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Objective To investigate human papillomavirus (HPV) genotypes in adult patients with plantar warts,and to explore their relationship with clinical features of adult plantar warts.Methods PCR,gene sequencing and TA-cloning technique were performed to detect HPV types in 221 patients with plantar warts.Meanwhile,clinical data were recorded,including patients' age,disease duration,symptoms and number of warts.Results Of 221 specimens,HPV DNA was detected in 215,with the HPV-positive rate being 97％.Single HPV infections were detected in 206 specimens (96％,206/215),among which,HPV types included HPV-27 (44.7％,92/206),-2 (12.1％,25/206),-57 (7.3％,15/206),-7 (Alpha genus;0.5％,1/206),-65 (Gamma genus;0.5％,1/206),-1 (33.5％,69/206) and-63 (Mu genus;1.5％,3/206).There were no significant differences in age,disease duration,number of warts,incidence of pain and gender among patients with HPV-27,-2 or-57 infections.Compared with patients with HPV-27 infection,patients with HPV-1 infection were more related to age ≤ 30 years,disease duration ≤ 1 year,number of warts ≤ 2 and high incidence of pain.Conclusion HPV-27,-2,-57 and-1 are the main types in adult patients with plantar warts,and HPV types are correlated with clinical features of adult plantar warts.
ABSTRACT
Objective To investigate human papillomavirus (HPV) genotypes in adult patients with plantar warts,and to explore their relationship with clinical features of adult plantar warts.Methods PCR,gene sequencing and TA-cloning technique were performed to detect HPV types in 221 patients with plantar warts.Meanwhile,clinical data were recorded,including patients' age,disease duration,symptoms and number of warts.Results Of 221 specimens,HPV DNA was detected in 215,with the HPV-positive rate being 97％.Single HPV infections were detected in 206 specimens (96％,206/215),among which,HPV types included HPV-27 (44.7％,92/206),-2 (12.1％,25/206),-57 (7.3％,15/206),-7 (Alpha genus;0.5％,1/206),-65 (Gamma genus;0.5％,1/206),-1 (33.5％,69/206) and-63 (Mu genus;1.5％,3/206).There were no significant differences in age,disease duration,number of warts,incidence of pain and gender among patients with HPV-27,-2 or-57 infections.Compared with patients with HPV-27 infection,patients with HPV-1 infection were more related to age ≤ 30 years,disease duration ≤ 1 year,number of warts ≤ 2 and high incidence of pain.Conclusion HPV-27,-2,-57 and-1 are the main types in adult patients with plantar warts,and HPV types are correlated with clinical features of adult plantar warts.
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Objective To establish a cell line stably expressing the human CD36 by using TA clo-ning and cell transfection technology and to analyze its application to the detection of anti-CD36 antibodies. Methods Total RNA was isolated from human platelets and then used to synthesize complementary DNA ( cDNA) . Sequence of the gene encoding CD36 on human platelets was obtained by PCR amplification. The recombinant vector was transformed into TOP10 E. coli after TA cloning. The positive recombinant pcDNA3. 1/V5-CD36 plasmid was screened out by blue-white selection and then sequenced. The correctly constructed plasmid coated with Effectene? Transfection Reagent was transferred into HEK293T cells. Fluo-rescence-activated cell sorting was performed to screen out the cell line that could stably express the CD36 on human platelets. The transfected cell line-based flow cytometry analysis and antibody capture assay ( ACA) were established and used for antibody detection in nine serum samples positive for anti-CD36 antibodies. Results The HEK293T cell line stably expressing the recombinant CD36 was successfully established. Compare with the monoclonal antibody immobilization of platelet antigens assay ( MAIPA) , anti-CD36 anti-bodies could be easily identified in nine serum samples by using the transfected cell line-based flow cytome-try analysis and ACA. Conclusion This study suggests that the HEK293T cells stably expressing the re-combinant CD36 could be used in flow cytometry analysis and ACA for the detection of anti-CD36 antibod-ies. It also paves the way for further researches on the mechanism of CD36 in other diseases.
