Lee A H, Suh Y S, Sung J H, Yang S H, Sung Y C
Department of Life Science, School of Environmental Engineering, Pohang University of Science and Technology, Korea.
Mol Cells. 1997 Aug 31;7(4):495-501.
Intramuscular injection of plasmid DNA is an efficient method to introduce a foreign gene into a live animal. We investigated several factors affecting the gene transfer efficiency and the following immune response by intramuscular injection of plasmid DNA. When the strength of several highly efficient viral promoters was compared in muscle by using the chloramphenicol acetyltransferase (CAT) gene as an indicator, cytomegalovirus (CMV) immediate early promoter was found to be stronger than any other viral promoters including Rous sarcoma virus (RSV), murine leukemia virus (SL3-3) and simian virus 40 (SV40) early promoters. Inclusion of adenovirus tripartite leader (TPL) sequences and a synthetic intron in the 5' untranslated region of mRNA moderately stimulated the CAT expression. On the other hand, the expression of encephalomyocarditis virus (EMCV) VP1 gene was greatly enhanced by the TPL sequences and an intron. The level of humoral immune response by intramuscular injection of various VP1 expression plasmids was compared. The seroconversion rate was highly dependent on the strength of the expression vector. However, the ratio of IgG1 and IgG2a immune response was not significantly variable depending on the strength of the expression vector. Also, the efficiency of the sindbis virus-based DNA vector was examined for the gene expression and immune response. Although a high level of CAT expression was obtained in muscle by using this system, VP1 was not produced as much as the conventional expression vectors. Furthermore, little humoral immune response was elicited by intramuscular injection of VP1-expressing sindbis vector, suggesting that this system was not superior to the conventional vector for DNA immunization.
肌肉注射质粒DNA是将外源基因导入活体动物的一种有效方法。我们通过肌肉注射质粒DNA研究了影响基因转移效率及后续免疫反应的几个因素。当以氯霉素乙酰转移酶（CAT）基因为指标比较几种高效病毒启动子在肌肉中的强度时，发现巨细胞病毒（CMV）立即早期启动子比其他任何病毒启动子都更强，包括劳氏肉瘤病毒（RSV）、鼠白血病病毒（SL3-3）和猿猴病毒40（SV40）早期启动子。在mRNA的5'非翻译区包含腺病毒三联前导序列（TPL）和一个合成内含子可适度刺激CAT表达。另一方面，脑心肌炎病毒（EMCV）VP1基因的表达通过TPL序列和一个内含子得到极大增强。比较了肌肉注射各种VP1表达质粒后的体液免疫反应水平。血清转化率高度依赖于表达载体的强度。然而，IgG1和IgG2a免疫反应的比例并不因表达载体的强度而有显著变化。此外，还检测了基于辛德毕斯病毒的DNA载体在基因表达和免疫反应方面的效率。尽管使用该系统在肌肉中获得了高水平的CAT表达，但VP1的产生量不如传统表达载体。此外，肌肉注射表达VP1的辛德毕斯载体引发的体液免疫反应很少，这表明该系统在DNA免疫方面并不优于传统载体。
