An HA-1-DC nucleic-acid vaccine was constructed to induce anti-leukemia effect after hematopoietic stem cell transplantation (HSCT). DCs were generated from HSCT donors in vitro, and its immunologic activity was assay...An HA-1-DC nucleic-acid vaccine was constructed to induce anti-leukemia effect after hematopoietic stem cell transplantation (HSCT). DCs were generated from HSCT donors in vitro, and its immunologic activity was assayed by using flow cytometry and mixed lymphocytes reaction. HA-1 gene was electroporated into the cultured DCs to construct a DC nucleic-acid vaccine. After transfection for 48 h, the expression of HA-1 protein could be detected by using Western blot. The DCs were cultured with syngenic lymphocytes to induce specific cytotoxic T lymphocytes (CTLs). The cytoxicity of the CTLs was detected by LDH assay. The results showed that The DCs derived from peripheral blood monocytes (PBMCs) expressed the phenotype of DCs, and were effective in stimulating proliferation of the allogenic lymphocytes. After electroporating for 48-h, HA-1 protein was detected by using Western blot. The cytotoxity of inducing CTLs was higher than the control group. It was suggested that minor histocompatibility antigen HA-1 could be considered as a target of immunotherapy against leukemia after HSCT.展开更多
Glutamate Dehydrogenase (GDH;EC 1.4.1.2) catalyzes the reversible amination of α-ketoglutarate to glutamate, and the polymerization of nucleoside triphosphate(s) to RNA. But the natural role of the reversible aminati...Glutamate Dehydrogenase (GDH;EC 1.4.1.2) catalyzes the reversible amination of α-ketoglutarate to glutamate, and the polymerization of nucleoside triphosphate(s) to RNA. But the natural role of the reversible amination reaction is the subject of an expanding conversation. The aim was to illuminate the natural role of GDH through its RNA synthetic activity. Stoichiometric combinations of mineral salts that targeted the GDH subunit compositions were applied to field-cultivated peanuts. GDH of seeds were made to synthesize RNA in the deamination and then in the amination direction. Free amino acids were analyzed by HPLC. Glutamate synthase (GOGAT) was assayed by photometry. Free amino acid yields in-creased from the control’s lowest (9.8 kg·ha–1) and amination-deamination ratio (0.05) through 12.0 - 23.0 kg·ha–1 in the K-, N+K+P+S-, Pi-, N+S-, S-treated peanuts with amination-deamination ratios between 0.6 and 10.0 until at the P+K-treated peanut which had the highest amino acid yield (52.4 kg·ha–1) and the highest amination-deamination ratio (61). The Km and Vmax values of GOGAT were within the normal range. Yields of free amino acids resulting from GDH aminating activity increased from <1.0 kg·ha–1 in the control, through 2.2 in the N+S-, 6.84 in the P+N-, 17.3 in the N-, to 42.6 kg·ha–1 in the P+K- treated peanut. These results show that the natural role of the GDH amination activity is to assimilate escalating multiples of the quantities of NH4+ ion as assimilated via the GS-GOGAT pathway. Peanut protein yields increased in parallel with GDH aminating activities and free amino acid yields such that the control peanut had the lowest protein (<26.0 kg·ha–1) and the yields increased exponentially (500 - 600 kg·ha–1) through the K-, P+S-, Pi-, N-treated to 910 kg·ha–1 in the P+K-treated peanut with the highest aminating activity of GDH. The ability of GDH aminating activity to escalate protein yields of food crops could be employed to address proteinenergy malnutrition syndrome of developi展开更多
