[Objective] The aim was to study the prokaryotic expression of P1 gene of foot-and-mouth disease virus（FMDV）type Asia 1and the preparation of its antiserum.[Method]The P1 gene of FMDV type Asia 1 was obtained by gen...[Objective] The aim was to study the prokaryotic expression of P1 gene of foot-and-mouth disease virus（FMDV）type Asia 1and the preparation of its antiserum.[Method]The P1 gene of FMDV type Asia 1 was obtained by gene cloning techniques,and then cloned into pET-32a（＋）plasmid;subsequently the recombinant plasmid was transformed into E.coli BL21（DE3）;after the IPTG induction and protein purification,SDS-PAGE analysis was carried out;the ultrasonic wave was use to lyse the cultivated recombinant strain,and after the isolation and purification,this fusion protein was utilized to immunize New Zealand rabbits so as to prepare P1 protein antiserum.[Result]The positive clones were obtained;SDS-PAGE result showed that the target band was appeared at 105 kD;Western blot analysis showed that the antisera could bind to the expressed P1 fusion protein specifically;the ELISA titer of the rabbit anti-FMDV-P1 sera was approximately 1∶5 120.[Conclusion]This study had provided foundations for FMDV serological diagnostic methods and genetically engineered vaccine.展开更多
Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse tr...Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHI and XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI, then the NOR1 gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1 was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analyzed with immunoblot. Results: The 1.25 kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography. The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1 is obtained in JM 105, and its antiserum can be prepared successfully.展开更多
A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading...A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET-21a(+) and over-expressed inE. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×105. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma. Key words saffron - carotenoids - phytoene desaturase - gene expression - antiserum - Western blot CLC number Q 781 - Q 786 Foundation item: Supported by the Doctoral Foundation of the Ministry of Education, P. R. China and the Young Science Foundation of Sichuan University (Grant 0020405505012)Biography: Bai Jie (1968-), female, Ph. D candidate, research direction: plant developmental biology and reproductive engineering.展开更多
Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene produ...Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. coli and purified by nickel-nitrilotriacetic acid (Ni 2+ -NTA) affinity resin column, finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.展开更多
The rapid epidemic of highly pathogenic A/H5N1 avian influenza virus by transmission from poultry to humans triggered global unrest in the pandemic of novel influenza. If a human trophic strain of avian influenza viru...The rapid epidemic of highly pathogenic A/H5N1 avian influenza virus by transmission from poultry to humans triggered global unrest in the pandemic of novel influenza. If a human trophic strain of avian influenza viruses replicates in livestock including pigs and chickens, it may have high infectivity and pathogenicity to humans. The most effective method of reducing the outbreaks of influenza would be prophylaxis with an effective vaccine as well as anti-viral drugs including Oseltamivir and Zanamivir hydrate. In this study, chicken antiserum against A/H5N1 virus was produced: the antisera from immunized adult chicken had a strong binding activity to A/H5N1 viral antigens by ELISA. Furthermore, the antiserum strongly inhibited hemaggregation of erythrocytes and cytopathic effects in MDCK cells, indicating a strong neutralization activity against A/H5N1 infections. Interestingly, the mortality rate of chicks inoculated with A/H5N1 virus was dramatically decreased with the antiserum injection. These results suggest that antiserum may be a potentially effective protective and therapeutic modality for A/H5N1 infection.展开更多
