated from patients with gastritis (n=17), atrophic gastritis (n=17), duodenal ulcer (n=16), intestinal metaplasia (n=16) and gastric cancer (n=18), were included. To determine the integrity of the cag pathogenicity island (cag PAI) we evaluated the presence of cagA, cagT, cagE, and cag10 genes by polymerase chain reaction. AGS gastric epithelial cellswere infected with each strain and assayed for translo-cation and tyrosine phosphorylation of CagA by western blot, secretion of interleukin-8 (IL-8) by enzyme-linked immuno sorbent assay after taking supernatants from cocultures and cell elongation induction. For cell elongation quantification, coculture photographs were taken and the proportion of "hummingbird" cells (>15 μm) was determined. RESULTS: Overall 72% (60/84) of the strains were found to harbor a functional cag PAI. Levels of phos-phorylated CagA were significantly higher for isolates from duodenal ulcer than the ones in strains from gas-tritis, atrophic gastritis, intestinal metaplasia and gastric cancer (49.1% ± 23.1% vs 21.1% ± 19.5%, P < 0.02; 49.1% ± 23.1% vs 26.2%±14.8%, P<0.045; 49.1% ± 23.1% vs 21.5% ± 19.5%, P<0.043 and 49.1% ± 23.1% vs 29.5% ± 27.1%, P < 0.047 respectively). We observed variable IL-8 expression levels ranging from 0 to 810 pg/mL and from 8.8 to 1442 pg/mL at 6 h and 30 h post-infection, respectively. cagPAI-defective strains did not induce detectable levels of IL-8 at 6 h post-infection. At 30 h post-infection all strains induced IL-8 expression in AGS cells, although cagPAI-defective strains induced significantly lower levels of IL-8 than strains with a functional cagPAI (57.1 ± 56.6 pg/mL vs 513.6 ± 338.6 pg/mL,P < 0.0001). We did not observe differences in the extent of cell elongation induction between strains with a functional or a defective cagPAI in 6 h cocultures. At 24 h post infection strains with functionalcagPAI showed high diversity in the extent of hummingbird phenotype induction ranging from 7% to 34%. cag PAI defective strains induced significantly lower levels of elongation than strains with functional cag-PAI with one or more than one EPIYA-C motif (15.1% ± 5.2%vs 18.9% ± 4.7%,P < 0.03; and 15.1% ± 5.2% vs 20.0% ± 5.1%, P < 0.003 respectively). No differences were observed in cellular elongation inductionor IL-8 expression among H. pylori strains bearing one and more than one EPIYA-C motifs, neither at 6 h nor at 24 h of coculture. There were no associations between the levels of induction of cell elongation or IL-8 expression and number of EPIYA motifs or pathology. CONCLUSION: The present work describes a lack of association between H. pylori CagA protein EPIYA motifs variations from Colombian isolates and disease-associated cellular responses.
基金Supported by the National Natural Science Foundation of China (30570178)
文摘The aim of the present work was to investigate the effects of osmoconditioning on chilling injury in chilling-sensitive soybean （Glycine max （L.） Merr. Zhonghuang No. 22） seeds during imbibition. Low temperatures reduced the germination rate and no seed germinated at 1 ℃. Osmoconditioning of seeds at 20℃ with a polyethylene glycol-8000 （PEG8000） solution at 1.5 MPa for 72 h followed by drying back to their initial moisture content （MC） reduced their chilling sensitivity. The phenylarsine oxide （PAO）, an inhibitor of protein tyrosinephosphatases, was used to investigate the possible involvement of phosphorylation-dephosphorylation of Tyr residues in the plasma membrane composition and function when seeds were osmoconditioned. The results showed the germination of osmoconditioned seeds decreased significantly when PAO was added in PEG solution after chilling treatment. PAO inhibited changes in composition of plasma membrane phospholipids and fatty acid induced by osmocondition, indicated that tyrosine protein phosphorylation is involved in the regulatory mechanisms of osmocondition-responsive chilling in soybean seeds. Western blot result further indicated that osmocondition treatment improved the activity of plasma membrane H^＋-ATPase after chilling treatment, but this effect was abolished by PAO. The possible regulation mechanism by Tyr protein phosphorylation is discussed.
文摘目的克隆腐食酪螨第13组变应原基因(Tyr p 13),表达纯化其重组蛋白,并进行免疫学特性鉴定,利用生物信息学软件分析该过敏原抗原表位等信息。方法挑取腐食酪螨,提取螨总RNA。逆转录-聚合酶链式反应(RTPCR)扩增cDNA,根据已知的Tyr p 13基因序列(GeneBank登录号AY710432.1)设计引物,PCR大量扩增目的基因。构建原核表达载体pET-32a(+)-Tyr p 13,转化感受态细胞E. coli Rosetta(DE3),用IPTG(异丙基-β-D-硫代半乳糖苷)诱导目的蛋白表达;层析纯化表达产物,免疫印迹(Western Blot)法检测纯化后的表达产物免疫学活性;通过生物信息学软件推测其抗原表位、构建进化树。结果克隆得到的序列经基因测序可知其长度约400 bp,其表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分子量约为14 kD,呈可溶性表达。免疫印迹结果显示,Tyr p 13能够与过敏患者血清IgE特异性结合,表明其具有过敏原性;表位分析进一步证实该过敏原具有免疫原性。结论经克隆表达、纯化后得到纯度较高的腐食酪螨Tyr p 13,为进一步开展临床特异性诊断和治疗提供参考。
