AIM To clone and identify the whole cDNA ofMXR7 gene and to find out its expression inhuman HCC,and normal tissues.METHODS The DNA primers were designed andsynthesized according to the whole cDNAsequence of MXR? gene....AIM To clone and identify the whole cDNA ofMXR7 gene and to find out its expression inhuman HCC,and normal tissues.METHODS The DNA primers were designed andsynthesized according to the whole cDNAsequence of MXR? gene.The cDNA of humanHCC was taken as the template while the cDNA ofMXR7 gene was synthesized by polymerasechain reaction（PCR）.Recombinant DNAconforming to reading frame was constructed byconnecting purified PCR product of the cDNA ofMXR? gene with expression vector pGEX-5X-1 offusion protein.The plasmid MXRT/pGEX-5X-1was identified by sequencing.Using <sup>32</sup>p labeledMXR? cDNA as probe,MXR7 mRNA expressionwas detected by Northern blot analysis in 12different human normal tissues,7 preoperativelyuntreated non-liver tumor tissues,30preoperatively untreated HCC,theparacancerous liver tissues and 12 normal livertissues samples.RESULTS Restriction enzyme and sequenceanalysis confirmed that the insertion sequence invector pGEX-5X-1 was the same as the cDNAsequence of MXR7 gene.Northern blot analysisshowed no expression of MXR? mRNA in 12 kindsof normal human tissues including liver,7 tumortissues in other sites and 12 normal livertissues,the frequencies of MXR7 mRNA expression in HCC and paracancerous livertissues were 76.6% and 13.3%,respectively.The frequency of MXR7 mRNA expression in HCCwithout elevation of serum AFP and in HCC【5cm was 90%（9/10）and 83.3%（5/6）,respectively.CONCLUSION MXR7 mRNA is highly expressedin human HCC,which is specific and occurs at anearly stage of HCC,suggesting MXR7 mRNA canbe a tumor biomarker for HCC.The detection ofMXR7 mRNA expression in the biopsied livertissue is helpful in discovering early subclinicalliver cancer in those with negative serum AFP.展开更多
To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid ...To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.展开更多
Objective To study the structure specificity of HPV16E6E7 gene from cervical carcinoma biopsies of patients in Shandong province. Methods The tissue DNAs were extracted from cervical carcinoma biopsies of 14 patients ...Objective To study the structure specificity of HPV16E6E7 gene from cervical carcinoma biopsies of patients in Shandong province. Methods The tissue DNAs were extracted from cervical carcinoma biopsies of 14 patients of Chinese from Shandong province. By PCR method using HPV multiple primers, the HPV types were identified in cervical carcinoma tissues, Using the tissue DNA of the 2 cases with the infection of HPV16 type only as templates, HPV16E6E7 gene was amplified by PCR respectively, then inserted the HPV16E6E7 gene into pALTER I vector, and obtained the recombinant plasmid pALTER HPV16E6E7. The constructs were sequenced using Sanger method, and then compared with the sequence of HPV16E6E7 gene of the prototype. Results Sequencing results showed that HPV16E6E7 gene in the 2 cases had sequence diversity from that of the prototype, and a total of five nucleotide exchanges were detected, four of these led to amino acid exchanges. Conclusion There are structure differences between the HPV16E6E7 of Chinese and that of the prototype.展开更多
Human papillomavirus 16 (HPV-16) is a major high-risk type causing cervical cancer. The E6 and E7 genes in HPV-16 are the major virulent genes in HPV, and we wanted to investigate the polymorphism of E6 and E7 genes o...Human papillomavirus 16 (HPV-16) is a major high-risk type causing cervical cancer. The E6 and E7 genes in HPV-16 are the major virulent genes in HPV, and we wanted to investigate the polymorphism of E6 and E7 genes of HPV-16 originated from Jingjiang, Jiangsu province. In research, HPV-16 sample was collected, and the E6 and E7 genes fragments were amplified by PCR assay. The sequences of E6 and E7 genes were used for phylogenetic analysis by Mega 5.0 software. By comparison, the major mutations of E6 gene were T178G (41.67%) and G658A (17%), as well as C58G, T61A and G188C (14%), T61C and C656A (11%). The mutation of E7 gene was mainly C491A and T935A (23%);G514C, G937C and G519C (11%). The 68 nucleotide site deficiency (69.4%) of E6 and 535 nucleotide site deficiency (23%) of E7 were dominant. In our study, we did not find the relevance between the E6 and E7 genes mutations and pathologic severity. But we think the E6 and E7 genes mutation of HPV-16 might affect vaccine prevention and treatment effect.展开更多
Introduction: Acute tubular necrosis (ATN) is the most prevalent cause of acute renal failure (ARF). Mesenchymal stem cell transplantation has been studied as a potential treatment for renal dysfunction due to ATN. In...Introduction: Acute tubular necrosis (ATN) is the most prevalent cause of acute renal failure (ARF). Mesenchymal stem cell transplantation has been studied as a potential treatment for renal dysfunction due to ATN. Inducible nitric oxide synthase (iNOS), bone morphogenetic protein-7 (BMP-7) and B-cell lymphoma 2 (Bcl-2) are surrogate markers of renal tubular epithelial regeneration and subsequent recovery of renal function following ATN. Methods: Serum creatinine (Scr) and blood urea nitrogen (BUN), as well as expression of iNOS, BMP-7 and Bcl-2 in gentamycin-induced ATN rat kidneys was investigated after human umbilical cord-derived mesenchymal stem cell (HUC-MSC) transplantation. Immunohistochemical staining was performed in 3 groups of rats: gentamycin-induced ATN treated with HUC-MSC, gentamycin-induced ATN without HUC-MSC, and untreated rats not receiving any treatments. Results: HUC-MSC transplantation led to a reduction in Scr and BUN in the kidneys of rats with gentamycin-induced ATN. Expression of iNOS in the HUC-MSC treated group occurred later and the expression levels were much lower during gentamycin-induced ATN compared to rats with ATN that were not treated with HUC-MSC. The expression of BMP-7 and Bcl-2 in the MSC-transplanted group was significantly increased compared to both control groups of rats with injured and healthy renal tubules. Conclusions: HUC-MSCs induce renal protection in a rat model of gentamycin-induced ATN, which is associated with reduced iNOS expression and up-regulation of Bcl-2 and BMP-7.展开更多
An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-dir...An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-directed mutagenesis at L57G, C113R for the E6 protein and C24G, E26G for the E7 protein for HPV16 ofE6E7 [patent pending (CN 101100672)]. The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice, but also maintained very good stability and antigenicity. These results suggests that the HPV16 omfE6E7 gene should undergo further study for application as a safe antigen-specific therapeutic vaccine for HPV16-associated tumors.展开更多
Objective To express human B7. 2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2 which c...Objective To express human B7. 2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2 which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-a. Tke fusion protein consisted of GST and hB7. 2(IgV+C) was identified by SDS-PAGE and Western blotting. T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated hy anti-CD3 antibody. Results The fusion protein GST-hB7. 2 (IgV+ C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7. 2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.展开更多
为探索建立持续稳定表达 h BD- 2的哺乳类动物型工程细胞的可能性 ,作者研究构建真核重组表达载体 p CMV.tag2 B/h BD- 2以进行 COS- 7细胞转染表达实验。将本室克隆获得的 h BD- 2全长 c DNA片段插入带有报告基因的真核表达质粒 p CMV....为探索建立持续稳定表达 h BD- 2的哺乳类动物型工程细胞的可能性 ,作者研究构建真核重组表达载体 p CMV.tag2 B/h BD- 2以进行 COS- 7细胞转染表达实验。将本室克隆获得的 h BD- 2全长 c DNA片段插入带有报告基因的真核表达质粒 p CMV.tag2 B构建出 h BD- 2的重组表达载体 p CMV.tag2 B/h BD- 2 ,并经 DNA测序证明h BD- 2 c DNA片段的插入方向和其全长 c DNA的碱基组成顺序均准确无误。通过脂质体转染法将 p CMV.tag2 B/h BD- 2导入 COS- 7细胞。RT- PCR法和免疫印迹法分别从 m RNA水平和蛋白质水平检测到被转染的 COS- 7细胞系能有效表达 h BD- 2。展开更多
目的检测抑制素在人肝癌细胞SMMC-7721及Huh-7中的基因表达,扩增抑制素全长编码序列。方法分别提取肝癌细胞SMMC-7721及Huh-7细胞总RNA,逆转录合成cDNA。使用Real time PCR,以GAPDH为内参检测抑制素的mRNA表达情况。采用RT-PCR方法扩增...目的检测抑制素在人肝癌细胞SMMC-7721及Huh-7中的基因表达,扩增抑制素全长编码序列。方法分别提取肝癌细胞SMMC-7721及Huh-7细胞总RNA,逆转录合成cDNA。使用Real time PCR,以GAPDH为内参检测抑制素的mRNA表达情况。采用RT-PCR方法扩增抑制素全长编码序列。结果 Real time PCR结果显示,SMMC-7721和Huh-7肝癌细胞株中均有抑制素的表达,且SMMC-7721细胞中抑制素表达量是Huh-7细胞中的6.652倍。RT-PCR方法扩增得到约835 bp的抑制素全长编码序列,条带特异,无非特异性扩增。结论人肝癌细胞株SMMC-7721中抑制素的表达量较高。展开更多
基金the National Natural Science Foundation of China,No.39770379the National Basic Research Project("973")SUGEN,USA.
文摘AIM To clone and identify the whole cDNA ofMXR7 gene and to find out its expression inhuman HCC,and normal tissues.METHODS The DNA primers were designed andsynthesized according to the whole cDNAsequence of MXR? gene.The cDNA of humanHCC was taken as the template while the cDNA ofMXR7 gene was synthesized by polymerasechain reaction（PCR）.Recombinant DNAconforming to reading frame was constructed byconnecting purified PCR product of the cDNA ofMXR? gene with expression vector pGEX-5X-1 offusion protein.The plasmid MXRT/pGEX-5X-1was identified by sequencing.Using <sup>32</sup>p labeledMXR? cDNA as probe,MXR7 mRNA expressionwas detected by Northern blot analysis in 12different human normal tissues,7 preoperativelyuntreated non-liver tumor tissues,30preoperatively untreated HCC,theparacancerous liver tissues and 12 normal livertissues samples.RESULTS Restriction enzyme and sequenceanalysis confirmed that the insertion sequence invector pGEX-5X-1 was the same as the cDNAsequence of MXR7 gene.Northern blot analysisshowed no expression of MXR? mRNA in 12 kindsof normal human tissues including liver,7 tumortissues in other sites and 12 normal livertissues,the frequencies of MXR7 mRNA expression in HCC and paracancerous livertissues were 76.6% and 13.3%,respectively.The frequency of MXR7 mRNA expression in HCCwithout elevation of serum AFP and in HCC【5cm was 90%（9/10）and 83.3%（5/6）,respectively.CONCLUSION MXR7 mRNA is highly expressedin human HCC,which is specific and occurs at anearly stage of HCC,suggesting MXR7 mRNA canbe a tumor biomarker for HCC.The detection ofMXR7 mRNA expression in the biopsied livertissue is helpful in discovering early subclinicalliver cancer in those with negative serum AFP.
基金a grant from National Natural Sciences Foundation of China (No. 30572065/ C03031103)
文摘To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.
基金Supported by National Natural Science Foundation of China(No.3 970 0 13 1)
文摘Objective To study the structure specificity of HPV16E6E7 gene from cervical carcinoma biopsies of patients in Shandong province. Methods The tissue DNAs were extracted from cervical carcinoma biopsies of 14 patients of Chinese from Shandong province. By PCR method using HPV multiple primers, the HPV types were identified in cervical carcinoma tissues, Using the tissue DNA of the 2 cases with the infection of HPV16 type only as templates, HPV16E6E7 gene was amplified by PCR respectively, then inserted the HPV16E6E7 gene into pALTER I vector, and obtained the recombinant plasmid pALTER HPV16E6E7. The constructs were sequenced using Sanger method, and then compared with the sequence of HPV16E6E7 gene of the prototype. Results Sequencing results showed that HPV16E6E7 gene in the 2 cases had sequence diversity from that of the prototype, and a total of five nucleotide exchanges were detected, four of these led to amino acid exchanges. Conclusion There are structure differences between the HPV16E6E7 of Chinese and that of the prototype.
文摘Human papillomavirus 16 (HPV-16) is a major high-risk type causing cervical cancer. The E6 and E7 genes in HPV-16 are the major virulent genes in HPV, and we wanted to investigate the polymorphism of E6 and E7 genes of HPV-16 originated from Jingjiang, Jiangsu province. In research, HPV-16 sample was collected, and the E6 and E7 genes fragments were amplified by PCR assay. The sequences of E6 and E7 genes were used for phylogenetic analysis by Mega 5.0 software. By comparison, the major mutations of E6 gene were T178G (41.67%) and G658A (17%), as well as C58G, T61A and G188C (14%), T61C and C656A (11%). The mutation of E7 gene was mainly C491A and T935A (23%);G514C, G937C and G519C (11%). The 68 nucleotide site deficiency (69.4%) of E6 and 535 nucleotide site deficiency (23%) of E7 were dominant. In our study, we did not find the relevance between the E6 and E7 genes mutations and pathologic severity. But we think the E6 and E7 genes mutation of HPV-16 might affect vaccine prevention and treatment effect.
文摘Introduction: Acute tubular necrosis (ATN) is the most prevalent cause of acute renal failure (ARF). Mesenchymal stem cell transplantation has been studied as a potential treatment for renal dysfunction due to ATN. Inducible nitric oxide synthase (iNOS), bone morphogenetic protein-7 (BMP-7) and B-cell lymphoma 2 (Bcl-2) are surrogate markers of renal tubular epithelial regeneration and subsequent recovery of renal function following ATN. Methods: Serum creatinine (Scr) and blood urea nitrogen (BUN), as well as expression of iNOS, BMP-7 and Bcl-2 in gentamycin-induced ATN rat kidneys was investigated after human umbilical cord-derived mesenchymal stem cell (HUC-MSC) transplantation. Immunohistochemical staining was performed in 3 groups of rats: gentamycin-induced ATN treated with HUC-MSC, gentamycin-induced ATN without HUC-MSC, and untreated rats not receiving any treatments. Results: HUC-MSC transplantation led to a reduction in Scr and BUN in the kidneys of rats with gentamycin-induced ATN. Expression of iNOS in the HUC-MSC treated group occurred later and the expression levels were much lower during gentamycin-induced ATN compared to rats with ATN that were not treated with HUC-MSC. The expression of BMP-7 and Bcl-2 in the MSC-transplanted group was significantly increased compared to both control groups of rats with injured and healthy renal tubules. Conclusions: HUC-MSCs induce renal protection in a rat model of gentamycin-induced ATN, which is associated with reduced iNOS expression and up-regulation of Bcl-2 and BMP-7.
基金National 863 Program(2007AA021107)Beijing Municipal Commission of Education Research Projects(KM201010005008)Doctoral Start-up Foundation of Beijing University of Technology
文摘An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-directed mutagenesis at L57G, C113R for the E6 protein and C24G, E26G for the E7 protein for HPV16 ofE6E7 [patent pending (CN 101100672)]. The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice, but also maintained very good stability and antigenicity. These results suggests that the HPV16 omfE6E7 gene should undergo further study for application as a safe antigen-specific therapeutic vaccine for HPV16-associated tumors.
基金This work was supported by the National Natural Science Foundation of China(No. 39470293).
文摘Objective To express human B7. 2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2 which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-a. Tke fusion protein consisted of GST and hB7. 2(IgV+C) was identified by SDS-PAGE and Western blotting. T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated hy anti-CD3 antibody. Results The fusion protein GST-hB7. 2 (IgV+ C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7. 2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.
文摘为探索建立持续稳定表达 h BD- 2的哺乳类动物型工程细胞的可能性 ,作者研究构建真核重组表达载体 p CMV.tag2 B/h BD- 2以进行 COS- 7细胞转染表达实验。将本室克隆获得的 h BD- 2全长 c DNA片段插入带有报告基因的真核表达质粒 p CMV.tag2 B构建出 h BD- 2的重组表达载体 p CMV.tag2 B/h BD- 2 ,并经 DNA测序证明h BD- 2 c DNA片段的插入方向和其全长 c DNA的碱基组成顺序均准确无误。通过脂质体转染法将 p CMV.tag2 B/h BD- 2导入 COS- 7细胞。RT- PCR法和免疫印迹法分别从 m RNA水平和蛋白质水平检测到被转染的 COS- 7细胞系能有效表达 h BD- 2。
文摘目的检测抑制素在人肝癌细胞SMMC-7721及Huh-7中的基因表达,扩增抑制素全长编码序列。方法分别提取肝癌细胞SMMC-7721及Huh-7细胞总RNA,逆转录合成cDNA。使用Real time PCR,以GAPDH为内参检测抑制素的mRNA表达情况。采用RT-PCR方法扩增抑制素全长编码序列。结果 Real time PCR结果显示,SMMC-7721和Huh-7肝癌细胞株中均有抑制素的表达,且SMMC-7721细胞中抑制素表达量是Huh-7细胞中的6.652倍。RT-PCR方法扩增得到约835 bp的抑制素全长编码序列,条带特异,无非特异性扩增。结论人肝癌细胞株SMMC-7721中抑制素的表达量较高。
