Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

AIM： To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS： Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type （wt） and mutant （mut） forms of the lumican gene were synthesized and amplified by polymerase chain reaction （PCR）. The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown- multiple cloning site （MCS）-/internal ribozyme entry site （IRES）/enhanced green fluorescent protein （EGFP）. Then the desired DNA fragments were integrated into the destination vector pAV.Desld yielding the final expression constructs pAV.Exld-CMV〉wt-lumican/IRES/ EGFP and pAV.Exld-cytomegalovirus （CMV） 〉mutlumican/IRES/EGFP, respectively.RESULTS： The adenovirus plasmids pAV.Exld-CMV〉 wt-lumican/IRES/EGFP and pAV.Exld-CMV 〉mutlumican/IRESlEGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION： We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumicangene in the pathogenesis of high myopia.

The Study of Food-Grade Induced Expression and Enzymatic Properties of L-Arabinose Isomerase from Lactobacillus plantarum WU14 with High D-Tagatose Yield

L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillus plantarum was isolated from the number of lactic acid bacteria from pickled vegetables. The crude L-arabinose isomerase activity of Lactobacillus plantarum WU14 with high D-tagatose yield was 13.95 U/mL under the optimal temperature 60&degC, pH 7.17 and substrate concentration 0.8 mol/L, and the conversion rate of 56.12% could be gained after 28 hours. Protein structure and specific of L-Arabinose Isomerase of Lactobacillus plantarum WU14 were researched. The results showed that L-arabinose isomerase is mainly composed of alpha helix and random coil. Then the recombinant L-AI gene was inserted into the food-grade expression vector pRNA48 and expressed in L. lactis NZ9000 successfully. The target protein expression reached the maximum amount when the induced concentration of nisin reaches 30 ng/mL after 12 h. And the crude enzyme activity of recombinant bacteria reached 6.21 U/mL under 60&degC. Otherwise the optimal conversion rate recombinant of L. lactis NZ9000/pRNA48-L-AI can reach 39.21% under the temperature of 50&degC, pH 7.17 and D-galactose concentration was 0.6 mol/L.

Expression of TIMP-3 Gene by Construction of a Eukaryotic Cell Expression Vector and Its Role in Reduction of Metastasis in a Human Breast Cancer Cell Line

The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 gene from the human placent by RT-PCR.TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector.Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent.Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined.The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis,PCR amplication and nucleotide sequencing.Western blotting showed that the transfected cells were able to express TIMP-3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully.Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453.Cellular & Molecular Immunology.2004;1(4):308-310.