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Objective:To clone the full-length glycosyltransferase genes （<italic>PpUGT</italic>1，<italic>PpUGT</italic>7） related to saponins biosynthesis in <italic>Paris polyphylla</italic> var. <italic>yunnanensis</italic>，and perform bioinformatics analysis，relative expression analysis and prokaryotic expression analysis. Method:Total RNA was isolated from <italic>P. polyphylla </italic>var. <italic>yunnanensis </italic>with use of the Eastep<sup>®</sup> Super Total RNA Extraction Kit and converted to cDNA. Specific primers were designed according to the transcriptome data to clone the full-length gene. Relevant software was then used for bioinformatic analysis of the protein sequences. The relative gene expression levels were detected by real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） and the prokaryotic expression vectors were built to heterologously express recombinant protein in <italic>Escherichia coli.</italic> Result:The open reading frame （ORF） of <italic>PpUGT</italic>1 was 1 827 bp，encoding 608 amino acids，and was predicted as a steroid glycosyltransferase；the ORF of <italic>PpUGT</italic>7 was 1 380 bp，encoding 459 amino acids，and was predicted as a triterpenoid glycosyltransferase. The calculated relative molecular mass of two proteins were 67.6 kDa and 51.3 kDa respectively，and both of them were hydrophilic proteins，no transmembrane domain，no signal peptides，both showing high similarity and conservativeness with homologous sequences. The results of Real-time PCR showed that the expression level of <italic>PpUGT</italic>1 was root>leaf>flower>stem；the expression level of <italic>PpUGT</italic>7 was stem>leaf>flower>root. In addition，PpUGTs proteins were expressed in <italic>E. coli</italic>. in a soluble form. Conclusion:The genes of <italic>PpUGT</italic>1 and <italic>PpUGT</italic>7 were cloned successfully. Real-time PCR showed the genes were expressed differently in different plant organs， and their recombinant proteins were successfully expressed in <italic>Escherichia coli</italic>. This study lays a foundation for functional characterization of PpUGTs and analysis of the biosynthesis pathway of saponins in <italic>Paris polyphylla </italic>var. <italic>yunnanensis</italic>.
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Objective To construct human yippee-like 5(YPEL5) gene eukaryotic expression recombinant plasmid and to express in esophageal carcinoma EC9706 cells .Methods The cDNA from human normal tissue was taken as a template and amplified to YPEL5 gene coding sequence with 366 bp in length .Then this se-quence was inserted into the multiple cloning site areas of eukaryotic expression vector pCDH-CD513B for ob-taining the eukaryotic expression vector pCDH-CD513B-Flag-YPEL5 .After the bacterial colony PCR identifi-cation ,it was sent to the corporation for testing the sequence .The successfully constructed recombinant plas-mid was transfected into human esophageal carcinoma EC9706 cells .The expression of PEL5 gene in EC9706 cells was detected by QRT-PCR and Western Blot .Results The YPEL5 gene segment with 366 bp in length was successfully amplified .pCDH-CD513B-Flag-YPEL5 recombinant plasmid was obtained by double enzyme digestion ,connection ,conversion and screening .The gene sequencing identification showed that the inserted gene sequence in recombinant plasmid was consistent with that in the GenBank .After 2 d of transfecting into EC9706 cells ,the QRT-PCR and Western Blot revealed that YPEL5 gene expression was significantly up-reg-ulated .Conclusion The pCDH-CD513B-Flag-YPEL5 eukaryotic expression vector is successfully constructed and is expressed in esophageal squamous cancer cell line EC9706 ,thus which lays a foundation for studying its function in the progression of esophageal cancer .
