Human Lactoferrin Gene Cloning, Site-specific Recombination Vector Construction And Cell Expression

Human lactoferrin (hLTF), also called lactotransefrrin, In large numbers in mammalian milk, aniron-binding Protein in host defense against in efetion and sevevre inflammation, Regulation of the cell's development and efrrumm etabolism, and so on.There of rethe research of rroducing recombinant hTLF by transgenic animal has many values of medical therapy and human health,Have broad market prospects and important theoretical value.ΦC31 integrase (ΦC31-int) belongs to the serine recombinase family that can catalyses the recombination reaction between the phage attachment site (attP) and the bacterial attachment site(attB). Studies prove thatΦC31-int is a valuable site-special integration tool enzyme because of its efficiency and safety. This integrase can perform and site-special chromosomal integration of aimed DNA into host genomes that results in long-term and efficient transgene expression, which also has the DNA capacity dominance. With further study, researchers know more about the mechanism and influence factors on the integration reaction mediated byΦC31-int and serial successful animal gene therapy studies by usingΦC31-int suggest the possible utility of nonvirus vectors asΦC31-int for constructing transgene animal model and gene therapy.In this research,hLTF (human lactoferrin) gene was cloned from human neutrophile granulocyte (hPMNs).And the vector pSC3/HLTF was constructed which contained eGFP gene and hLTF gene co-expression wth the eucaryon expression vector pDB2 that contained attB side and eGFP gene.The Vector was cotransfection withΦC31-int expression vector pCMV-int into HEK293cell cultured in vitro and the expression cell strains were abtained.High-level expression vector was provided for the neo resistance gene filter with several days and detection,in order to attest that this projet was feasible and in reason.The results of research is as following:1.Human hLTF (human lactoferrin) gene was cloned from human neutrophile granulocyte (hPMNs) totle RNA with RT-PCR,which was 2136 bp and comprised intact coding regions.Sequence analysis demonstrated that its homology with the relative region of human lysozyme on GenBank was 99.9%.Within the open reading frame,three site-mutations occured which could not alter protein's function.2.According to sequence analysis, cut down and attachall kind of phage vector and DNA fragement,get the shuttle vectors pSC3/hLTF.3.The vector pSC3/hLTF and pCMV-int was transfected into 293 cells cultured in research transfect method. After 7day's G418 fastness screening,the expression of report gene eGFP mainly in cytoplasm was observed under fluoroscope.It's proved that the report gene, eGFP expressed in this cultured system.The positive cloned cells were studied by RT-PCR,the result indicated that the vector had integrated into 293 cellular chromatin.Positive cells were proliferated and induced by hormone.The result of RT-PCR analysis on cultured cell showed that transfected cells could expressed the exogenic protein.