Construction and expression of nucleic acid vaccine pVAX1-h-alpha S1–140 coding human alpha-synuclein

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Abstract

BACKGROUND:

The deposition of α -synuclein (α -syn) aggregates is a neuropathological feature of Parkinson's disease. It remains impossible to involve α -syn aggregation in the treatment of Parkinson's disease. A nucleic acid vaccine will provide a new pathway to immunotherapy for Parkinson's disease.

OBJECTIVE:

To construct a recombinant eukaryotic expression vector pVAX1 coding human α -syn and to observe its expression level in COS-7 cells.

DESIGN AND SETTING:

The present bioengineering and molecular biology experiment was performed at Department of Neurology, First Affiliated Hospital of Chongqing Medical University & Chongqing Key Laboratory of Neurology.

MATERIALS:

The eukaryotic expression plasmid pVAX1, human embryonic brain tissue, healthy human blood cells, and COS-7 cells were purchased from Promega Company, USA.

METHODS:

The full-length CDS sequence of the human α -syn gene was amplified by RT-PCR, which contained restriction sites for the enzymes Kpn I, Xba I and Kozak consensus sequence. Then the PCR products and eukaryotic expression vector pVAX1 were digested with Kpn I and Xba I simultaneously, and were extracted and ligated by T4 ligase. The recombinant constructs pVAX1-h α -S1–140 were transformed into competent E. coli TOP 10 cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis, and DNA sequencing. The constructs were then tested for protein expression in COS-7 cells by RT-PCR and Western blotting.

MAIN OUTCOME MEASURES:

Identification of an eukaryotic expression vector containing the human α -syn gene, pVAX1-h α -S1–140, and detection of the expression in mammalian cell COS-7.

RESULTS:

The pVAX1 vector was successfully cloned with human α -syn in the correct orientation and in-frame. The DNA vaccine constructs pVAX1-h α -S1–140 with the human α -syn gene were shown to be expressed in COS-7 cells. Human α -syn was successfully expressed in the mammalian cell line and was detected by RT-PCR and western blotting.

CONCLUSION:

Nucleic acid vaccine pVAX1-h α S1–140 was successfully constructed and expressed in COS-7 cells.

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