In this work we present the preparation and functionalization of gold nanoparticles (AuNPs) for the detection of presence of gp63 glycoprotein in the surface of Leishmania genus parasites. AuNPs were prepared by induc...In this work we present the preparation and functionalization of gold nanoparticles (AuNPs) for the detection of presence of gp63 glycoprotein in the surface of Leishmania genus parasites. AuNPs were prepared by induced laser ablation in a clean and biologically suitable media. The nanoparticles were functionalized with anti-gp63 lgG antibody in order to study the interaction with the glycoprotein component gp63 (63 kDa) present on the membrane surface of Leishmania genus parasites. The functionalized AuNPs showed potential as a spectrometric indicator of the parasite existence, both by the detection of the presence of the gp63 in solution and through the specific interaction with the parasites in vitro. The specificity of the study opens a new line of research on the use of modified nanoparticles in the development of a fast and easy assay for Leishmaniosis diagnostics.展开更多
The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis. Inbred BALB/c mice were immunized subcutaneously twice at an interval of...The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis. Inbred BALB/c mice were immunized subcutaneously twice at an interval of three weeks with pcDNA3.1 (+) encoding T cell epitopes of gp63 and Hsp70 individually and in combination. Animals were challenged intracardially with 107 promastigotes ofLeishmania donovani 10 days post immunization and sacrificed 1,2 and 3 months post challenge. The immunized animals revealed a significant reduction (P 〈 0.05) in splenic and hepatic parasite burden as compared to the infected controls. Maximum reduction in parasite load (P 〈 0.05) was observed in animals treated with a combination ofpcDNA/gp63 and pcDNA/Hsp70. These animals also showed heightened DTH response, increased IgG2a, elevated Thl cytokines (IFN-γ and IL-2) and reduced IgG 1 and IL-10 levels. Thus, mice immunized with the cocktail vaccine exhibited significantly greater protection in comparison to those immunized with individual antigens.展开更多
本文报道应用从国外引进的由硕大利什曼原虫 DNA克隆的原虫细胞表面糖蛋白 GP6 3基因与 p Bluescript M13质粒构建的重组质粒 p AS2 6转化大肠菌 XL1- Blue进行生产性表达及免疫学鉴定的结果。每 2 5 0 m L L B培养物可获取融合蛋白包涵...本文报道应用从国外引进的由硕大利什曼原虫 DNA克隆的原虫细胞表面糖蛋白 GP6 3基因与 p Bluescript M13质粒构建的重组质粒 p AS2 6转化大肠菌 XL1- Blue进行生产性表达及免疫学鉴定的结果。每 2 5 0 m L L B培养物可获取融合蛋白包涵体 5 3mg。表达产物在降解条件下与β半乳糖苷酶分离后的分子量为 5 5 k D。重组 GP6 3及其免疫家兔血清与细菌来源的β半乳糖苷酶有低滴度交叉。与婴儿利什曼原虫 90 1株前鞭毛体及其免疫血清和黑热病人血清 EL ISA反应滴度很高。表明重组 GP6展开更多
Using pseudorabies virus Ea strain as material,we inserted LacZ gene expression cassette into gE gene.After blue plaque and plaque purification,a recombinant virus PRVEa TK+-/gE+-/LacZ++ generated.Utilizing EcoR I sit...Using pseudorabies virus Ea strain as material,we inserted LacZ gene expression cassette into gE gene.After blue plaque and plaque purification,a recombinant virus PRVEa TK+-/gE+-/LacZ++ generated.Utilizing EcoR I site in LacZ gene, digested PRVEa TK+-/gE+-/LacZ++ genome DNA was cotransfected into PK-15 cells with plasmid pFBBS,then PRVEa TK+-/gE+-/gp63+- generated after plaque purification.Four pairs of primers amplification demonstrated the virus was pure TK+-/gE+-/gp63+- mutant virus.PCR product sequence indicates there were 205bp deletion in TK gene;1247bp deletion in gE,gp63 and intergenic region of PRVEa TK+-/gE+-/gp63+- mutant virus genome DNA.Inoculation to Balb/C mice with PRVEa TK+-/gE+-/gp63+- indicates the virulence is reduced greatly.展开更多
目的选取婴儿利什曼原虫的Pepck和Gp63的优势表位基因,构建Pepck-Gp63二联优势表位的原核与真核重组表达载体并表达蛋白。方法用计算机分析预测磷酸烯醇丙酮酸羧基酶(phosphoenolpyruvate carboxylase,PEPCK)的二级结构及HLA表位,筛选...目的选取婴儿利什曼原虫的Pepck和Gp63的优势表位基因,构建Pepck-Gp63二联优势表位的原核与真核重组表达载体并表达蛋白。方法用计算机分析预测磷酸烯醇丙酮酸羧基酶(phosphoenolpyruvate carboxylase,PEPCK)的二级结构及HLA表位,筛选出优势表位。根据本实验室之前对表面蛋白酶63(glycoprotein of 63×10^(3),GP63)用相同的方法分析而筛选的表位结果,把Pepck和Gp63的优势表位基因通过重叠PCR和酶切连接反应构建出重组原核表达质粒pET32a-Pepck-Gp63,并诱导其在大肠杆菌中表达;构建出重组真核表达质粒pVAX1-Pepck-Gp63,用脂质体转染法把真核质粒转染到NIH3T3细胞中表达。结果Pepck存在多个优势表位;测序结果表明二联优势表位基因Pepck-Gp63连接成功;PEPCK-GP63蛋白在大肠杆菌中以包涵体形式表达并在SDS-PAGE电泳-考马斯亮蓝染色的结果中呈现相对分子质量为74×10^(3)大小的条带;细胞免疫荧光中被pVAX1-Pepck-Gp63转染的NIH3T3细胞呈阳性。结论成功构建重组原核表达质粒pET32a-Pepck-Gp63和重组真核表达质粒pVAX1-Pepck-Gp63,并分别在大肠杆菌和NIH3T3细胞中验证重组质粒能表达相应目的蛋白,为后续的免疫策略研究提供初步实验基础。展开更多
文摘In this work we present the preparation and functionalization of gold nanoparticles (AuNPs) for the detection of presence of gp63 glycoprotein in the surface of Leishmania genus parasites. AuNPs were prepared by induced laser ablation in a clean and biologically suitable media. The nanoparticles were functionalized with anti-gp63 lgG antibody in order to study the interaction with the glycoprotein component gp63 (63 kDa) present on the membrane surface of Leishmania genus parasites. The functionalized AuNPs showed potential as a spectrometric indicator of the parasite existence, both by the detection of the presence of the gp63 in solution and through the specific interaction with the parasites in vitro. The specificity of the study opens a new line of research on the use of modified nanoparticles in the development of a fast and easy assay for Leishmaniosis diagnostics.
