Tyrosine Hydroxylase 재조합 단백질의 제작과 특이항체의 생산

Abstract

Parkinson's disease (PD) with resting tremor, rigidity, bradykinesia, leading to dementia was first documented by James Parkinson in 1817. The cause of PD is not clearly known but epidemic encephalitis, slow virus, genetic and environmental background were suggested and recently, reactive oxyradicals. Anatomically, these factors could cause progressive degeneration of dopaminergic neurons of substantia nigra pars compacta (SNc) which ultimately cause denervation and dopamine deficiency in striatal region. This deficiency can result in a relative enhancement of the action of acetylcholine, the excitatory neurotransmitter. Based on these findings, surgical and chemical treatment were tried but with side effects and reduced effectiveness on time, which invoked the development of gene therapy. In order to facilitate the research on tyrosine hydroxylase (TH) which is the prime candidate for PD gene therapy, we have raised a polyclonal antibody against TH. A cDNA fragment corresponding to amino acid #91 to #286 of TH was PCR amplified and subcloned into bacterial expression vector, pRSETb after thorough DNA sequence analysis. The expression of recombinant protein of about 30 kD derived from the partial TH in pRSETb reached peak at approximately 1 hour post IPTG treatment. Approximately 500 ㎍ of recombinant protein was purified from 500 ml culture using His-bind resin (Novagn). 100 ㎍ of antigen emulsified in Freund's complete adjuvant was used to immunize rabbit (intramuscular). Four to six weeks after the primary immunization, the rabbit was boosted with 100 ㎍ of antigen emulsified in Freund's incomplete adjuvant followed by a second boost in three to four weeks. Production and specificity of the antibody produced was confirmed by western blot analysis.