Nucleic acid constructs for gene expression
A nucleic acid construct, a technology for constructs, applied in the fields of molecular biology and immunology, which can solve problems such as large differences in responses
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[0137] Preparation of viral genomic nucleic acid
[0138] Generally, viral genomic nucleic acids for use in the present invention can be obtained from a genomic library of a particular virus of choice. Other methods may also be used, such as PCR amplification of selected regions of the viral genome in several fragments or a single fragment, or obtained from existing clones of sub-regions of the viral genome.
[0139] Viral genomic libraries can be generated by any method known in the art. In many embodiments of the invention, the viral genomic nucleic acid in the constructs of the invention can be, or can be derived from, a fragment from a genomic library. Various sources are available for genomic DNA. Genomic DNA is commercially available, eg, from sources such as Advanced Biotechnologies Inc (ABI) and Clonetech, Inc. Another standard source is genomic DNA isolated directly from the virus of choice.
[0140] Viral genomic nucleic acids used in the constructs of the invent...
Embodiment 1
[0209] Example 1: Construction of OP23-6 Constructs
[0210] An HSV-2 construct containing all four immediate early genes but lacking the genome sequence of an unrelated virus was constructed. The starting point for construction of this vector was a cosmid containing three EcoRI fragments spanning nucleotides 110,931 to 147,530 from the HSV-2MS strain of the HSV-2 genome based on the published sequence (strain HG52). Gene sequences are also shown in published sequences.
[0211] Cosmids were partially digested with EcoRI and religated and constructs with only 28,000 fragments (110,931-139,697) were selected. The molecule was named OP-23. Six changes were made from this molecule to remove most of the non-essential sequences from the viral genomic nucleic acid. Change as follows:
[0212] 1. Bst1107I and ScaI digestion of cosmids and religation (to remove the ampicillin resistance gene) to generate OP23-1.
[0213] 2. NsiI digestion and religation to remove the SV40 origin ...
Embodiment 2
[0220] Example 2: Generation of a multi-antigen HSV1 vaccine lacking unrelated sequences
[0221] A similar type of vaccine against HSV-1 as described in Example 1 can be prepared against HSV-2.
[0222] HSV-1 was partially digested with EcoRI to generate genomic fragments, and cosmids containing these fragments were subsequently generated using the superCos kit from Stratagene. Cosmids containing sequences from 110,095 to 146,694 of the HSV-1 genome were then selected. Several digestions and religations can then be performed to generate the final compact vector expressing the 4 immediate early genes from HSV-1 but lacking most of the non-essential intervening sequences:
[0223] The following steps were performed to generate the desired constructs:
[0224] 1. Cosmid HSV1 (43392bp) was digested with ScaI and NdeI and religated to obtain OPhsv1-1.
[0225] 2. OPhsv1-1 (39694bp) was digested with AflII and ClaI and religated to obtain OPhsv1-2.
[0226] 3. Digest and religa...
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