According to the data of banana transcriptome sequencing, an E3 ubiquitin-protein ligase gene was cloned by RT-PCR method using the cDNA sample of banana leaves. The complete ORF of E3 ubiquitin-protein ligase is 681 ...According to the data of banana transcriptome sequencing, an E3 ubiquitin-protein ligase gene was cloned by RT-PCR method using the cDNA sample of banana leaves. The complete ORF of E3 ubiquitin-protein ligase is 681 bp long and its encoded protein showed 100% sequence identity to homologue RING-H2 finger protein (XP_009407047.1) of Musa_acuminata. Bioinformatic analysis indicated that E3 ubiquitin-protein ligase contains the Ring finger domain in C terminus and eight cross-brace motifs are found in the domain. The target gene was digested by EcoR V and EcoR I, and was inserted into prokaryotic expression vector pET-32a of the same digestions to obtain the plasmid pET32a-E3 ubiquitin-protein ligase. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3), and induced at 25°C with 0.4 mmol/L IPTG for 6 hours. The soluble fusion protein was expressed and high purity fusion protein was obtained by Ni<sup>2+</sup>-NTA agarose affinity chromatography purification. The fusion protein was injected into mice 3 times to prepare the antiserum. Western blot analysis showed a specific protein band was detected in total protein sample of banana leaves, but not for the samples of wild-type Nicotiana benthamiana (N.B.) and wild-type Arabidopsis thaliana (A.T.), implying the antiserum was specific to banana E3 ubiquitin-protein ligase.展开更多
By reverse transcription-polymerase chain reaction (RT-PCR), cDNA fragment of wheat yellow mosaic vir黶 (WYMV) RNA2 encoding 72 ku protein has been synthesized and cloned into plasmid pET30a(+) for overexpression in p...By reverse transcription-polymerase chain reaction (RT-PCR), cDNA fragment of wheat yellow mosaic vir黶 (WYMV) RNA2 encoding 72 ku protein has been synthesized and cloned into plasmid pET30a(+) for overexpression in prokaryotic celis. BL21(DE3) pLys S of E.coli transformed with the recombinant plasmid pETP72 containing the fragment has been induced to express the 72 ku protein on high level. The produced protein has been purified from sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for its antiserum preparation. in western-blotting analysis, the antibodies reacted with the 72 ku protein expressed in E.coli.展开更多
花生过敏蛋白Ara h 3广泛存在于各类花生制品中,少量摄入即可引发过敏反应,严重威胁花生过敏患者的健康。精准地识别Ara h 3便于开展后续免疫学检测,因此,制备高纯度、高特异性的抗体显得尤为重要。以花生脱脂粉为原料,通过HiTrap Q HP...花生过敏蛋白Ara h 3广泛存在于各类花生制品中,少量摄入即可引发过敏反应,严重威胁花生过敏患者的健康。精准地识别Ara h 3便于开展后续免疫学检测,因此,制备高纯度、高特异性的抗体显得尤为重要。以花生脱脂粉为原料,通过HiTrap Q HP阴离子交换层析柱和HiLoad Superdex 200 pg制备级分子筛层析柱,制备高纯度的花生致敏蛋白Ara h 3,将其与CNBr-activated SepharoseTM 4B进行偶联,利用不同洗脱液对血清样品中的非特异性及特异性物质进行分离,制备高纯度、高特异性的抗Ara h 3多克隆抗体。结果表明:Gly-HCl洗脱的蛋白质量浓度为0.6 mg/mL;SDS-PAGE分析显示,多克隆抗体中的非特异性物质已被有效去除;免疫印迹和ELISA试验证明,纯化后的抗体仍具有较高的特异性,其效价达到1∶51200。上述结果表明抗体成功纯化,为花生过敏原Ara h 3的检测研究奠定了基础。展开更多
该研究采用高毒力持留基因型单增李斯特菌819-2菌株全菌蛋白免疫SPF级Balb/C小鼠制备抗血清,利用免疫蛋白质组学对菌株胞外蛋白质组进行分析,旨在挖掘筛选单增李斯特菌强免疫原性蛋白作为特异性抗体制备的候选抗原。超声法提取819-2菌...该研究采用高毒力持留基因型单增李斯特菌819-2菌株全菌蛋白免疫SPF级Balb/C小鼠制备抗血清,利用免疫蛋白质组学对菌株胞外蛋白质组进行分析,旨在挖掘筛选单增李斯特菌强免疫原性蛋白作为特异性抗体制备的候选抗原。超声法提取819-2菌株全菌蛋白免疫SPF级Balb/C小鼠制备抗血清,四次免疫后经间接ELISA测定效价达1:512000。脱氧胆酸钠(DOC)-10%(m/V)TCA沉淀法提取单增李斯特菌819-2菌株胞外蛋白,利用免疫蛋白质组学和LC-MS/MS技术挖掘并鉴定具有强免疫反应的蛋白点,结果表明双向电泳图谱成功获得85个蛋白点,并成功鉴定了P60、InlC、MltG、Enolase和假定蛋白YxeA family protein等5个强免疫原性蛋白,研究结果为基于强免疫原性蛋白制备特异性抗体用于食品及其加工环境中单增李斯特菌富集及快速检测技术研制提供了数据基础。展开更多
基金Supported by National Transgenic Major Program of China(2009ZX08007-006B)the National Natural Science Foundation of China(31072160)+2 种基金Science and Technique Foundation of Shandong Province(2009GG20002032)Natural Science Foundation ofShandong Province(Y2008D20)an Open Issue of State Key Laboratory of Veterinary Biotechnology Fund(SKLVBF200806)~~
文摘[Objective] The aim was to study the prokaryotic expression of P1 gene of foot-and-mouth disease virus（FMDV）type Asia 1and the preparation of its antiserum.[Method]The P1 gene of FMDV type Asia 1 was obtained by gene cloning techniques,and then cloned into pET-32a（＋）plasmid;subsequently the recombinant plasmid was transformed into E.coli BL21（DE3）;after the IPTG induction and protein purification,SDS-PAGE analysis was carried out;the ultrasonic wave was use to lyse the cultivated recombinant strain,and after the isolation and purification,this fusion protein was utilized to immunize New Zealand rabbits so as to prepare P1 protein antiserum.[Result]The positive clones were obtained;SDS-PAGE result showed that the target band was appeared at 105 kD;Western blot analysis showed that the antisera could bind to the expressed P1 fusion protein specifically;the ELISA titer of the rabbit anti-FMDV-P1 sera was approximately 1∶5 120.[Conclusion]This study had provided foundations for FMDV serological diagnostic methods and genetically engineered vaccine.
文摘Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHI and XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI, then the NOR1 gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1 was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analyzed with immunoblot. Results: The 1.25 kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography. The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1 is obtained in JM 105, and its antiserum can be prepared successfully.
文摘A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET-21a(+) and over-expressed inE. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×105. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma. Key words saffron - carotenoids - phytoene desaturase - gene expression - antiserum - Western blot CLC number Q 781 - Q 786 Foundation item: Supported by the Doctoral Foundation of the Ministry of Education, P. R. China and the Young Science Foundation of Sichuan University (Grant 0020405505012)Biography: Bai Jie (1968-), female, Ph. D candidate, research direction: plant developmental biology and reproductive engineering.
基金The startup fund of the hundred talents program of the Chinese academy of science(20071010- 141)National natural science foundation of China (30870120)+2 种基金Open research fund program of the state key laboratory of virology of China (2007003, 2009007)Hubei province natural science foundation of innovation groups project (2008CDA013)Major state basic research development program (973 Program) of China (2010CB 530105)
文摘Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. coli and purified by nickel-nitrilotriacetic acid (Ni 2+ -NTA) affinity resin column, finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.
文摘The rapid epidemic of highly pathogenic A/H5N1 avian influenza virus by transmission from poultry to humans triggered global unrest in the pandemic of novel influenza. If a human trophic strain of avian influenza viruses replicates in livestock including pigs and chickens, it may have high infectivity and pathogenicity to humans. The most effective method of reducing the outbreaks of influenza would be prophylaxis with an effective vaccine as well as anti-viral drugs including Oseltamivir and Zanamivir hydrate. In this study, chicken antiserum against A/H5N1 virus was produced: the antisera from immunized adult chicken had a strong binding activity to A/H5N1 viral antigens by ELISA. Furthermore, the antiserum strongly inhibited hemaggregation of erythrocytes and cytopathic effects in MDCK cells, indicating a strong neutralization activity against A/H5N1 infections. Interestingly, the mortality rate of chicks inoculated with A/H5N1 virus was dramatically decreased with the antiserum injection. These results suggest that antiserum may be a potentially effective protective and therapeutic modality for A/H5N1 infection.
文摘According to the data of banana transcriptome sequencing, an E3 ubiquitin-protein ligase gene was cloned by RT-PCR method using the cDNA sample of banana leaves. The complete ORF of E3 ubiquitin-protein ligase is 681 bp long and its encoded protein showed 100% sequence identity to homologue RING-H2 finger protein (XP_009407047.1) of Musa_acuminata. Bioinformatic analysis indicated that E3 ubiquitin-protein ligase contains the Ring finger domain in C terminus and eight cross-brace motifs are found in the domain. The target gene was digested by EcoR V and EcoR I, and was inserted into prokaryotic expression vector pET-32a of the same digestions to obtain the plasmid pET32a-E3 ubiquitin-protein ligase. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3), and induced at 25°C with 0.4 mmol/L IPTG for 6 hours. The soluble fusion protein was expressed and high purity fusion protein was obtained by Ni<sup>2+</sup>-NTA agarose affinity chromatography purification. The fusion protein was injected into mice 3 times to prepare the antiserum. Western blot analysis showed a specific protein band was detected in total protein sample of banana leaves, but not for the samples of wild-type Nicotiana benthamiana (N.B.) and wild-type Arabidopsis thaliana (A.T.), implying the antiserum was specific to banana E3 ubiquitin-protein ligase.
文摘By reverse transcription-polymerase chain reaction (RT-PCR), cDNA fragment of wheat yellow mosaic vir黶 (WYMV) RNA2 encoding 72 ku protein has been synthesized and cloned into plasmid pET30a(+) for overexpression in prokaryotic celis. BL21(DE3) pLys S of E.coli transformed with the recombinant plasmid pETP72 containing the fragment has been induced to express the 72 ku protein on high level. The produced protein has been purified from sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for its antiserum preparation. in western-blotting analysis, the antibodies reacted with the 72 ku protein expressed in E.coli.
文摘花生过敏蛋白Ara h 3广泛存在于各类花生制品中,少量摄入即可引发过敏反应,严重威胁花生过敏患者的健康。精准地识别Ara h 3便于开展后续免疫学检测,因此,制备高纯度、高特异性的抗体显得尤为重要。以花生脱脂粉为原料,通过HiTrap Q HP阴离子交换层析柱和HiLoad Superdex 200 pg制备级分子筛层析柱,制备高纯度的花生致敏蛋白Ara h 3,将其与CNBr-activated SepharoseTM 4B进行偶联,利用不同洗脱液对血清样品中的非特异性及特异性物质进行分离,制备高纯度、高特异性的抗Ara h 3多克隆抗体。结果表明:Gly-HCl洗脱的蛋白质量浓度为0.6 mg/mL;SDS-PAGE分析显示,多克隆抗体中的非特异性物质已被有效去除;免疫印迹和ELISA试验证明,纯化后的抗体仍具有较高的特异性,其效价达到1∶51200。上述结果表明抗体成功纯化,为花生过敏原Ara h 3的检测研究奠定了基础。
文摘该研究采用高毒力持留基因型单增李斯特菌819-2菌株全菌蛋白免疫SPF级Balb/C小鼠制备抗血清,利用免疫蛋白质组学对菌株胞外蛋白质组进行分析,旨在挖掘筛选单增李斯特菌强免疫原性蛋白作为特异性抗体制备的候选抗原。超声法提取819-2菌株全菌蛋白免疫SPF级Balb/C小鼠制备抗血清,四次免疫后经间接ELISA测定效价达1:512000。脱氧胆酸钠(DOC)-10%(m/V)TCA沉淀法提取单增李斯特菌819-2菌株胞外蛋白,利用免疫蛋白质组学和LC-MS/MS技术挖掘并鉴定具有强免疫反应的蛋白点,结果表明双向电泳图谱成功获得85个蛋白点,并成功鉴定了P60、InlC、MltG、Enolase和假定蛋白YxeA family protein等5个强免疫原性蛋白,研究结果为基于强免疫原性蛋白制备特异性抗体用于食品及其加工环境中单增李斯特菌富集及快速检测技术研制提供了数据基础。