基金support provided by the Indian Council of Medical ResearchDepartment of Health Research,India for providing financial support for this study under project ref.5/8-7(77)/2006-ECD-||
文摘The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis. Inbred BALB/c mice were immunized subcutaneously twice at an interval of three weeks with pcDNA3.1 (+) encoding T cell epitopes of gp63 and Hsp70 individually and in combination. Animals were challenged intracardially with 107 promastigotes ofLeishmania donovani 10 days post immunization and sacrificed 1,2 and 3 months post challenge. The immunized animals revealed a significant reduction (P 〈 0.05) in splenic and hepatic parasite burden as compared to the infected controls. Maximum reduction in parasite load (P 〈 0.05) was observed in animals treated with a combination ofpcDNA/gp63 and pcDNA/Hsp70. These animals also showed heightened DTH response, increased IgG2a, elevated Thl cytokines (IFN-γ and IL-2) and reduced IgG 1 and IL-10 levels. Thus, mice immunized with the cocktail vaccine exhibited significantly greater protection in comparison to those immunized with individual antigens.
文摘本文报道应用从国外引进的由硕大利什曼原虫 DNA克隆的原虫细胞表面糖蛋白 GP6 3基因与 p Bluescript M13质粒构建的重组质粒 p AS2 6转化大肠菌 XL1- Blue进行生产性表达及免疫学鉴定的结果。每 2 5 0 m L L B培养物可获取融合蛋白包涵体 5 3mg。表达产物在降解条件下与β半乳糖苷酶分离后的分子量为 5 5 k D。重组 GP6 3及其免疫家兔血清与细菌来源的β半乳糖苷酶有低滴度交叉。与婴儿利什曼原虫 90 1株前鞭毛体及其免疫血清和黑热病人血清 EL ISA反应滴度很高。表明重组 GP6
文摘Using pseudorabies virus Ea strain as material,we inserted LacZ gene expression cassette into gE gene.After blue plaque and plaque purification,a recombinant virus PRVEa TK+-/gE+-/LacZ++ generated.Utilizing EcoR I site in LacZ gene, digested PRVEa TK+-/gE+-/LacZ++ genome DNA was cotransfected into PK-15 cells with plasmid pFBBS,then PRVEa TK+-/gE+-/gp63+- generated after plaque purification.Four pairs of primers amplification demonstrated the virus was pure TK+-/gE+-/gp63+- mutant virus.PCR product sequence indicates there were 205bp deletion in TK gene;1247bp deletion in gE,gp63 and intergenic region of PRVEa TK+-/gE+-/gp63+- mutant virus genome DNA.Inoculation to Balb/C mice with PRVEa TK+-/gE+-/gp63+- indicates the virulence is reduced greatly.
文摘目的选取婴儿利什曼原虫的Pepck和Gp63的优势表位基因,构建Pepck-Gp63二联优势表位的原核与真核重组表达载体并表达蛋白。方法用计算机分析预测磷酸烯醇丙酮酸羧基酶(phosphoenolpyruvate carboxylase,PEPCK)的二级结构及HLA表位,筛选出优势表位。根据本实验室之前对表面蛋白酶63(glycoprotein of 63×10^(3),GP63)用相同的方法分析而筛选的表位结果,把Pepck和Gp63的优势表位基因通过重叠PCR和酶切连接反应构建出重组原核表达质粒pET32a-Pepck-Gp63,并诱导其在大肠杆菌中表达;构建出重组真核表达质粒pVAX1-Pepck-Gp63,用脂质体转染法把真核质粒转染到NIH3T3细胞中表达。结果Pepck存在多个优势表位;测序结果表明二联优势表位基因Pepck-Gp63连接成功;PEPCK-GP63蛋白在大肠杆菌中以包涵体形式表达并在SDS-PAGE电泳-考马斯亮蓝染色的结果中呈现相对分子质量为74×10^(3)大小的条带;细胞免疫荧光中被pVAX1-Pepck-Gp63转染的NIH3T3细胞呈阳性。结论成功构建重组原核表达质粒pET32a-Pepck-Gp63和重组真核表达质粒pVAX1-Pepck-Gp63,并分别在大肠杆菌和NIH3T3细胞中验证重组质粒能表达相应目的蛋白,为后续的免疫策略研究提供初步实验基础。