Tolpyralate is a benzoylpyrazole, 4-hydroxyphenyl-pyruvate dioxygenase inhibitor, and a herbicide registered for use in corn. The efficacy of tolpyralate plus atrazine to provide full-season residual control of glypho...Tolpyralate is a benzoylpyrazole, 4-hydroxyphenyl-pyruvate dioxygenase inhibitor, and a herbicide registered for use in corn. The efficacy of tolpyralate plus atrazine to provide full-season residual control of glyphosate-resistant (GR) Canada fleabane in corn is not known under Ontario environmental conditions. Five field trials were completed over a two-year period (2018-19) in south-western Ontario on farms with confirmed GR Canada fleabane [Conyza canadensis (L.) Cronq.] populations to determine if tolpyralate + atrazine provides full-season residual control of GR Canada fleabane in corn. Corn injury was less than 10% with all treatments. At 4 weeks after application (WAA), tolpyralate (30 g·ai·ha−1), tolpyralate (40 g·ai·ha−1), and atrazine (560 g·ai·ha−1) controlled GR Canada fleabane 64, 78 and 72%, respectively. A tank mix of tolpyralate + atrazine at both rates improved GR Canada fleabane control to 94%. Saflufenacil/dimethenamid-p, mesotrione + atrazine, and dicamba/atrazine controlled GR Canada fleabane 99, 95 and 92%, respectively. At 8 WAA, tolpyralate (30 g·ai·ha−1), tolpyralate (40 g·ai·ha−1) and atrazine (560 g·ai·ha−1) controlled GR Canada fleabane 83, 88, and 83%, respectively (Table 2). The tank mixes of tolpyralate (30 g·ai·ha−1) + atrazine (560 g·ai·ha−1) and tolpyralate (40 g·ai·ha−1) + atrazine (560 g·ai·ha−1) controlled GR Canada fleabane 94, and 97%, respectively, 8 WAA which was similar to saflufenacil/dimethenamid-p, mesotrione + atrazine and dicamba/atrazine. There was no treatment difference for corn yield. Based on these results, tolpyralate (40 g·ha−1), tolpyralate (30 g·ha−1) + atrazine and tolpyralate (40 g·ha−1) + atrazine, applied PRE, provided similar control of GR Canada fleabane as current industry standards at 8 WAA.展开更多
目的原核表达流感病毒(influenza virus,IV)球状头部结构域HA1基因,优化其诱导表达条件,并进行蛋白纯化,以期获得具有良好免疫原性的IV HA1蛋白。方法将Influenza A virus[A/Saratov/CRIE-250/2017(H3N2)]、Influenza A virus[A/Hebei/F...目的原核表达流感病毒(influenza virus,IV)球状头部结构域HA1基因,优化其诱导表达条件,并进行蛋白纯化,以期获得具有良好免疫原性的IV HA1蛋白。方法将Influenza A virus[A/Saratov/CRIE-250/2017(H3N2)]、Influenza A virus[A/Hebei/F076/2018(mixed)]和Influenza A virus[A/USA/LAN_(P5)_HA/2018(H1N1)]进行截短,取63~286头状结构域氨基酸序列,按照大肠埃希菌常用密码子进行优化,合成基因命名为17Sa、Hebei和USA,并对基因17Sa进行点突变命名为基因17Sa变。将4个基因分别克隆至原核表达载体pET-28a(+),构建重组质粒,将重组质粒转化至E.coli BL21(DE3)感受态细胞,IPTG诱导表达,优化蛋白表达条件,并利用His标签镍离子蛋白纯化柱纯化蛋白。结果成功插入长度为762 bp的目的基因,重组质粒经双酶切(NheⅠ/XhoⅠ)鉴定构建正确。表达的重组蛋白USA相对分子质量约为26000,17Sa、p17Sa和Hebei相对分子质量约为28000,均可与鼠抗His抗体特异性结合。重组蛋白均以包涵体形式表达,在诱导温度37℃,诱导8 h表达量最高。纯化的重组蛋白17Sa、17Sa变、Hebei和USA纯度分别为90%、85%、95%和80%。结论通过原核表达系统成功表达并纯化了目的蛋白,该蛋白具有良好的反应原性,为流感抗体检测和新型疫苗的开发奠定了基础。展开更多
