AIM To investigate the activation, expression of c src gene and its role in the carcinogenetic process of human cardia adenocarcinoma (CA). METHODS Fifty six cases of CA, 34 cases of normal, 36 cases of protiferative ...AIM To investigate the activation, expression of c src gene and its role in the carcinogenetic process of human cardia adenocarcinoma (CA). METHODS Fifty six cases of CA, 34 cases of normal, 36 cases of protiferative epithelia adjacent to carcinoma, and 20 cases of lymph node metastases of CA were studied for PP60 c src , the expression product of c src gene immunohistochemically by using the specific monoclonal antibody, Mab327. RESULTS The positive rates of PP60 c src in the normal epithelia, protiferative epithelia, CA and lymph node metastases were 29 4% (10/*!34), 94 4% (34/*!36), 71 4% (40/*!56) and 60 0% (12/*!20) , respectively, among them, the differences of the positive rates were statistically significant ( P <0 01) . The expression levels of PP60 c src in CA and proliferative epithelia were significantly higher than that in the normal epithelia ( P <0 01) . The PP60 c src positive rates in the papillary, tubular, poorly differentiated and mucous adenocarcinoma were 75 0% (6/*!8) , 81 8% (18/*!22) , 50 0% (10/*!20) and 100 0% (6/*!6) , respectively, whereas those of tubular and mucous adenocarcinomas were significantly higher than those of papillary and poorly differentiated adenocarcinomas ( P <0 05) , and the PP60 c src expression levels of tubular and mucous adenocarcinomas were also significantly higher than those of papillary and poorly differentiated adenocarcinomas ( P <0 01) . CONCLUSION The activation and expression of c src gene are associated with the initiation and development of human CA; the protein amount of PP60 c src increased during the process of carcinogenesis; and PP60 c src expression is also related to lymph node metastases.展开更多
To make up an engineered E.coli strain for expression of antigenic determinants of pp150 of human cytomegalovirus,the gene encoding the 420-752 amino acid peptides of pp150 was amplified from human cytomegalovirus AD1...To make up an engineered E.coli strain for expression of antigenic determinants of pp150 of human cytomegalovirus,the gene encoding the 420-752 amino acid peptides of pp150 was amplified from human cytomegalovirus AD169 by RT-PCR and cloned into Nde I/BamHⅠ sites in pET30a.The recombinant plasmid was transformed into E.coli BL21(DE3)and a foreign protein about 35 kD were detected by SDS-PAGE and Western blotting assay.The antiserum prepared by using prokaryotic expressed product as antigen turned to be against pp150 specifically and had low neutralization effect to HCMV.展开更多
There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene; 1.8-kb mRNA transcript gene family in the genome of Marek's disease virus (MDV). In this study, enhanced green fluorescenc...There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene; 1.8-kb mRNA transcript gene family in the genome of Marek's disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) reporter plamids, pP(pp38)-EGFP; pP(1.8-kb)-EGFP, were constructed under this bi-directional promoter in two directions. The two plasmids were transfected into uninfected chicken embryo fibroblast (CEF), MDV clone rMd5 infected CEF (rMd5-CEF); pp38-deleted derivative rMd5Δpp38 infected CEF (rMd5Δpp38-CEF) respectively. Transfection analysis showed that EGFP was only expressed in rMd5-CEF,; no EGFP could be detected in uninfected CEF or rMd5Δpp38-CEF, implying that pp38 was a factor influencing the activity of the promoter. The pp38-expressing recombinant plasmid pcDNA-pp38 was constructed to co-transfect CEF or rMd5Δpp38-CEF with pP(pp38)-EGFP or pP(1.8-kb)-EGFP. In this case, EGFP could be detected only in rMd5Δpp38-CEF but still not in uninfected CEF, implying that pp38 needs other protein(s) to work together for the complete trans-acting activity. Another MDV gene, 24 kd phosphorylated protein pp24 gene was cloned into pcDNA3.1 as a pp24-expressing recombinant plasmid pcDNA-pp24. When uninfected CEF was co-transfected with pcDNA-pp38, pcDNA-pp24; EGFP expressing plasmids pP(pp38)-EGFP or pP(1.8-kb)-EGFP, the EGFP could be detected. These results indicated that pp38; pp24 could enhance the activity of the promoter when they worked together. DNA mobility shift assay showed that pp38 would bind to the bi-directional promoter with the co-existing of pp24, although neither of them alone influenced mobility of the promoter DNA. All the above suggested that MDV pp38 could transactivate the bi-directional promoter when combined with pp24. The results also indicated that the activity of the promoter in the direction of 1.8-kb mRNA was significantly stronger than that of pp38 direction.展开更多
