Hsv gene expression vector and bxb1 integrase-mediated recombination system for high-throughput cloning

A genetically modified herpes simplex virus (HSV) gene expression vector for expressing one or more heterologous antigen, comprising:

• an HSV-2 or HSV-1 genome comprising a complete deletion of glycoprotein G-encoding gene, glycoprotein J-encoding gene, glycoprotein D-encoding gene, and glycoprotein I-encoding gene;
• a heterologous nucleic acid comprising an expression cassette and inserted in a region of the genome from which the glycoprotein G-encoding gene, glycoprotein J-encoding gene, glycoprotein D-encoding gene, and glycoprotein I-encoding gene have been deleted;
• an attL sequence;
• and
• an attR sequence,
• wherein the attL sequence is adjacent to a first end of the expression cassette and the attR sequence is adjacent to a second end of the expression cassette, and
• wherein the expression cassette comprises in operable communication a promoter, one or more gene encoding the one or more heterologous antigen, and optionally, a selectable marker.

A method of producing a herpes simplex virus (HSV) gene expression vector comprising a gene encoding a heterologous protein, the method comprising:

• providing a genetically modified HSV-1 or HSV-2 genome comprising
• a complete deletion of glycoprotein G-encoding gene, glycoprotein J-encoding gene, glycoprotein D-encoding gene, and glycoprotein I-encoding gene, and
• a first recombination sequence inserted in a region of the genome from which the glycoprotein G-encoding gene, glycoprotein J-encoding gene, glycoprotein D-encoding gene, and glycoprotein I-encoding gene have been deleted;
• providing a heterologous nucleic acid comprising a second recombination sequence and an expression cassette comprising the gene encoding the heterologous protein, wherein the heterologous protein is chosen from dengue virus serotype 2 NS1 antigen, M. tuberculosis gene DnaK, M. tuberculosis groEL-2, M. tuberculosis cut4, M. tuberculosis cut7, M. tuberculosis 1ppx, and M. tuberculosis pstS1;
• an attL sequence;
• and
• an attR sequence,
• wherein the attL sequence is adjacent to a first end of the expression cassette and the attR sequence is adjacent to a second end of the expression cassette, and
• wherein the expression cassette comprises in operable communication a promoter and at least one gene encoding a heterologous protein;
• contacting the genome and the heterologous nucleic acid in the presence of a bacteriophage Bxb1 serine integrase under conditions in which sequence-specific recombination occurs between the first recombination sequence and the second recombination sequence to provide a recombined HSV genome;
• and
• transfecting a complementing cell with the recombined HSV genome to obtain the HSV gene expression vector,
• wherein the first recombination sequence comprises a Bxb1 attB sequence from Mycobacterium smegmatis identified in SEQ ID NO:
• 1 and the second recombination sequence comprises an attP sequence identified in SEQ ID NO:
• 2 from bacteriophage Bxb1.