Method for cloning and expressing target gene by homologous recombination

Abstract

A method for cloning and expressing a target gene by homologous recombination, and more particularly a method for cloning and expressing a target gene by homologous recombination, wherein a host cell transformed with a recombinant vector and a plasmid containing a recombinase system is introduced with a linear DNA fragment comprising a target gene and a sequence having homology to the recombinant vector. Because complicated genetic engineering steps, such as the restriction enzyme treatment and ligation of a vector and a target gene, are not required, the cloning of a gene can be performed without needing a high degree of skill, and enzyme cost can be reduced. The inventive method can be effectively used for the massive, high-speed cloning and protein expression of genes, and the disclosed pRMT-iTGR system can be used as an analytical means for improving high-efficiency recombinase.

1 . A method for cloning and expressing a target gene by homologous recombination, the method comprising the steps of: 
 (a) preparing a recombinant vector containing (i) a first selection marker gene having deletions of a ribosome recognition sequence and a start codon, and thus having no expression ability, (ii) a second selection marker gene that can be expressed, (iii) a promoter and (iv) a forward ribosome recognition sequence, which is constructed such that, if a iTGR( i nsert for  T arget  G ene and  R BS) fragment is inserted between the forward ribosome recognition sequence and the first selection marker gene, the target gene and the first selection marker gene will be expressed;    (b) preparing a microorganism for homologous recombination by introducing the recombinant vector into a microorganism comprising a plasmid containing a homologous recombination-promoting gene;    (c) introducing a linear DNA fragment into the microorganism for homologous recombination, the linear DNA fragment comprising: 
 (i) a base sequence of a target gene,  
 (ii) a base sequence located at the 5′-terminus of the target gene which has a forward ribosome recognition sequence of the recombinant vector prepared in the step (a) and a homologous sequence to a portion of the region downstream of the promoter,  
 (iii) a base sequence located at the 3′-terminus of the target gene which has the backward ribosome recognition sequence of the first selection marker gene of the recombinant vector prepared in the step (a), start codon and a homologous sequence to 5′-terminus of the first selection marker gene; and  
   (d) selecting a transformed microorganism introduced with the target gene using the first selection marker gene and the second selection marker gene.    
     
     
         2 . The method for cloning and expressing a target gene by homologous recombination according to  claim 1 , wherein the iTGR fragment comprises the target gene, the backward ribosome recognition sequence of the first selection marker gene, and a translational start codon.  
     
     
         3 . The method for cloning and expressing a target gene by homologous recombination according to  claim 1 , wherein the promoter is T7 promoter, and the first selection marker gene is a chloramphenicol-resistant gene (cat).  
     
     
         4 . The method for cloning and expressing a target gene by homologous recombination according to  claim 1 , wherein the homologous recombination-promoting gene is a Redα/β or RecE/T gene.  
     
     
         5 . The method for cloning and expressing a target gene by homologous recombination according to  claim 1 , wherein the step (d) comprises selecting a microorganism expressing the first selection marker gene as the microorganism introduced with the target gene.  
     
     
         6 . The method for cloning and expressing a target gene by homologous recombination according to  claim 1 , wherein the selection step (d) comprises selecting a microorganism growing in the presence of the first selection marker as the microorganism introduced with the target gene.  
     
     
         7 . A recombinant vector containing (a) a first selection marker gene having deletions of a backward ribosome recognition sequence and a start codon, and thus having no expression ability, (b) a second selection marker gene that can be expressed, (c) a promoter and (d) a forward ribosome recognition sequence, which is constructed such that, if a iTGR fragment is inserted between the forward ribosome recognition sequence and the first selection marker gene, the iTGR fragment and the first selection marker gene will be expressed.  
     
     
         8 . The recombinant vector according to  claim 7 , wherein the iTGR fragment comprises a target gene, the backward ribosome recognition sequence of the first selection marker gene, and a start codon.  
     
     
         9 . The recombinant vector according to  claim 7 , wherein the promoter is T7 promoter, and the first selection marker gene is a chloramphenicol-resistant gene (cat).  
     
     
         10 . A linear DNA fragment comprising: 
 (a) a base sequence of a target gene;    (b) a base sequence located at the 5′-terminus of the target gene which has a forward ribosome recognition sequence of the recombinant vector of  claim 7  and a homologous sequence to a portion of the region downstream of the promoter; and    (c) a base sequence located at the 3′-terminus of the target gene which has the backward ribosome recognition sequence of the first selection marker gene of the recombinant vector of  claim 7 , start codon and a homologous sequence to 5′-terminus of the first selection marker gene.    
     
     
         11 . A microorganism for homologous recombination, transformed with the recombinant vector of  claim 7  and a plasmid containing a homologous recombination-promoting gene.  
     
     
         12 . The microorganism for homologous recombination according to  claim 11 , wherein the homologous recombination-promoting gene is a Redα/β or RecE/T gene.  
     
     
         13 . A primer for homologous recombination, which is set forth in SEQ ID NO: 7 and contains a backward ribosome recognition sequence, a start codon and a sequence of the 5′-terminus of chloramphenicol-resistant gene (cat).