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
AIM： To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS： Gateway recombinant cloning technology was used to co...AIM： To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS： Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type （wt） and mutant （mut） forms of the lumican gene were synthesized and amplified by polymerase chain reaction （PCR）. The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown- multiple cloning site （MCS）-/internal ribozyme entry site （IRES）/enhanced green fluorescent protein （EGFP）. Then the desired DNA fragments were integrated into the destination vector pAV.Desld yielding the final expression constructs pAV.Exld-CMV〉wt-lumican/IRES/ EGFP and pAV.Exld-cytomegalovirus （CMV） 〉mutlumican/IRES/EGFP, respectively.RESULTS： The adenovirus plasmids pAV.Exld-CMV〉 wt-lumican/IRES/EGFP and pAV.Exld-CMV 〉mutlumican/IRESlEGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION： We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumicangene in the pathogenesis of high myopia.展开更多
To clone and sequence the human tissue kallikrein gene of Chinese, and to construct eukaryotic expression recombinant of KK, total RNA was extracted from human pancreas and human tissue kallikrein gene cDNA was amplif...To clone and sequence the human tissue kallikrein gene of Chinese, and to construct eukaryotic expression recombinant of KK, total RNA was extracted from human pancreas and human tissue kallikrein gene cDNA was amplified by PCR after reverse-transcription by using Oligo（dT） primer. The original kallikrein cDNA was recovered and filled with Klenow enzyme and inserted into KS plasmid. After restriction endonuclease digestion, KK cDNA was sequenced by ABI 377 analyzer. Then the KK gene was amplified from pBluescript KSKK and inserted into pcDNA3. A sequence comparison showed that the cloned kallikrein gene was only one nucleotide different from that reported in the Genbank. The coding amino acid was Asp in the Genbank gene, while the coding amino acid of Chinese kallikrein gene was Asn. The KK cDNA fragment was inserted into the eukaryotic expression vector pcDNA3. The cloned kallikrein gene and the pcDNA3KK can be used for further study in gene therapy.展开更多
Introducing PCR products into plasmids vectors is key for molecular techniques. Ideally cloning vectors are easy to construct, modify and propagate, neither require advanced techniques nor special equipment or reagent...Introducing PCR products into plasmids vectors is key for molecular techniques. Ideally cloning vectors are easy to construct, modify and propagate, neither require advanced techniques nor special equipment or reagents and efficiently incorporate PCR products at close to zero empty vector background. We provide an easy to engineer self-made cloning vector, neither requiring sophisticated tools or techniques nor advanced cloning knowledge. Through recombination we obtained the pUC18ccdB vector, carrying the ccdB suicide gene within the pUC18 backbone. When SmaI cleaved (within the ccdB) vector was T4 ligated with small (0.2 kbp) and intermediate (1.3 to 2.2 kbp) blunt end PCR-products and transformed into E. coli, the amount of clones with incorporated PCR product was comparable to commercial PCR-cloning kits and at a close to zero PCR product negative background. In conclusion we present a simple, versatile and cheap approach to an efficient “home made” PCR-cloning vector that allows integration of crude blunt end PCR products at close to zero background.展开更多
To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1)encoded by Epstein-Barr virus(pshLMP1),and to study the inhibition functi...To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1)encoded by Epstein-Barr virus(pshLMP1),and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells,we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis,RT-PCR and western blot.pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method.Furthermore,the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors.According to our research,we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells,and also provides a novel application of RNA interference technology against-EBV.展开更多
BACKGROUND： We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter- 1 （GLUT 1） i...BACKGROUND： We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter- 1 （GLUT 1） in rats, OBJECTIVE： This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-X^TM system. DESIGN： An in vitro cell-based experiment. SETTING： This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS： Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5 a and human embryonic kidney 293 cells （HEK293 cells） used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody （Chemicon, U.S.A.） and primers （Shanghai Boya Bioengineering Co., Ltd） were also used. METHODS： E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES： Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively. RESULTS： Results demonstrated that recombinant adenoviral vectors successfully expressed GLUT1 protein, with a relative molecular mass of 55000 in HEK293 cells. These results suggest that recombinant adenoviral vectors obtained by homologous bacterial recombination feature high efficiency, rapidness, and simplicity. CONCLUSION： We successfully amplified the rat GLUT1 gene and constructed replication-defective adenoviral vectors expressing GLUT1. The replication-defective adenoviral vectors proved to successfully express the target gene in HEK293 cells.展开更多
A full-length cDNA sequence of xyloglucan endotransglycosylase gene （XET）, abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromoso...A full-length cDNA sequence of xyloglucan endotransglycosylase gene （XET）, abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame （ORF） encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment ofAcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism ofAcXET gene during wood formation.展开更多