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For more economical and efficient DNA clonging, pFL-XS-T, a Biobrick-T vector was constructed based on pMD18-T vector, carrying clonging regions of XbaⅠ-XcmⅠ-XcmⅠ-SpeⅠ. The results revealed that PCR products could be conveniently inserted into pFL-XS-T vevtor digested by XcmⅠby means of TA cloning. The positive frequency of recombination can meet the experimental requirements and all the plasmids obtained meet Biobrick standard. Moreover, the pFL-XS-T is compatible with other Biobrick parts, and serves as a vector for functional DNA fragments screening.
Subject(s)
Cloning, Molecular , DNA , Genetic Vectors , Plasmids , Polymerase Chain Reaction , Synthetic BiologyABSTRACT
Objective: To construct a novel plasmid as Salmonella enterica serovar typhimurium (S. typhimurium) sipC gene knockouts candidate. Methods: In this research, 5' upstream and 3' downstream regions of S. typhimurium sipC gene and kanamycin gene were PCR amplified. Each of these DNA fragment was cloned into pGEM T-easy vector. The construct was confirmed by PCR and restriction digest. Results: PCR amplified 320, 206 and 835 bp DNA fragments were subcloned into pET-32 vector resulting with a plasmid called pET-32-sipC up-kan- sip C down. Conclusions: The new plasmid (pET-32-sipC up-kan- sip C down) is useful for genetic engineering and for future manipulation of S. typhimurium sipC gene.
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Objective: To construct a novel plasmid as Salmonella enterica serovar typhimurium (S. typhimurium) sipC gene knockouts candidate. Methods: In this research, 50 upstream and 30 downstream regions of S. typhimurium sipC gene and kanamycin gene were PCR amplified. Each of these DNA fragment was cloned into pGEM T-easy vector. The construct was confirmed by PCR and restriction digest. Results: PCR amplified 320, 206 and 835 bp DNA fragments were subcloned into pET-32 vector resulting with a plasmid called pET-32-sipC up-kan-sip C down. Conclusions: The new plasmid (pET-32-sipC up-kan-sip C down) is useful for genetic engineering and for future manipulation of S. typhimurium sipC gene.
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ObjectiveTo investigate the methylation of the promoter region in miRNA in pancreatic cancer cell line PANC1 and normal pancreatic tissue,to discover the miRNA with hypermethylation associated with pancreatic cancer.MethodsThe genomic DNA of PANC1 and normal pancreatic tissue was extracted,and fractured by ultrasound.Methylation DNA fragments were obtained by 5-methyl of pyrimidine nucleoside antibodies and immunomagnetic beads.The hypermethylation miRNA differentially expressed between PANC1 and normal pancreatic tissue was selected by using methylation DNA chip.BSP ( bisulfite genomic sequencing PCR) and TA clone sequencing was performed for further validation.The genomic DNA of pancreatic cancer cell lines BXPC3,CFPAC1,PANC1 and SW1990 was extracted.The COBRA (combined bisulfite restriction analysis) was used to validate differentially expressed hypermethylation miRNA.ResultsEight differentially expressed hypermethylation miRNAs were screened from the DNA methylation chips,then five of them were selected for sequencing.The methylation status of miRNA-615,-663,-663b was significantly higher in the PANC1 than in normal tissues (60.6％ vs 7.6％,88.8％ vs 22.2％,94.4％ vs 13.0％ ) ; the methylation status of miRNA-675 was not significantly different between PANC1 and normal pancreatic tissue (76.0％ vs 100％ ).Due to large error in sequencing,miRNA1826 was excluded.The results of COBRA confirmed all the 4 miRNAs were highly methylated in PANC1 ; except for miRNA-675,other 3 miRNAs were highly methylated in BxPC,miRNA-663,miRNA-663b were highly methylated in CFPAC1,while miRNA-615,miRNA-663 were highly methylated in SW1990.ConclusionsHypermethylation miRNAs were differentially expressed between pancreatic cancer cell lines and normal pancreatic tissue,among them,highly methylated miRNA-663 was possibly associated with pancreatic cancer.