The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction wit...The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction with IPTG.The expressed HA protein was identified by SDS-PAGE and Western blotting which showed the protein to be 42kDa and was immunoreactive.The purified HA protein was used to establish the indirect ELIS A for detection of the antibodies,specifically against the H3 subtype of SIV.The assay has excellent specificity,sensitivity and reproducibility.When 96 serum samples,randomly collected from the field,were evaluated in parallel by this new ELISA using recombinant HA1 and a routine HI test,the coincidental rate between the two tests was 86.5%.These results show that the recombinant HAl-based ELISA is specific,sensitive and easy to perform for the serological diagnosis of SIV infection.展开更多
文摘An HA-1-DC nucleic-acid vaccine was constructed to induce anti-leukemia effect after hematopoietic stem cell transplantation (HSCT). DCs were generated from HSCT donors in vitro, and its immunologic activity was assayed by using flow cytometry and mixed lymphocytes reaction. HA-1 gene was electroporated into the cultured DCs to construct a DC nucleic-acid vaccine. After transfection for 48 h, the expression of HA-1 protein could be detected by using Western blot. The DCs were cultured with syngenic lymphocytes to induce specific cytotoxic T lymphocytes (CTLs). The cytoxicity of the CTLs was detected by LDH assay. The results showed that The DCs derived from peripheral blood monocytes (PBMCs) expressed the phenotype of DCs, and were effective in stimulating proliferation of the allogenic lymphocytes. After electroporating for 48-h, HA-1 protein was detected by using Western blot. The cytotoxity of inducing CTLs was higher than the control group. It was suggested that minor histocompatibility antigen HA-1 could be considered as a target of immunotherapy against leukemia after HSCT.
文摘Glutamate Dehydrogenase (GDH;EC 1.4.1.2) catalyzes the reversible amination of α-ketoglutarate to glutamate, and the polymerization of nucleoside triphosphate(s) to RNA. But the natural role of the reversible amination reaction is the subject of an expanding conversation. The aim was to illuminate the natural role of GDH through its RNA synthetic activity. Stoichiometric combinations of mineral salts that targeted the GDH subunit compositions were applied to field-cultivated peanuts. GDH of seeds were made to synthesize RNA in the deamination and then in the amination direction. Free amino acids were analyzed by HPLC. Glutamate synthase (GOGAT) was assayed by photometry. Free amino acid yields in-creased from the control’s lowest (9.8 kg·ha–1) and amination-deamination ratio (0.05) through 12.0 - 23.0 kg·ha–1 in the K-, N+K+P+S-, Pi-, N+S-, S-treated peanuts with amination-deamination ratios between 0.6 and 10.0 until at the P+K-treated peanut which had the highest amino acid yield (52.4 kg·ha–1) and the highest amination-deamination ratio (61). The Km and Vmax values of GOGAT were within the normal range. Yields of free amino acids resulting from GDH aminating activity increased from <1.0 kg·ha–1 in the control, through 2.2 in the N+S-, 6.84 in the P+N-, 17.3 in the N-, to 42.6 kg·ha–1 in the P+K- treated peanut. These results show that the natural role of the GDH amination activity is to assimilate escalating multiples of the quantities of NH4+ ion as assimilated via the GS-GOGAT pathway. Peanut protein yields increased in parallel with GDH aminating activities and free amino acid yields such that the control peanut had the lowest protein (<26.0 kg·ha–1) and the yields increased exponentially (500 - 600 kg·ha–1) through the K-, P+S-, Pi-, N-treated to 910 kg·ha–1 in the P+K-treated peanut with the highest aminating activity of GDH. The ability of GDH aminating activity to escalate protein yields of food crops could be employed to address proteinenergy malnutrition syndrome of developi
文摘Tolpyralate is a benzoylpyrazole, 4-hydroxyphenyl-pyruvate dioxygenase inhibitor, and a herbicide registered for use in corn. The efficacy of tolpyralate plus atrazine to provide full-season residual control of glyphosate-resistant (GR) Canada fleabane in corn is not known under Ontario environmental conditions. Five field trials were completed over a two-year period (2018-19) in south-western Ontario on farms with confirmed GR Canada fleabane [Conyza canadensis (L.) Cronq.] populations to determine if tolpyralate + atrazine provides full-season residual control of GR Canada fleabane in corn. Corn injury was less than 10% with all treatments. At 4 weeks after application (WAA), tolpyralate (30 g·ai·ha−1), tolpyralate (40 g·ai·ha−1), and atrazine (560 g·ai·ha−1) controlled GR Canada fleabane 64, 78 and 72%, respectively. A tank mix of tolpyralate + atrazine at both rates improved GR Canada fleabane control to 94%. Saflufenacil/dimethenamid-p, mesotrione + atrazine, and dicamba/atrazine controlled GR Canada fleabane 99, 95 and 92%, respectively. At 8 WAA, tolpyralate (30 g·ai·ha−1), tolpyralate (40 g·ai·ha−1) and atrazine (560 g·ai·ha−1) controlled GR Canada fleabane 83, 88, and 83%, respectively (Table 2). The tank mixes of tolpyralate (30 g·ai·ha−1) + atrazine (560 g·ai·ha−1) and tolpyralate (40 g·ai·ha−1) + atrazine (560 g·ai·ha−1) controlled GR Canada fleabane 94, and 97%, respectively, 8 WAA which was similar to saflufenacil/dimethenamid-p, mesotrione + atrazine and dicamba/atrazine. There was no treatment difference for corn yield. Based on these results, tolpyralate (40 g·ha−1), tolpyralate (30 g·ha−1) + atrazine and tolpyralate (40 g·ha−1) + atrazine, applied PRE, provided similar control of GR Canada fleabane as current industry standards at 8 WAA.
文摘目的原核表达流感病毒(influenza virus,IV)球状头部结构域HA1基因,优化其诱导表达条件,并进行蛋白纯化,以期获得具有良好免疫原性的IV HA1蛋白。方法将Influenza A virus[A/Saratov/CRIE-250/2017(H3N2)]、Influenza A virus[A/Hebei/F076/2018(mixed)]和Influenza A virus[A/USA/LAN_(P5)_HA/2018(H1N1)]进行截短,取63~286头状结构域氨基酸序列,按照大肠埃希菌常用密码子进行优化,合成基因命名为17Sa、Hebei和USA,并对基因17Sa进行点突变命名为基因17Sa变。将4个基因分别克隆至原核表达载体pET-28a(+),构建重组质粒,将重组质粒转化至E.coli BL21(DE3)感受态细胞,IPTG诱导表达,优化蛋白表达条件,并利用His标签镍离子蛋白纯化柱纯化蛋白。结果成功插入长度为762 bp的目的基因,重组质粒经双酶切(NheⅠ/XhoⅠ)鉴定构建正确。表达的重组蛋白USA相对分子质量约为26000,17Sa、p17Sa和Hebei相对分子质量约为28000,均可与鼠抗His抗体特异性结合。重组蛋白均以包涵体形式表达,在诱导温度37℃,诱导8 h表达量最高。纯化的重组蛋白17Sa、17Sa变、Hebei和USA纯度分别为90%、85%、95%和80%。结论通过原核表达系统成功表达并纯化了目的蛋白,该蛋白具有良好的反应原性,为流感抗体检测和新型疫苗的开发奠定了基础。
基金supported by the Chinese National S&T Plan(2004BA519A55)
文摘The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction with IPTG.The expressed HA protein was identified by SDS-PAGE and Western blotting which showed the protein to be 42kDa and was immunoreactive.The purified HA protein was used to establish the indirect ELIS A for detection of the antibodies,specifically against the H3 subtype of SIV.The assay has excellent specificity,sensitivity and reproducibility.When 96 serum samples,randomly collected from the field,were evaluated in parallel by this new ELISA using recombinant HA1 and a routine HI test,the coincidental rate between the two tests was 86.5%.These results show that the recombinant HAl-based ELISA is specific,sensitive and easy to perform for the serological diagnosis of SIV infection.