Objective: To clone tumor suppressor gene Fhit from normal human appendix’s tissue and construct its cloning vector. Methods: Extract human total RNA with TRIzol reagent and amplification the Fhit gene with reverse t...Objective: To clone tumor suppressor gene Fhit from normal human appendix’s tissue and construct its cloning vector. Methods: Extract human total RNA with TRIzol reagent and amplification the Fhit gene with reverse transcription-PCR (RT-PCR), then clone Fhit into a pGEM-T easy cloning vector, lastly identify the pGEM-Fhit vector with polymerase chain reaction (PCR), endonuclease digestion and DNA sequencing. Results: Fhit complementary DNA (cDNA) was cloned into pGEM-T vector, and DNA sequencing was the same as reported. Conclusions: The cloning vector pGEM-Fhit was constructed successfully and builds up a good foundation going deep into the study of Fhit.展开更多
To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was a...To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.展开更多
Objective:To construct the recombinant adenovirus vectors of calretinin(CALB_2) gene and small interfering RNA(siRNA),for over-expression or knock-down of CALB_2,as the basis of functional investigation of CALB_2 in t...Objective:To construct the recombinant adenovirus vectors of calretinin(CALB_2) gene and small interfering RNA(siRNA),for over-expression or knock-down of CALB_2,as the basis of functional investigation of CALB_2 in testicular Leydig cells. Methods:The cDNA sequence of CALB_2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB_2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB_2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB_2.The recombinant AdCMV-CALB_2 was further packaged and amplificated in AD293 cells.The expression of CALB_2 protein in AD293 cells was detected by Western blotting.CALB_2 protein was over-expressed in mouse Leydig cell line(MLTC-1 cells) by the constructed AdCMV-CALB_2. CALB_2 gene siRNA recombinant adenovirus vector(Ad-H1-siRNA/CALB_2 was also constructed simultaneously. Its efficacy was detected in AD293 cells by Western blotting. Results:The CALB_2 gene recombinant adenovirus vector AdCMV-CALB_2 and the CALB_2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB_2 were constructed successfully by endonulease digestion and sequencing. AD293 cells infected with AdCMV-CALB_2 or Ad-H1-SiRNA/CALB_2 significantly expressed GFP protein. The expression of CALB_2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB_2 plasmids, while the expression of CALB_2 protein was down-regulated by 60%in the CALB_2 cells infected with Ad-H1-SiRNA/CALB_2. MLTC-1 cells did not markedly express CALB_2 protein,while MLTC-1 cells infected with AdCMV-CALB_2 expressed CALB_2 protein at a high level. Conclusions:The recombinant adenovirus vectors of AdCMV-CALB_2 and Ad-H1-SiRNA/CALB_2 were successfully constructed.Both vectors effectively expressed in AD293.CALB_2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB_2.These vectors of CALB_2 gene and Leydig cell line are useful tools for investigating the testicular function.展开更多
Objective:To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA(SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels. Methods:The cDNA sequence of SET w...Objective:To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA(SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels. Methods:The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction(RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET.The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells.The expression of SET in AD293 cells was detected by Western blot.In addition,we constructed SET gene SiRNA recombinant adenovirus vector(Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results:The recombinant adenovirus vectors,both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET,were proven to be constructed successfully by the evidence of endonulease digestion and sequencing.AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP.The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector.On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion:The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells.It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.展开更多
In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lu...In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lung fibro-blast (HLF) was used for isolation of total RNA. Then the total RNA was purified and reverse- transcribed into cDNA using an oligo (dT) primer. A full PCR fragment for hKGF was generated and cloned. Restriction digestion and nucleo-tide sequence analysis validated the complete hKGF transcription. The hKGF cDNA fragment was inserted into pEGFP-C2 vector by means of recombinant DNA technology and verified by restriction analysis and sequencing. We have constructed pEGFP-C2-hKGF encoding the green fluorescent protein (GFP). Furthermore, hKGF had the effect on AEC II proliferation. These results suggest that the potential appli-cation of a hKGF plasmid of gene expression should be useful for sustained AEC proliferation, and its in vivo efficacy needs to be validated. Keywords: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 Guangdong Province(No.2015A030310158No.2014A030313359)+1 种基金the Fundamental Research Funds for the Central Universities(No.21611446)the Scientific and Cultivation Foundation of the First Affiliated Hospital of Jinan University(No.2015201)
文摘AIM： To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS： Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type （wt） and mutant （mut） forms of the lumican gene were synthesized and amplified by polymerase chain reaction （PCR）. The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown- multiple cloning site （MCS）-/internal ribozyme entry site （IRES）/enhanced green fluorescent protein （EGFP）. Then the desired DNA fragments were integrated into the destination vector pAV.Desld yielding the final expression constructs pAV.Exld-CMV〉wt-lumican/IRES/ EGFP and pAV.Exld-cytomegalovirus （CMV） 〉mutlumican/IRES/EGFP, respectively.RESULTS： The adenovirus plasmids pAV.Exld-CMV〉 wt-lumican/IRES/EGFP and pAV.Exld-CMV 〉mutlumican/IRESlEGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION： We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumicangene in the pathogenesis of high myopia.