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Background and purpose: Peroxiredoxin Ⅱ (PrxⅡ) has the activity of peroxidase. The relevant studies found it played an important role in gastric cancer. This study aimed to investigate the expression of PrxⅡ in human gastric cancer tissues and cells, analyze its relationship with clinicopathological characteristics, and explore the relationship between PrxⅡ and the prognosis and the development of gastric cancer. Methods: The expression of PrxⅡmRNA and protein in gastric cancer tissues and the paired adjacent normal tissues from 45 patients was detected by real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot. The same methods were used to detect the expression of PrxⅡ mRNA and protein in GES-1, MGC-803, MKN-45 and MKN-28. Tissue mi-croarray and immunohistochemistry were used to detect the expression of PrxⅡ protein in gastric cancer tissues and the paired adjacent normal tissues from 116 patients. The relationship between the results and clinicopathological char-acteristics was analyzed. The prognosis was analyzed. Results: According to results of RTFQ-PCR and Western blot, we found that PrxⅡ mRNA and protein in gastric cancer tissues were significantly higher than that in adjacent normal tissues (P<0.05). PrxⅡ mRNA and protein in gastric cancer cells were higher than that in normal gastric cells (P<0.01).Immunohistochemistry revealed that the expression of PrxⅡ protein in gastric cancer tissues (76.7％) was also significantly higher (P<0.01) than that in adjacent normal tissues (30.1％). The expression of PrxⅡ protein is significantly related to tumor size, histological differentiation, depth of invasion, TNM stage and lymph node metastasis (P<0.05), but had no significant relationship with the gender, age, tumor location and distant metastasis. Survival in patients with higher PrxⅡ expression significantly shorter than in those with lower expression (P<0.01). PrxⅡ is an independent prognostic factor of gastric cancer (P<0.05). Conclusion: PrxⅡ promotes the development of gastric cancer. It is one of the adverse prognostic factors of gastric cancer and may serve as a new therapeutic target for gastric cancer.
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Objective The aim of this study was to analyze of the prevalence of Norovirus in the foodborne disease surveillance population in Baiyin City in 2015,and provide scientific basis for the prediction,early warning,prevention and control of foodborne diseases caused by Norovirus.Methods A questionnaire survey was conducted on the cases of foodborne disease surveillance.Fecal specimen were collected,and Norovirus GⅠ and GⅡ were detected by real time fluorescence quantitative polymerase chain reaction.Results Three hundred and forty-four foodborne disease cases were reported,78 cases were positive,and the detection rate was 22.7％.Five cases were positive for Norovirus GⅠ,and 71 cases for GⅡ.Two cases were positive for both GⅠ and GⅡ.Forty-six cases were male and 32 cases were female.The oldest patient was 83 years old,and the youngest was only 3 months with an average age of 20.3.Conclusion Norovirus was one of the main foodborne pathogens in the city,and the main epidemic was GⅡ in autumn and winter.Public education and health monitoring should be strengthened.
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Objective To compare the expression of serum miR-100 in patients with esophageal cancer and healthy person ,and explore the value of miR-100 in diagnosis for esophageal cancer .Methods Real-time fluorescent quantitative polymerase chain reac-tion was used to detecting miR-100 in 40 esophageal cancer patients(study group) and 50 healthy person(control group) .Results The expression of miR-100 in the study group and control group were 6 .399 ± 3 .541 ,2 .625 ± 1 .515 respective ,the expression in the study group was significant higher than that of the control group(t= 9 .07 ,P< 0 .05) .The under area of receiver operating char-acteristic curve of miR-100 in diagnosis for esophageal cancer was 0 .832(95% confidence interval was 0 .731 - 0 .934) ,when the Cut off value was 5 .285 ,the sensitivity and specificity of miR-100 in diagnosis for esophageal cancer were 65% and 95% . Conclusion Serum miR-100 in esophageal cancer patients is higher than that in healthy person ,which might be a new molecular markers in diagnosis for esophageal caner .
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Objective To develop a quick and sensitive real-time fluorescent quantitative polymerase chain reaction(RT-PCR) method for detecting human GAPDH gene .Methods According to the published GAPDH gene(NC_000012) mRNA sequence in GeneBank ,a pair of primers was designed in the conserved region .After optimization of reaction system and condition ,the method for detection of human GAPDH gene by SYBR Green RT-PCR was established .Results The measuring range lower limit of GAP-DH gene could reach 15 copies per microlitre and there was a nice linear relationship in statistics between the Ct value and the con-centration gradient of standard plasmid DNA specimen was from 1 .5 × 101 to 1 .5 × 107 per microlitre(r=0 .992) .The melting curve present a single and clear peck and the Tm value was (84 .5 ± 0 .2)℃ .Conclusion The method established in this research is rapid and sensitive ,which provides a methodological basis for quantitative analysis of human functional and etiological gene using GAP-DH as reference gene .
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Objective To evaluate the cefoxitin screening test in detecting(MRSA)with the real-time fluorescent quantitative polymerase chain reaction(RT-PCR)for detecting mecA gene as the golden standard.Methods Staphylococcus aureus strains iso-lated from various bacterial infection specimens were collected,which were amplified by RT-PCR and detected by the cefoxitin screening test respectively.The results were compared.Results In 121strains of MRSA,66 strains carrying mecA gene were detec-ted by RT-PCR(P >0.05),accounting for 54.55%(66/121).There was no statsitcal difference in the detection results between the cefoxitin screening test and RT-PCR.The sensitivity of the cefoxitin screening test was 93.93%,the specificity was 81.82%,the positive predictive value was 84.93% and negative predictive value was 91.83%.Conclusion The RT-PCR technique can accurate-ly and rapidly detect MRSA.
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ObjectiveTo investigate the expression of a disintegrin-like and metalloproteinase 8 ( ADAM8 ) in breast cancer and in normal breast tissues and its negative regulation role in tumorigenesis and progress of breast cancer.MethodsThe expression of ADAM8 in breast cancer and normal breast tissues was detected by immunohistochemistry (IHC),qRT-PCR,and Western blot.The relation between ADAM8 expression and the clinicopathological parameters of breast cancer patients was analyzed.ResultsADAM8 was expressed in breast cancer and normal breast tissues.The expression of ADAM8 mRNA and protein was significantly lower in breast cancer than in normal breast tissues (qRT-PCR:P =0.015,IHC:P =0.044,Western blot:P =0.000).The expression rate of ADAM8 was related to lymph node metastasis,tumor stage and tumor size,although the difference had no statistical significance.IHC results showed that ADAM8 expression level was lower in stage Ⅲ + Ⅳthan in stage Ⅰ + Ⅱ ( P =0.574 ).qRT-PCR showed ADAM8 mRNA expression was lower in stage Ⅱb + Ⅲ than in stage Ⅰ + Ⅱ a ( P =0.247).ADAM8 expression was lower in the breast cancer tissues with lymph node metastasis than in those without lymph node metastasis (P =0.560 by IHC and P =0.592 by qRT-PCR).ADAM8 expression was lower in tumors whose size ＞ 2 cm than in tumors whose size ≤ 2 cm,however,the difference had no statistical significance ( P ＞ 0.05 ).ConclusionsADAM8 is significantly lower-expressed in breast cancer than in normal breast tissues,which is associated with clinical stages and lymph node metastasis.The reduced expression of ADAM8 may play a role in the pathogenesis and progress of breast cancer.
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Objective To study the role of Foxp3 + CD4 + CD25 + regulatory T cells(Foxp3 + CD4 + CD25 + Tregs) and Foxp3mRNA in pathogenesis of passive transferred myasthenia gravis(PTMG) by detecting their expression in a PTMG model.Methods Sixteen C57BL/6 young female mice were divided into model group and control group.A PTMG model was established by injecting Ringer's solution containing 1.0 mg/kg mAb35 into abdominal cavity.Mice in control group were injected with Ringer's solution not containing mAb35.Levels of CD4 + CD25 + T cells and Foxp3 + CD4 + CD25 + Tregs in spleen cells were measured by flow cytometry.Expression of Foxp3 mRNA in spleen cells was detected by real-time fluorescent quantitative polymerase chain reaction(RT-FQ-PCR).Serum level of interleukin-2 and interferon-? was detected by ELISA.Results The ratio of CD4 + CD25 + T cells and CD4 + T cells was higher in model group(8.82 ? 0.74)% than in control group(9.89 ? 0.88)%(P