Objective To investigate the polymorphism of human cytomegalovirus (HCMV) UL150 open reading frame (ORF) in low-passaged clinical isolates, and to study the relationship between the polymorphism and different pathogen...Objective To investigate the polymorphism of human cytomegalovirus (HCMV) UL150 open reading frame (ORF) in low-passaged clinical isolates, and to study the relationship between the polymorphism and different pathogenesis of congenital HCMV infection. Methods PCR was performed to amplify the entire HCMV UL150 ORF region of 29 clinical isolates, which had been proven containing detectable HCMV-DNA using fluorescence quantitative PCR. PCR amplification products were sequenced directly, and the data were analyzed. Results Totally 25 among 29 isolates were amplified, and 18 isolates were sequenced successfully. HCMV UL150 ORF sequences derived from congenitally infected infants were high variability. The UL150 ORF in all 18 clinical isolates shifted backward by 8 nucleotides leading to frame-shift, and contained a single nucleotide deletion at nucleotide position 226 compared with that of Toledo strain. The nucleotide diversity was 0.1% to 6.8% and the amino acid diversity was 0.2% to 19.2% related to Toledo strain. However, the nucleotide diversity was 0.1% to 6.4% and amino acid diversity was 0.2% to 8.3% by compared with Merlin strain. Compared with Toledo, 4 new cysteine residues and 13 additional posttranslational modification sites were observed in UL150 putative proteins of clinical isolates. Moreover, the UL150 putative protein contained an additional transmembrane helix at position of 4-17 amino acid related to Toledo. Conclusion HCMV UL150 ORF and deduced amino acid sequences of clinical strains are hypervariability. No obvious linkage between the polymorphism and different pathogenesis of congenital HCMV infection is found.展开更多
文摘AIM To investigate the activation, expression of c src gene and its role in the carcinogenetic process of human cardia adenocarcinoma (CA). METHODS Fifty six cases of CA, 34 cases of normal, 36 cases of protiferative epithelia adjacent to carcinoma, and 20 cases of lymph node metastases of CA were studied for PP60 c src , the expression product of c src gene immunohistochemically by using the specific monoclonal antibody, Mab327. RESULTS The positive rates of PP60 c src in the normal epithelia, protiferative epithelia, CA and lymph node metastases were 29 4% (10/*!34), 94 4% (34/*!36), 71 4% (40/*!56) and 60 0% (12/*!20) , respectively, among them, the differences of the positive rates were statistically significant ( P <0 01) . The expression levels of PP60 c src in CA and proliferative epithelia were significantly higher than that in the normal epithelia ( P <0 01) . The PP60 c src positive rates in the papillary, tubular, poorly differentiated and mucous adenocarcinoma were 75 0% (6/*!8) , 81 8% (18/*!22) , 50 0% (10/*!20) and 100 0% (6/*!6) , respectively, whereas those of tubular and mucous adenocarcinomas were significantly higher than those of papillary and poorly differentiated adenocarcinomas ( P <0 05) , and the PP60 c src expression levels of tubular and mucous adenocarcinomas were also significantly higher than those of papillary and poorly differentiated adenocarcinomas ( P <0 01) . CONCLUSION The activation and expression of c src gene are associated with the initiation and development of human CA; the protein amount of PP60 c src increased during the process of carcinogenesis; and PP60 c src expression is also related to lymph node metastases.
