Prokaryotic expression of functional PTEN in Escherichia coli and preparation of polyclonal antibody / 生物工程学报

PTEN, a dual-specificity phosphatase, exerts its tumor-suppressive effects through the inhibition of cell cycle progression and cell immigration, therefore could be an important candidate for tumor-suppression. As study on prokaryotic expression of PTEN and its anti-tumor functions has not been reported, the present study aims at an efficient expression of functional PTEN in Escherichia coli and the investigation of its tumor-suppression activity. PTEN cDNA cloned in our lab previously was recombined into prokaryotic expression vector pET-44a(+) to construct pET-PTEN (pEP) and pET-Nus-PTEN (pENP). PTEN was fused with 6 x His tag in pEP, and with Nus in pENP, which could be useful for a stable and soluble expression. The recombinant vectors were transformed into both BL21 (DE3) (BL) and Rosetta-gami (DE3) pLysS (RG). The former is a normal expression host while the latter is optimized for expression of eukaryotic genes and folding of target proteins. On the induction of 0.5mmol/L IPTG, 55kD and 118kD specific protein bands were observed, corresponding to His-PTEN and Nus-PTEN fusion proteins, respectively. Western blot analysis showed the recombinant fusion proteins could react with PTEN polyclonal antibody. The recombinant HTEN was expressed both in soluble fraction and inclusion body. Higher expression levels of recombinant PTEN were obtained in BL (His-PTEN: 10.3%; NusA-PTEN: 18.7%), whereas the higher percentages of soluble recombinant proteins were observed in RG (His-PTEN: 4.7%; Nus-PTEN: 6.6%). The obtained recombinant fusion proteins were purified by affinity chromatography and were showed to be homogeneous in SDS-PAGE. In tumor-suppression experiments, His-PTEN was proved to have significant inhibition on growth of mice solid tumor with an inhibitory ratio of 58.76%, and on the proliferation of DU-145 tumor cells with an inhibitory ratio of 46.16%. The cell cycle progression of DU-145 tumor cells was also arrested from G0/G1 to S phase. His-PTEN from RG was proved to have significantly higher tumor-suppression activity than that from BL, indicating that there may be some advantages for eukaryotic genes to be expressed in the former host. This is the report of functional recombinant PTEN expressed in Escherichia coli. Purified His-PTEN was used for immunizing Kunming mice, and ascitic polyclonal antibodies raised against His-PTEN were generated using sarcoma 180 cells. At 1:2000 dilution, the antibodies could interact with PTEN by western blot. The present study has laid a foundation for application of PTEN in cancer therapy.

Polymorphism of CYP1A1 gene Msp I site in the Mongolian and Han nationality populations of Inner Mongolia of China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the polymorphism of CYP1A1 gene Msp I site in the Mongolian and Han nationality populations of Inner Mongolia.</p><p><b>METHODS</b>The PCR-restriction fragment length polymorphism(PCR-RFLP) technique was used to analyze the genotypes of CYP1A1 gene Msp I site in 80 subjects of Mongolian nationality and 120 subjects of Han nationality among whom there is no blood relationship each other.</p><p><b>RESULTS</b>The genotype frequency of CYP1A1 gene Msp I site showed that the wild-type, heterozygote, homozygous variants were 35.0%, 48.7%, 16.3% and 33.3%, 52.5%, 14.2% respectively distributions of Mongolian nationality and Han nationality population, and the Chi-square tests showed that there was no significant difference between the two groups.</p><p><b>CONCLUSION</b>The genotype frequency distributions of CYP1A1 gene Msp I site did not exhibit the obvious difference between Mongolian nationality and Han nationality population of Inner Mongolia.</p>

Influence of smoke tar on mRNA expression of aromatic hydrocarbon receptor and cytochrome P4501A1 gene of mice lungs / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the influence of the smoke tar on the expression of aromatic hydrocarbon receptor (AHR) and the cytochrome P4501Al (CYP1A1) gene of mice lungs.</p><p><b>METHODS</b>The smoke tar of 5.29, 10.58 and 15.87 mg/kg was administered intraperitoneally in mice respectively. RNA of mice lungs was got with RNA kit. RT-PCR technique was used for determining AHR and CYP1A1 gene expression with beta-actin as control.</p><p><b>RESULTS</b>The AHR gene expression level was (0.554 +/- 0.023) for the mice intraperitoneally administered with 5.29 mg/kg smoke tar for 72 hours with the significant difference in gene expression level compared with the Tween-80 group (0.484 +/- 0.045) (P < 0.05). The AHR gene expression levels were (0.555 +/- 0.014), (0.606 +/- 0.051), and (0.566 +/- 0.014), (0.684 +/- 0.069) for the mice intraperitoneally administered with 10.58 and 15.87 mg/kg smoke tar for 48 hours and 72 hours respectively with the significant difference in gene expression level compared with the Tween-80 group (0.486 +/- 0.060, 0.484 +/- 0.045) (P < 0.05, P < 0.01). The CYP1Al gene expression levels were (1.535 +/- 0.021), (1.643 +/- 0.046) and (1.624 +/- 0.056), (1.739 +/- 0.038) respectively with the significant difference compared with the Tween-80 group (l.436 +/- 0.016, 1.404 +/- 0.036) (P < 0.01).</p><p><b>CONCLUSION</b>The smoke tar can regulate up the expression of AHR and CYP1A1 gene at a certain dosage and time. The regulation of the smoke tar for the expression of AHR was earlier than for that of CYP1A1.</p>