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.

Identification and characterization of integron mediated antibiotic resistance in pentachlorophenol degrading bacterium isolated from the chemostat

A bacterial consortium was developed by continuous enrichment of microbial population isolated from sediment core of pulp and paper mill effluent in mineral salts medium（MSM） supplemented with pentachlorophenol（PCP） as sole source of carbon and energy in the chemostat.The consortia contained three bacterial strains.They were identified as Escherichia coli,Pseudomonas aeruginosa and Acinetobacter sp.by 16S rRNA gene sequence analysis.Acinetobacter sp.readily degraded PCP through the formation of tetrachloro-p-hydroquinone（TecH）,2-chloro-1,4-benzenediol and products of ortho ring cleavage detected by gas chromatograph/mass spectrometer（GC-MS）.Out of the three acclimated PCP degrading bacterial strains only one strain,Acinetobacter sp.showed the presence of integron gene cassette as a marker of its stability and antibiotic resistance.The strain possessed a 4.17 kb amplicon with 22 ORF＇s.The plasmid isolated from the Acinetobacter sp.was subjected to shotgun cloning through restriction digestion by BamHI,HindIII and SalI,ligated to pUC19 vector and transformed into E.coli XLBlue1α,and finally selected on MSM containing PCP as sole source of carbon and energy with ampicillin as antibiotic marker.DNA sequence analysis of recombinant clones indicated homology with integron gene cassette and multiple antibiotic resistance genes.

人c－fos原癌基因cDNA克隆及核酸序列分析

本研究应用逆转录ＰＣＲ方法从Ｊｕｒｋａｔ传代细胞株获得一３８０ｂｐ的人ｃ一ｆｏｓ原癌基因ｃＤＮＡ片段，并将其克隆到具有双向转录启动子的载体质粒ｐＧＥＭ３Ｚｆ（＋）中。核酸序列分析表明，所克隆的ｃＤＮＡ片段包括人ｃ－ｆｏｓ基因外显子２的大部分，外显子３的全部及外显子４的一部分。该重组质粒可分别用于制备ｃ－ｆｏｓｃＤＮＡ探针和ＲＮＡ探针，为ｃ－ｆｏｓ基因结构、表达调控等方面的研究奠定了工作基础。

Cloning of a novel phytase from an anaerobic rumen bacterium, Mitsuokella jalaludinii, and its expression in Escherichia coli

The ful length phytase gene of Mitsuokel a jalaludini was successful y cloned and was found to be 1 047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7 phytase gene of M. jalaludini with various microbial phytase gene sequences showed that it was not similar to those from other bacteria except Selenomonas ruminatium, thus suggesting that they may both express a new class of phytase. The PHY7 phytase gene was subsequently subcloned into bacterial expression vector, pET32a, for expression in Escherichia coli strain Ro-setta-gami. Expression of the recombinant phytase gene was optimised and characterised. The recombinant phytase was estimated to be approximately 55 kDa by SDS-PAGE analysis. The recombinant phytase exhibited optimum activity at 55°C, pH 4.5 and showed good pH stability from pH 3.5 to 5.5 (>78%relative activity). Metal ions such as Ca2+, Mg2+, and K+were found to exert signiifcant stimulatory effect on the recombinant phytase activity while Cu2+, Fe3+, and Zn2+greatly inhibited the enzyme activity. The recombinant phytase showed moderate resistance to trypsin proteolysis, but susceptible to pepsin proteolysis. The results of the study showed that several characteristics of recombinant phytase were slightly different from the native enzyme. Unfavourable characteristics such as reduced pH stability and metal ion effects should be taken into consideration during feed enzyme formulation.

Rapid generation of recombinant Pseudomonas putida secondary metabolite producers using yTREX

Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture.Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts.We have previously established the TREX system which facilitates the transfer,integration and expression of biosynthetic gene clusters in various bacterial hosts.Here,we describe the yTREX system,a new tool adapted for one-step yeast recombinational cloning of gene clusters.We show that with yTREX,Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition.Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning,transfer and expression of the respective biosynthesis genes from Serratia marcescens.Furthermore,the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum,producing pathway metabolites violacein,deoxyviolacein,prodeoxyviolacein,and deoxychromoviridans.Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype.Finally,the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa.All compounds accumulated to substantial titers in the mg range.We thus corroborate here the suitability of P.putida for the biosynthesis of diverse natural products,and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters,applicable for natural compound discovery and combinatorial biosynthesis.