基金This project is supported by a program of the 15th Fiveyear Plan of Scientific Research of Bureau of Science and Technology of Guangdong Province (No. 2001c11801)
文摘To clone and sequence the human tissue kallikrein gene of Chinese, and to construct eukaryotic expression recombinant of KK, total RNA was extracted from human pancreas and human tissue kallikrein gene cDNA was amplified by PCR after reverse-transcription by using Oligo（dT） primer. The original kallikrein cDNA was recovered and filled with Klenow enzyme and inserted into KS plasmid. After restriction endonuclease digestion, KK cDNA was sequenced by ABI 377 analyzer. Then the KK gene was amplified from pBluescript KSKK and inserted into pcDNA3. A sequence comparison showed that the cloned kallikrein gene was only one nucleotide different from that reported in the Genbank. The coding amino acid was Asp in the Genbank gene, while the coding amino acid of Chinese kallikrein gene was Asn. The KK cDNA fragment was inserted into the eukaryotic expression vector pcDNA3. The cloned kallikrein gene and the pcDNA3KK can be used for further study in gene therapy.
文摘Introducing PCR products into plasmids vectors is key for molecular techniques. Ideally cloning vectors are easy to construct, modify and propagate, neither require advanced techniques nor special equipment or reagents and efficiently incorporate PCR products at close to zero empty vector background. We provide an easy to engineer self-made cloning vector, neither requiring sophisticated tools or techniques nor advanced cloning knowledge. Through recombination we obtained the pUC18ccdB vector, carrying the ccdB suicide gene within the pUC18 backbone. When SmaI cleaved (within the ccdB) vector was T4 ligated with small (0.2 kbp) and intermediate (1.3 to 2.2 kbp) blunt end PCR-products and transformed into E. coli, the amount of clones with incorporated PCR product was comparable to commercial PCR-cloning kits and at a close to zero PCR product negative background. In conclusion we present a simple, versatile and cheap approach to an efficient “home made” PCR-cloning vector that allows integration of crude blunt end PCR products at close to zero background.
文摘To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1)encoded by Epstein-Barr virus(pshLMP1),and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells,we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis,RT-PCR and western blot.pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method.Furthermore,the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors.According to our research,we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells,and also provides a novel application of RNA interference technology against-EBV.
基金the National Natural Science Foundation of China, No. 39900048the Natural Science Foundation of Guangdong Province, No.010721
文摘BACKGROUND： We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter- 1 （GLUT 1） in rats, OBJECTIVE： This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-X^TM system. DESIGN： An in vitro cell-based experiment. SETTING： This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS： Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5 a and human embryonic kidney 293 cells （HEK293 cells） used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody （Chemicon, U.S.A.） and primers （Shanghai Boya Bioengineering Co., Ltd） were also used. METHODS： E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES： Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively. RESULTS： Results demonstrated that recombinant adenoviral vectors successfully expressed GLUT1 protein, with a relative molecular mass of 55000 in HEK293 cells. These results suggest that recombinant adenoviral vectors obtained by homologous bacterial recombination feature high efficiency, rapidness, and simplicity. CONCLUSION： We successfully amplified the rat GLUT1 gene and constructed replication-defective adenoviral vectors expressing GLUT1. The replication-defective adenoviral vectors proved to successfully express the target gene in HEK293 cells.
