A simple and universal ligation mediated fusion of genes based on hetero-staggered PCR for generating immunodominant chimeric proteins.
We developed a simple T4 DNA ligase mediated strategy for inframe splicing of two or more cohesive genes generated by hetero-staggered PCR and directionally cloning the spliced product bearing sticky overhangs in to a correspondingly cut vector. For this, two pairs of primers are used in two different parallel PCRs, for generation of each cohesive gene product. We exemplified this strategy by splicing two major super-antigen genes of Staphylococcus aureus, namely, staphylococcal enterotoxin A (sea), and toxic shock syndrome toxin (tsst-1) followed by its directional cloning into pre-digested pRSET A vector. The fusion gene encoding chimeric recombinant SEA-TSST protein (32kDa) was expressed in E. coli BL21(DE3) host strain. The recombinant chimeric protein retained the antigenicity of both toxins as observed by the strong immunoreactivity with commercial antibodies against both SEA and TSST-1 toxin components by Western blot analysis. We observed that the present method for gene splicing with cohesive ends is simple since it does not require elaborate standardization and a single fusion product is obtained consistently during nested PCR with forward primer of first gene and reverse primer of second gene. For comparison, we fused the same genes using splicing by overlap extension PCR (SOE-PCR) and consistently obtained DNA smearing and multiple non-specific bands even after several rounds of PCRs from gel excised product. Moreover, the newly described method requires only two to six complimentary sticky ends between the genes to be spliced, in contrast to long stretch of overlapping nucleotides in case of SOE-PCR.
Defence Food Research Laboratory, Mysore, 570011, India.
Prakash K
PK
Reddy
Shylaja
S
Ramlal
Murali H
MH
Sripathy
Harshvardhan
H
Batra
D000907:Antibodies, Bacterial; D019677:Artificial Gene Fusion / Q000379:methods*; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D011088:DNA Ligases; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004768:Enterotoxins / Q000235:genetics / Q000276:immunology; D016056:Immunodominant Epitopes / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012326:RNA Splicing / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology*; D013211:Staphylococcus aureus / Q000235:genetics / Q000276:immunology; D018089:Superantigens / Q000235:genetics / Q000276:immunology
D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D017931:DNA Primers; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004768:Enterotoxins; D016056:Immunodominant Epitopes; D011993:Recombinant Fusion Proteins; D018089:Superantigens; C030107:enterotoxin F, Staphylococcal; C026066:enterotoxin A, Staphylococcal; D011088:DNA Ligases