文摘To make up an engineered E.coli strain for expression of antigenic determinants of pp150 of human cytomegalovirus,the gene encoding the 420-752 amino acid peptides of pp150 was amplified from human cytomegalovirus AD169 by RT-PCR and cloned into Nde I/BamHⅠ sites in pET30a.The recombinant plasmid was transformed into E.coli BL21(DE3)and a foreign protein about 35 kD were detected by SDS-PAGE and Western blotting assay.The antiserum prepared by using prokaryotic expressed product as antigen turned to be against pp150 specifically and had low neutralization effect to HCMV.
基金supported by the National Natural Science Foundation of China(Grant Nos.30300450&30070544).
文摘There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene; 1.8-kb mRNA transcript gene family in the genome of Marek's disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) reporter plamids, pP(pp38)-EGFP; pP(1.8-kb)-EGFP, were constructed under this bi-directional promoter in two directions. The two plasmids were transfected into uninfected chicken embryo fibroblast (CEF), MDV clone rMd5 infected CEF (rMd5-CEF); pp38-deleted derivative rMd5Δpp38 infected CEF (rMd5Δpp38-CEF) respectively. Transfection analysis showed that EGFP was only expressed in rMd5-CEF,; no EGFP could be detected in uninfected CEF or rMd5Δpp38-CEF, implying that pp38 was a factor influencing the activity of the promoter. The pp38-expressing recombinant plasmid pcDNA-pp38 was constructed to co-transfect CEF or rMd5Δpp38-CEF with pP(pp38)-EGFP or pP(1.8-kb)-EGFP. In this case, EGFP could be detected only in rMd5Δpp38-CEF but still not in uninfected CEF, implying that pp38 needs other protein(s) to work together for the complete trans-acting activity. Another MDV gene, 24 kd phosphorylated protein pp24 gene was cloned into pcDNA3.1 as a pp24-expressing recombinant plasmid pcDNA-pp24. When uninfected CEF was co-transfected with pcDNA-pp38, pcDNA-pp24; EGFP expressing plasmids pP(pp38)-EGFP or pP(1.8-kb)-EGFP, the EGFP could be detected. These results indicated that pp38; pp24 could enhance the activity of the promoter when they worked together. DNA mobility shift assay showed that pp38 would bind to the bi-directional promoter with the co-existing of pp24, although neither of them alone influenced mobility of the promoter DNA. All the above suggested that MDV pp38 could transactivate the bi-directional promoter when combined with pp24. The results also indicated that the activity of the promoter in the direction of 1.8-kb mRNA was significantly stronger than that of pp38 direction.
基金Supported by the National Natural Science Foundation of China(30170986,30371492).
文摘Objective To investigate the polymorphism of human cytomegalovirus (HCMV) UL150 open reading frame (ORF) in low-passaged clinical isolates, and to study the relationship between the polymorphism and different pathogenesis of congenital HCMV infection. Methods PCR was performed to amplify the entire HCMV UL150 ORF region of 29 clinical isolates, which had been proven containing detectable HCMV-DNA using fluorescence quantitative PCR. PCR amplification products were sequenced directly, and the data were analyzed. Results Totally 25 among 29 isolates were amplified, and 18 isolates were sequenced successfully. HCMV UL150 ORF sequences derived from congenitally infected infants were high variability. The UL150 ORF in all 18 clinical isolates shifted backward by 8 nucleotides leading to frame-shift, and contained a single nucleotide deletion at nucleotide position 226 compared with that of Toledo strain. The nucleotide diversity was 0.1% to 6.8% and the amino acid diversity was 0.2% to 19.2% related to Toledo strain. However, the nucleotide diversity was 0.1% to 6.4% and amino acid diversity was 0.2% to 8.3% by compared with Merlin strain. Compared with Toledo, 4 new cysteine residues and 13 additional posttranslational modification sites were observed in UL150 putative proteins of clinical isolates. Moreover, the UL150 putative protein contained an additional transmembrane helix at position of 4-17 amino acid related to Toledo. Conclusion HCMV UL150 ORF and deduced amino acid sequences of clinical strains are hypervariability. No obvious linkage between the polymorphism and different pathogenesis of congenital HCMV infection is found.
