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209 PCR-Based Methods as an Alternative Tool for Generating Humanized Oligonucleotide
S. Sirivichayakul*, T. Tirawatnapong, K. Ruxrungtham, S. Lorenzen, R. Oelrichs, and P. Phanuphak
Chulalongkorn Univ., Bangkok, Thailand
Background: : DNA vaccination has been widely attempted for various infectious agents such as hepatitis B, herpes simplex, and HIV viruses. Humanized DNA is one of the most interesting methods to increase the immune response. The concept of humanized DNA is to change the viral DNA coding sequence to those most commonly used in human without changing the amino acid sequences of the protein they encode. The humanized construct of gag DNA has been reported of being more immunogenic in mice than the unaltered one. Whereas most of the humanized DNA constructs were synthesized, in this report we used the PCR-based method to generate an HIV envelope vaccine for further testing.
Methods: A 297-bp humanized DNA of the HIV subtype E envelope V3 region was created by PCR technique. This 297-bp region includes V3, CTL, and T-helper epitopes, CD4 binding site, and neutralizing epitopes. Three primers of 134-, 138-, and 125-mer in length with 25-mer overlapping regions with one another were designed and synthesized by a commercially available company. The first primer, which is 134-mer long, includes EcoRI site, Kozak sequence (to increase expression) and ATG starting codon. The third one, which is 125-mer long, includes TAG stop codon and XbaI site. These 3 primers were mixed altogether without any DNA template in a conventional PCR reagents with Taq DNA polymerase and then subjected to nested PCR with another pair of primers that cover the whole 297-bp long PCR. The correct sequence of the PCR product was verified by sequencing reaction and then was cloned into pCI expression vector. COS-7 cells were transfected with the 297-bp cloned plasmid for 48 hours. RNA was extracted from the transfected cells and then subjected to RT-PCR and nested PCR.
Results: The 297-bp PCR product was detected from transfection experiment, which confirmed that this 297-bp humanized DNA construct produced by PCR could be expressed in vitro. The immunogenicity of this DNA construct needs to be performed and compared with the native one.
Conclusions: The PCR-based method can be used as an alternative tool to generate 297-bp humanized oligonucleotide. This method is more economical and more convenient than the one obtained by automatic synthesizer.
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