Preparation and identification of the oligonucleotide chip for detecting the virulence-associated genotypes and drug resistance of Helicobacter pylori / 中国医学科学院学报

<p><b>OBJECTIVE</b>To develop an oligonucleotide microarray-based method for the simultaneous detection of Helicobacter pylori (Hp) infection, virulence-associated genotypes, and drug resistance.</p><p><b>METHODS</b>Hp was classified into cagA + and cagA- genotypes based on its virulence. Clarithromycin-resistance of Hp was identified by existence of point mutations in 23S rRNA. We constructed an oligonucleotide microarray chip to simultaneously diagnose Hp infection and detect its virulence-associated genotypes and mutations associated with clarithromycin-resistance. The diagnostic accuracy of the constructed microarray was tested with templates of wild type and mutated type.</p><p><b>RESULTS</b>The oligonucleotide chip accurately detected cagA + and cagA- genotypes of Hp, as well as four common point mutations in 23S rRNA related to clarithromycin-resistance.</p><p><b>CONCLUSION</b>Oligonucleotide microarray chip can be used to diagnose Hp infection and test its virulence-associated genotypes and drug resistance simultaneously.</p>

Purification of Trypsin by Using AOT/Isooctane Reverse Micellar Extraction and Overcoming Denaturation and Precipitation in the Process / 中国生物工程杂志

Trypsin was purified from crude material of pig pancreas by AOT/isooctane reversed micellar system.The influence of the main operating parameters such as ethanol concentration in forward and backward extraction,pH,KCl and AOT concentration,temperature were investigated.Forward and backward extraction recovery of trypsin reached almost 90% and neared 100%,respectively.Finally,about 88% of total yield was obtained,and the specific activity of trypsin was increased to over 1800U/mg with purification factor of 5 folds more.In AOT-isooctane reverse micellar extraction system,denaturation or precipitation of proteins always occured due to strong electrostatic interaction between AOT-proteins molecules.It had been resolved by adding ethanol into reverse micellar system,and no denaturation was observed.Otherwise,the phase separation time was shortened significantly because of ethanol added.It was only 10 minutes or less to reach phases separation after forward and backward extraction.If this method can be applied in industry,efficiency will be greatly improved.

Expression and purification of recombinant human interleukin 4 in Escherichia coli / 生物工程学报

Human interleukin 4 (IL-4) cDNA was optimized and synthesized according to E. coli preferred codon. A recombinant expression plasmid pET-30a (+)/rhIL-4 was constructed with the target cDNA inserted between Nde I and EcoR I sites, which can translate the mature IL-4 protein with an extra methionine residue at N-terminal. The expression vector was transformed into E. coli BL21 (DE3). The rhIL-4 protein was expressed in the inclusion body. By using the optimized fermentation conditions, the high expression level was achieved with the expression level as high as 35% of total protein obtained. A purification strategy has been designed which includes Q-Sepharose and SP-Sepharose ion-exchange chromatography and dialysis renaturation. The rhIL-4 was purified with the purity more than 98% and the yield of 40 mg per liter fermentation culture achieved. Western blot proved that the purified protein is IL-4. Amino acid sequencing revealed that N-terminal 16 residue sequence is identical to the theoretical sequence. Biological activity assay on TF-1 cells demonstrated that the rhIL-4 is active with an activity of 2.5 x 10(6) AU/mg. This study promises large scale production of rhIL-4.

Detection of P53 and K-ras gene mutations in lung cancer with oligonucleotide chip / 生物工程学报

Different factors including hybridization solution components, hybridization temperature, and the concentration and proportion of the labelled primer, which affected the sensitivity and specificity of single mutation identification, were exploited. Asymmetric PCR increased the hybridization sensitivity, and the asymmetric multi-PCR did not affect the specificity, while the sensitivity was improved a little. Among 30 lung cancer samples detected with the oligonucleotide microarray, 12 was found P53 gene mutations and 5 had K-ras gene mutations. The P53 gene mutations identified by the oligonucleotide microarray was proved 80% same as the sequencing results. The obvious statistical relations of K-ras and P53 gene mutations with tumor type, tumor stage and smoking were not obtained because of less samples and mutation sites.

Study on the Effect of Dark Tea Fermentation Liquid with Eurotium cristatum on the Activity of Digestive Enzyme / 微生物学通报

The effect of dark tea fermentation liquid with Eurotium Cristatum on the activity of digestive enzyme was researched, as fellowing amylase, protease, lipase. The results showed that dark tea fermentation liquid with Eurotium Cristatum may increase remarkably the activity of ?-amylase and protease, but decrease efficiently the activity of lipase. The fermentation liquid improves the digestion and absorption of starch and protein, but inhibits the decomposition and absorption of fat, so it can explain the mechanism of Fu-brick tea's health functions.