基金supported by the National Natural Science Foundation of China (Grant No. 30901158)the Key Project of Chinese Ministry of Education (Grant No. 104243)
文摘A full-length cDNA sequence of xyloglucan endotransglycosylase gene （XET）, abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame （ORF） encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment ofAcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism ofAcXET gene during wood formation.
文摘Objective: To clone tumor suppressor gene Fhit from normal human appendix’s tissue and construct its cloning vector. Methods: Extract human total RNA with TRIzol reagent and amplification the Fhit gene with reverse transcription-PCR (RT-PCR), then clone Fhit into a pGEM-T easy cloning vector, lastly identify the pGEM-Fhit vector with polymerase chain reaction (PCR), endonuclease digestion and DNA sequencing. Results: Fhit complementary DNA (cDNA) was cloned into pGEM-T vector, and DNA sequencing was the same as reported. Conclusions: The cloning vector pGEM-Fhit was constructed successfully and builds up a good foundation going deep into the study of Fhit.
文摘To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
基金supported by projects from China National Nature Foundation (81170559)Jiangsu Health Foundation(XK02200901-NG09)
文摘Objective:To construct the recombinant adenovirus vectors of calretinin(CALB_2) gene and small interfering RNA(siRNA),for over-expression or knock-down of CALB_2,as the basis of functional investigation of CALB_2 in testicular Leydig cells. Methods:The cDNA sequence of CALB_2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB_2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB_2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB_2.The recombinant AdCMV-CALB_2 was further packaged and amplificated in AD293 cells.The expression of CALB_2 protein in AD293 cells was detected by Western blotting.CALB_2 protein was over-expressed in mouse Leydig cell line(MLTC-1 cells) by the constructed AdCMV-CALB_2. CALB_2 gene siRNA recombinant adenovirus vector(Ad-H1-siRNA/CALB_2 was also constructed simultaneously. Its efficacy was detected in AD293 cells by Western blotting. Results:The CALB_2 gene recombinant adenovirus vector AdCMV-CALB_2 and the CALB_2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB_2 were constructed successfully by endonulease digestion and sequencing. AD293 cells infected with AdCMV-CALB_2 or Ad-H1-SiRNA/CALB_2 significantly expressed GFP protein. The expression of CALB_2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB_2 plasmids, while the expression of CALB_2 protein was down-regulated by 60%in the CALB_2 cells infected with Ad-H1-SiRNA/CALB_2. MLTC-1 cells did not markedly express CALB_2 protein,while MLTC-1 cells infected with AdCMV-CALB_2 expressed CALB_2 protein at a high level. Conclusions:The recombinant adenovirus vectors of AdCMV-CALB_2 and Ad-H1-SiRNA/CALB_2 were successfully constructed.Both vectors effectively expressed in AD293.CALB_2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB_2.These vectors of CALB_2 gene and Leydig cell line are useful tools for investigating the testicular function.
基金Projects from China National Department of Science and Technology(81070465)a project from China Jiangsu Province (BM2008151)
文摘Objective:To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA(SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels. Methods:The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction(RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET.The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells.The expression of SET in AD293 cells was detected by Western blot.In addition,we constructed SET gene SiRNA recombinant adenovirus vector(Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results:The recombinant adenovirus vectors,both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET,were proven to be constructed successfully by the evidence of endonulease digestion and sequencing.AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP.The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector.On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion:The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells.It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.
文摘In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lung fibro-blast (HLF) was used for isolation of total RNA. Then the total RNA was purified and reverse- transcribed into cDNA using an oligo (dT) primer. A full PCR fragment for hKGF was generated and cloned. Restriction digestion and nucleo-tide sequence analysis validated the complete hKGF transcription. The hKGF cDNA fragment was inserted into pEGFP-C2 vector by means of recombinant DNA technology and verified by restriction analysis and sequencing. We have constructed pEGFP-C2-hKGF encoding the green fluorescent protein (GFP). Furthermore, hKGF had the effect on AEC II proliferation. These results suggest that the potential appli-cation of a hKGF plasmid of gene expression should be useful for sustained AEC proliferation, and its in vivo efficacy needs to be validated. Keywords:
