Given the sequence of Chs gene promoter from Arabidopsis thaliana reported in GenBank (AF248988), a pair of specific PCR primers was designed with the Primer Premier 5.0 software. PCR products of about 0.5 kb were s...Given the sequence of Chs gene promoter from Arabidopsis thaliana reported in GenBank (AF248988), a pair of specific PCR primers was designed with the Primer Premier 5.0 software. PCR products of about 0.5 kb were successfully amplified with the genome DNA of A. thaliana as a DNA template and Taq polymerase as DNA polymerase. The purified PCR products were ligated to the pMD18-T vector. The sequencing result showed that the Chs promoter from A. thaliana was 531 bp long. Sequence alignment analysis based on the DNAMAN software revealed that the sequence similarity between the cloned promoter and target promoter (AF248988) was up to 100%. Online PLACE analysis indicated that the Chs promoter contained cis-elements such as TATA-box, CAAT-box, pollen-box, G-box, ACGT-containing element, R response element, Myb recognition element and TACPyAT-box. At the same time, a plant expression vectorpAtChs::GUS which fused the Chs promoter and the marker gene GUS was successfully constructed.展开更多
EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was...EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was selected as a bioreactor for the production of an edible EV71 vaccine designed for the VP1 capsid protein.Using molecular biology techniques,the fusion gene EV71-VP1 was cut from vector PGEX-4T-2,a vector containing the p2300-EV71 gene with CaMV35S promoter and TL regulatory elements was constructed,and the hypocotyl and cotyledons of tomato were transformed using Agrobacterium(EHA105)-mediated method,screened,elongated and rooted,and finally 20 resistant tomato plants were obtained.Five transgenic positive seedlings were obtained by digestion and PCR assay,among which three plants were detected by RT-PCR to be capable of transcriptional translation at the RNA level.The experimental results aimed to explore new material support for the preparation of transgenic plant oral vaccines against EV71 infection and provide a theoretical basis for accelerating the development of transgenic plant vaccines in the future.展开更多
[Objectives]This study was conducted to obtain porcine gastrin-releasing peptide(GRP)fusion protein.[Methods]The constructed pET32a(+)-GRP plasmid was transformed into Escherichia coli BL21(DE3)competent cells to obta...[Objectives]This study was conducted to obtain porcine gastrin-releasing peptide(GRP)fusion protein.[Methods]The constructed pET32a(+)-GRP plasmid was transformed into Escherichia coli BL21(DE3)competent cells to obtain the pET32a(+)-GRP-BL21(DE3)fusion protein expression strain,which was induced with 0.5 mM IPTG at 25℃and 150 r/min for 12 h,and the His-tagged GRP fusion protein was detected by SDS-Page gel electrophoresis and Western Blot.[Results]After optimizing the IPTG-induced expression conditions,it was confirmed that the porcine GRP fusion protein was obtained,and the porcine GRP fusion protein was soluble,stable and highly active.[Conclusions]This study lays a foundation for the subsequent preparation of anti-pig GRP antibodies.展开更多
The Pseudopleuronectes americanus antifreeze protein gene was synthesized and control sequences were added such as 35S promoter and nos terminator that can facilitate the transcription and Ω sequence and Kozak sequen...The Pseudopleuronectes americanus antifreeze protein gene was synthesized and control sequences were added such as 35S promoter and nos terminator that can facilitate the transcription and Ω sequence and Kozak sequence that can improve the expression in translation level, the high expression cassette of antifreeze protein was constructed. This cassette was connected to pBI121.1 and finally got the high expression vector pBRTSAFP introduced into the maize callus. The expression of gus gene that linked to the antifreeze protein gene was detected, and the results was that the gus gene can express strongly and instantaneously.展开更多
[Objective] This study aimed to construct RNAi expression vector against r/boflavin synthase (RS) gene. [Method] By using the primers designed based on RS gone coding sequence that was screened from Arabidopsis cDNA...[Objective] This study aimed to construct RNAi expression vector against r/boflavin synthase (RS) gene. [Method] By using the primers designed based on RS gone coding sequence that was screened from Arabidopsis cDNA library, the 476 bp cDNA fragment of RS was amplified from pGADTT-RS recombinant plasmid, and then cloned into pUCm-T vector to obtain pUCm-RS. Two RS fragments (476 bp) were obtained through digesting pUCm-RS with restriction enzymes PstI/BamHI and PstI / Xhol, and then respectively connected into vector pBSSK-in to form pBSSK-RS-in-RS, in which the two RS fragments were inverted re- peats. Finally, the transform unit RS-intron-RS, got by digesting vector pBSSK-RS-in-RS with Sac I and Kpn I, was ligated into expression vector pCAMBIA1301 to obtain the RS gene silencing vector. [ Result] The restriction enzyme digestion sequencing analysis proved that the RS gene silencing vector was successfully con- structed. [ Conclusion] This study may provide a basic material for further studies on the bio-function of RS gene and the mechanism of signal transduction induced by HpaGxoo in plant.展开更多
[ Objective] This study aimed to construct the prokaryotic expression vector for Arab/dops/s At4g12500 gene. [ Method] Genomic DNA of wild-type Ar- abidopsis Cold) was extracted as the template for amplification of A...[ Objective] This study aimed to construct the prokaryotic expression vector for Arab/dops/s At4g12500 gene. [ Method] Genomic DNA of wild-type Ar- abidopsis Cold) was extracted as the template for amplification of At4g12.500 gene with PCR technology. The target fragment was double-digested with BamH I and Xho I, and ligated with prokaryotic expression vector pET-28a. [Result] Prokaryotic expression vector pEI28a-At4gI2500 was successfully constructed and trans- formed into Escherichia coli expression strain BL21 (DE3). [ Conclusion] This study laid the foundation for subsequent expression and functional analysis of At4g12500 gene.展开更多
Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase ( COX) activity in mouse Sertoli cell line TM4. Methods The coding region of CoxTa2 was amplified from mouse Sertoli cell l...Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase ( COX) activity in mouse Sertoli cell line TM4. Methods The coding region of CoxTa2 was amplified from mouse Sertoli cell line TM4 by RT-PCR. PCR product was展开更多
Brucella spp. are pathogenic to humans and domestic animal. Nowadays,there is no effective vaccine and control strategy in China. So,it is necessary to research effective vaccines for prevention and treatment of this ...Brucella spp. are pathogenic to humans and domestic animal. Nowadays,there is no effective vaccine and control strategy in China. So,it is necessary to research effective vaccines for prevention and treatment of this disease. In order to deal with these,we isolated and identified the type of Brucella in Darhan Muminggan Joint Banner and Siziwang Banner of Inner Mongolia. Totally 26 samples of sheep blood which were positive in serological test,one sample of spleen from aborted and one sample of secretion from birth canal were isolated,and the 16 S r DNA genes of positive samples were sequenced. Phylogenetic analysis proved that there were four isolates similar to B. melitensis. As an important diagnostic antigens of B. melitensis,the BP26 gene was amplified. The BP26 gene was cloned into vector p ET24a( +) and conducted sequence analysis. The BP26 gene was 900 bp,with an open reading frame of 753 bp. The homology of BP26 gene with the vaccine strain M5 was 100%,and that with S2 and A19 vaccine strains was 99. 99%. These finding supported the development of BP26-based specific serodiagnostic test and vaccine for B. melitensis in China.展开更多
AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning t...AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning the human αB-crystallin cDNA according to the nucleotide sequence of the human αBcrystallin, constructing the pET-28/CRYAB prokaryotic expression plasmid by restriction enzyme digestion method, and stably expressing transformed into the Escherichia coli(E. coli) DH5 alpha. The recombinant human αB-crystallin was purified by Q sepharose. By enzyme digestion analysis, Western blotting and sequencing, the recombinant human αB-crystallin was identified and the activity of its molecular protein was detected.RESULTS: Compared with the gene bank(GeneBank), the cloned human sequence of human αB-crystallin cDNA has the same open reading frame. Identification and sequencing of the cloned human αB-crystallin cDNA in prokaryotic expression vector confirmed the full length sequence, and the vector was constructed successfully. The E. coli containing plasmid pET-28/CRYAB induced by isopropyl-β-D-thiogalactoside successfully expressed the human αB-crystallin. Insulin confirmed that the recombinant human αB-crystallin has a molecular chaperone activity. CONCLUSION: The prokaryotic expression vector pET-28/CRYAB of recombinant human αB-crystallin issuccessfully constructed, and the recombinant human αBcrystallin with molecular chaperone activity is obtained, which lay a foundation for the research and application of the recombinant human αB-crystallin and its chaperone activity.展开更多
Xylem-specific promoter could regulate efficient expression of foreign genes in xylem. Deletion analysis method was applied to obtain 5' deletion promoter fragments MU2 andMU4 with different distances from transcript...Xylem-specific promoter could regulate efficient expression of foreign genes in xylem. Deletion analysis method was applied to obtain 5' deletion promoter fragments MU2 andMU4 with different distances from transcription start site of xylem-specific promoter MDCcsAP through PCR procedure. Then CaMV35S promoter in plant expression vector pBI121 was replaced by amplified fragments. The recombinant plasmids fused with corresponding gene segments and GUS reporter gene were obtained and transformed into Agrobacterium. The results of the tobacco transient transformation test showed that MU2 andMU4 had the promoter function, and both of their activity were significantly higher than that of CaMV35S promoter, and they had the characteristics of xylem tissue specificity as well. Research on xy- lem-specific promoter structure and function could lay a foundation for the determination of necessary components to the xylem tissue specificity and cis-acting ele- ments with induction activity, so as to enable the regulation of efficient expression of exogenous genes in specific time and specific tissues in plants through these cis-acting elements.展开更多
The concept of group construction vector and independent construction vector for visual cryptography is proposed, and the method based on construction vector is presented for con-structing basis matrices. The general ...The concept of group construction vector and independent construction vector for visual cryptography is proposed, and the method based on construction vector is presented for con-structing basis matrices. The general solutions to construction vectors and the general solutions to k out of n visual cryptographic schemes are obtained. Using the construction vectors, everyone can construct visual cryptographic schemes simply and efficaciously according to the formulas. The concept and the general solutions to construction vector present a good idea for researches on visual cryptographic schemes, including structural properties, the bound of pixel expansion and contrast, and optimal construction.展开更多
Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmi...Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmid,then the amplified fragment was clonded into the p IRES2-ZsG reen1 vector using展开更多
英语复杂介词应界定为词还是短语,学界未有定论。本文以构式化理论为视角,借助语义向量空间的定量方法,考察临界复杂介词in the face of的固化过程。本研究发现:1)在过去200年间,该构式的能产性和图式性增加,而组构性降低。从基于使用...英语复杂介词应界定为词还是短语,学界未有定论。本文以构式化理论为视角,借助语义向量空间的定量方法,考察临界复杂介词in the face of的固化过程。本研究发现:1)在过去200年间,该构式的能产性和图式性增加,而组构性降低。从基于使用的视角来看,这种变化趋势不仅反映了构式固化程度的增强,同时也表明其逐渐向介词范畴的演进;2)该构式的固化过程并不是单向的线性发展,而是表现为构式节点间横向和纵向关系的变化;3)该构式固化的动因有二:一是高能产性导致的组块效应,二是识解层面的语义简化。展开更多
Human erythropoietin gene was ligated into the mammalian high efficiency expression vector pSV2 dhfr by standard procedures of filling in, trimming, ligation and transformation to construct a high efficiency expressi...Human erythropoietin gene was ligated into the mammalian high efficiency expression vector pSV2 dhfr by standard procedures of filling in, trimming, ligation and transformation to construct a high efficiency expression vector. After the expression vector pSEPO25 was transfected into CHO dhfr - cells, the cell line which could express high levels of EPO was successfully selected. The result lays the foundation for production of EPO by genetic biotechnology.展开更多
基金supported by the National Natural Science Foundation of China (Grant No.30740013)the Key Laboratory for Genetics and Breeding in Forestry Trees and Ornamental Plants,Ministry of Education (03-05)
文摘Given the sequence of Chs gene promoter from Arabidopsis thaliana reported in GenBank (AF248988), a pair of specific PCR primers was designed with the Primer Premier 5.0 software. PCR products of about 0.5 kb were successfully amplified with the genome DNA of A. thaliana as a DNA template and Taq polymerase as DNA polymerase. The purified PCR products were ligated to the pMD18-T vector. The sequencing result showed that the Chs promoter from A. thaliana was 531 bp long. Sequence alignment analysis based on the DNAMAN software revealed that the sequence similarity between the cloned promoter and target promoter (AF248988) was up to 100%. Online PLACE analysis indicated that the Chs promoter contained cis-elements such as TATA-box, CAAT-box, pollen-box, G-box, ACGT-containing element, R response element, Myb recognition element and TACPyAT-box. At the same time, a plant expression vectorpAtChs::GUS which fused the Chs promoter and the marker gene GUS was successfully constructed.
基金Supported by the Natural Science Foundation of Heilongjiang Province(LH2021C032)。
文摘EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was selected as a bioreactor for the production of an edible EV71 vaccine designed for the VP1 capsid protein.Using molecular biology techniques,the fusion gene EV71-VP1 was cut from vector PGEX-4T-2,a vector containing the p2300-EV71 gene with CaMV35S promoter and TL regulatory elements was constructed,and the hypocotyl and cotyledons of tomato were transformed using Agrobacterium(EHA105)-mediated method,screened,elongated and rooted,and finally 20 resistant tomato plants were obtained.Five transgenic positive seedlings were obtained by digestion and PCR assay,among which three plants were detected by RT-PCR to be capable of transcriptional translation at the RNA level.The experimental results aimed to explore new material support for the preparation of transgenic plant oral vaccines against EV71 infection and provide a theoretical basis for accelerating the development of transgenic plant vaccines in the future.
基金Supported by National Natural Science Foundation of China(31802149)China Postdoctoral Science(2019M651985)+2 种基金Natural Science Foundation of Jiangsu Province(BK20180919)Scientific Research Fund of Nanjing General Hospital of Nanjing Military Command(2016033)Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)。
文摘[Objectives]This study was conducted to obtain porcine gastrin-releasing peptide(GRP)fusion protein.[Methods]The constructed pET32a(+)-GRP plasmid was transformed into Escherichia coli BL21(DE3)competent cells to obtain the pET32a(+)-GRP-BL21(DE3)fusion protein expression strain,which was induced with 0.5 mM IPTG at 25℃and 150 r/min for 12 h,and the His-tagged GRP fusion protein was detected by SDS-Page gel electrophoresis and Western Blot.[Results]After optimizing the IPTG-induced expression conditions,it was confirmed that the porcine GRP fusion protein was obtained,and the porcine GRP fusion protein was soluble,stable and highly active.[Conclusions]This study lays a foundation for the subsequent preparation of anti-pig GRP antibodies.
基金Supported by National Transgenic Plant Research and.Industrialization Foundation(J00-B-003-04)
文摘The Pseudopleuronectes americanus antifreeze protein gene was synthesized and control sequences were added such as 35S promoter and nos terminator that can facilitate the transcription and Ω sequence and Kozak sequence that can improve the expression in translation level, the high expression cassette of antifreeze protein was constructed. This cassette was connected to pBI121.1 and finally got the high expression vector pBRTSAFP introduced into the maize callus. The expression of gus gene that linked to the antifreeze protein gene was detected, and the results was that the gus gene can express strongly and instantaneously.
基金Supported by the Doctor Fund of Langfang Teachers College ( LSZB201002)
文摘[Objective] This study aimed to construct RNAi expression vector against r/boflavin synthase (RS) gene. [Method] By using the primers designed based on RS gone coding sequence that was screened from Arabidopsis cDNA library, the 476 bp cDNA fragment of RS was amplified from pGADTT-RS recombinant plasmid, and then cloned into pUCm-T vector to obtain pUCm-RS. Two RS fragments (476 bp) were obtained through digesting pUCm-RS with restriction enzymes PstI/BamHI and PstI / Xhol, and then respectively connected into vector pBSSK-in to form pBSSK-RS-in-RS, in which the two RS fragments were inverted re- peats. Finally, the transform unit RS-intron-RS, got by digesting vector pBSSK-RS-in-RS with Sac I and Kpn I, was ligated into expression vector pCAMBIA1301 to obtain the RS gene silencing vector. [ Result] The restriction enzyme digestion sequencing analysis proved that the RS gene silencing vector was successfully con- structed. [ Conclusion] This study may provide a basic material for further studies on the bio-function of RS gene and the mechanism of signal transduction induced by HpaGxoo in plant.
文摘[ Objective] This study aimed to construct the prokaryotic expression vector for Arab/dops/s At4g12500 gene. [ Method] Genomic DNA of wild-type Ar- abidopsis Cold) was extracted as the template for amplification of At4g12.500 gene with PCR technology. The target fragment was double-digested with BamH I and Xho I, and ligated with prokaryotic expression vector pET-28a. [Result] Prokaryotic expression vector pEI28a-At4gI2500 was successfully constructed and trans- formed into Escherichia coli expression strain BL21 (DE3). [ Conclusion] This study laid the foundation for subsequent expression and functional analysis of At4g12500 gene.
文摘Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase ( COX) activity in mouse Sertoli cell line TM4. Methods The coding region of CoxTa2 was amplified from mouse Sertoli cell line TM4 by RT-PCR. PCR product was
基金Supported by Natural Science Foundation of Inner Mongolia(2014MS0356)
文摘Brucella spp. are pathogenic to humans and domestic animal. Nowadays,there is no effective vaccine and control strategy in China. So,it is necessary to research effective vaccines for prevention and treatment of this disease. In order to deal with these,we isolated and identified the type of Brucella in Darhan Muminggan Joint Banner and Siziwang Banner of Inner Mongolia. Totally 26 samples of sheep blood which were positive in serological test,one sample of spleen from aborted and one sample of secretion from birth canal were isolated,and the 16 S r DNA genes of positive samples were sequenced. Phylogenetic analysis proved that there were four isolates similar to B. melitensis. As an important diagnostic antigens of B. melitensis,the BP26 gene was amplified. The BP26 gene was cloned into vector p ET24a( +) and conducted sequence analysis. The BP26 gene was 900 bp,with an open reading frame of 753 bp. The homology of BP26 gene with the vaccine strain M5 was 100%,and that with S2 and A19 vaccine strains was 99. 99%. These finding supported the development of BP26-based specific serodiagnostic test and vaccine for B. melitensis in China.
基金Supported by National Natural Science Foundation of China Grant (No.81270996)Science and Technology Project Foundation of Hainan Province (No.ZDYF201631)Health Science and Technology Innovation Project Foundation of Sanya (No.2016YW22)
文摘AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning the human αB-crystallin cDNA according to the nucleotide sequence of the human αBcrystallin, constructing the pET-28/CRYAB prokaryotic expression plasmid by restriction enzyme digestion method, and stably expressing transformed into the Escherichia coli(E. coli) DH5 alpha. The recombinant human αB-crystallin was purified by Q sepharose. By enzyme digestion analysis, Western blotting and sequencing, the recombinant human αB-crystallin was identified and the activity of its molecular protein was detected.RESULTS: Compared with the gene bank(GeneBank), the cloned human sequence of human αB-crystallin cDNA has the same open reading frame. Identification and sequencing of the cloned human αB-crystallin cDNA in prokaryotic expression vector confirmed the full length sequence, and the vector was constructed successfully. The E. coli containing plasmid pET-28/CRYAB induced by isopropyl-β-D-thiogalactoside successfully expressed the human αB-crystallin. Insulin confirmed that the recombinant human αB-crystallin has a molecular chaperone activity. CONCLUSION: The prokaryotic expression vector pET-28/CRYAB of recombinant human αB-crystallin issuccessfully constructed, and the recombinant human αBcrystallin with molecular chaperone activity is obtained, which lay a foundation for the research and application of the recombinant human αB-crystallin and its chaperone activity.
基金Supported by National Science Foundation of China(31000305)Fund from Education Department of Hebei Province(QN2015182)+1 种基金Innovation Fund for Forestry Discipline of Hebei Agricultural University(LXXK2014-1)Fund for Overseas Research and Study by Young and Middle-aged Backbone Teachers in Hebei Agriculture University(JWYX2015)
文摘Xylem-specific promoter could regulate efficient expression of foreign genes in xylem. Deletion analysis method was applied to obtain 5' deletion promoter fragments MU2 andMU4 with different distances from transcription start site of xylem-specific promoter MDCcsAP through PCR procedure. Then CaMV35S promoter in plant expression vector pBI121 was replaced by amplified fragments. The recombinant plasmids fused with corresponding gene segments and GUS reporter gene were obtained and transformed into Agrobacterium. The results of the tobacco transient transformation test showed that MU2 andMU4 had the promoter function, and both of their activity were significantly higher than that of CaMV35S promoter, and they had the characteristics of xylem tissue specificity as well. Research on xy- lem-specific promoter structure and function could lay a foundation for the determination of necessary components to the xylem tissue specificity and cis-acting ele- ments with induction activity, so as to enable the regulation of efficient expression of exogenous genes in specific time and specific tissues in plants through these cis-acting elements.
文摘The concept of group construction vector and independent construction vector for visual cryptography is proposed, and the method based on construction vector is presented for con-structing basis matrices. The general solutions to construction vectors and the general solutions to k out of n visual cryptographic schemes are obtained. Using the construction vectors, everyone can construct visual cryptographic schemes simply and efficaciously according to the formulas. The concept and the general solutions to construction vector present a good idea for researches on visual cryptographic schemes, including structural properties, the bound of pixel expansion and contrast, and optimal construction.
文摘Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmid,then the amplified fragment was clonded into the p IRES2-ZsG reen1 vector using
文摘英语复杂介词应界定为词还是短语,学界未有定论。本文以构式化理论为视角,借助语义向量空间的定量方法,考察临界复杂介词in the face of的固化过程。本研究发现:1)在过去200年间,该构式的能产性和图式性增加,而组构性降低。从基于使用的视角来看,这种变化趋势不仅反映了构式固化程度的增强,同时也表明其逐渐向介词范畴的演进;2)该构式的固化过程并不是单向的线性发展,而是表现为构式节点间横向和纵向关系的变化;3)该构式固化的动因有二:一是高能产性导致的组块效应,二是识解层面的语义简化。
文摘Human erythropoietin gene was ligated into the mammalian high efficiency expression vector pSV2 dhfr by standard procedures of filling in, trimming, ligation and transformation to construct a high efficiency expression vector. After the expression vector pSEPO25 was transfected into CHO dhfr - cells, the cell line which could express high levels of EPO was successfully selected. The result lays the foundation for production of EPO by genetic biotechnology.